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Hallie Leighton had dense breasts — a fact she discovered only in her late 30s, via a mammogram. She grew up in an Ashkenazi family in New York, pursued a career in writing and worked with organizations promoting peace between Israelis and Arabs. By 2013 she was making a documentary on her father Jan Leighton, an actor who set the record as an actor for appearing in the most roles (2,407 according to the 1985 Guinness Book of World Records). She was never able to complete it. She died that year, at the age of 42.
Every woman in Leighton’s family had breast cancer, so she began getting annual mammograms at 35 — five years earlier than the recommended age. In 2009 the results of Leighton’s mammogram came in as “negative” or “normal”; by 2013 she was bedridden, undergoing her final days of chemotherapy.
When Leighton was first diagnosed in 2010, her doctor told her, “You have breast cancer, and it was there in 2009.” The tumor in Leighton’s breast went undiscovered until it was palpable — and at that point, the cancer was already in stage 4.
“Happygram,” a documentary which exposes some of the shortcomings in mammography, chronicles Leighton’s struggle with cancer and the implications of having dense breasts.
“Most women simply aren’t informed that they have dense breast tissue,” said Leighton’s best friend Julie Marron. She wrote and directed the documentary, which is currently screening at film festivals around the country.
Breast density is defined by the relative amount of fat in relation to the amount of connective and epithelial tissue (tissue that lines blood vessels and cavities). When more than 50% of breast tissue is connective and epithelial tissue, instead of fatty tissue, the breasts are considered dense. Mammography is the only way to determine breast density.
“If you have dense breasts, what looks dense on a mammogram looks the same as a cancer would look. It tends to confuse or confound the physician, and reduces the sensitivity of the mammogram,” said Gerald Kolb, founder and president of The Breast Group, which counsels clients on different technologies in breast care. “Hallie Leighton’s breasts looked like snowballs; there was no chance they were going to find anything with the mammogram.”
Forty percent of women who are screened for breast cancer have dense breast tissue. These women also account for more than 70% of all invasive cancers. “Mammograms are not very effective screening tools for these women, as they miss between 50% and 75% of all invasive cancers in dense breast tissue,” Marron said. “This is obviously a very critical issue when you are dealing with a population that is more likely to develop cancer.”
Ashkenazi women are even more at risk. They are 1.6 times more likely than the general population to have dense breast tissue, according to Kolb. Moreover, one in 40 Ashkenazi women will test positive for one or both of BRCA gene mutations responsible for breast cancer. For the general population, that number is between one in 350 and one in 800.The BRCA 1 or 2 genes don’t cause cancer, they fight cancer, Kolb says. But if the gene is mutated, the body is not as well equipped to fight the cancer.
“A woman with a BRCA mutation has a lifetime risk of around 33% to 87%, depending on the gene and mutation,” Marron said. “Compare this to a lifetime risk of 12% for developing breast cancer for the overall population.” BRCA gene mutations can be inherited from either or both parents, and therefore they can be present in men as well as in women.
Breast density and BRCA gene mutations are not directly related, but both independently present an increased susceptibility to breast cancer.
“The biggest risk is that a doctor is not going to find the cancer when it’s really small,” Kolb said. When a tumor is detected at a centimeter or smaller, there’s a 95% cure rate. But if the cancer is the size of a golf ball by the time it’s detected, Kolb says, the woman has a 60% chance of living for five years, and then her mortality increases dramatically.
The good news is that mammography isn’t the only method of detecting breast cancer; the bad news is that very few people know this. “What we’re trying to do in the density movement is give women enough information so they can ask appropriate questions of a doctor,” Kolb said.
Kolb advises high-risk women to get a genetic risk analysis, which can be performed by a genetic counselor or a radiologist. He advises getting the risk analysis as early as age 25, but doing so is a personal decision. Not every woman is emotionally prepared to know the results.
“Mammography is a starting point,” said Dr. Dennis McDonald, a California-based women’s imager. Additionally, doctors recommend that women with dense breasts get an MRI, which McDonald says is reserved for high-risk women. It’s an expensive, invasive and time-consuming procedure that requires the injection of fluid in order to read the MRI. As of yet, doctors do not know the side effects of getting an annual MRI.
“A doctor should have started [Leighton] on an MRI right away. She was high risk and they chose to just monitor with a mammogram,” Kolb said. “That’s insufficient.”
Breast ultrasound is another alternative for women with dense breast tissue. “Most of the time, breast density doesn’t present a problem [with ultrasounds],” McDonald said. Though the ultrasound is effective in detecting cancer, he says the downside is that radiologists are often not that comfortable with the technology, simply because they have little experience with it. There are also a lot of false positives, he adds, which result in unnecessary exams or biopsies.
As “Happygram” documents, informing women of their breast density and of alternatives to mammography is a highly charged political issue.
“The whole breast cancer industry has grown up around mammograms,” Marron said. “Physicians weren’t educated on [breast density], deliberately so to a certain extent, and refused to inform patients on this issue, which is really outrageous if you think about it.” Marron says that doctors are required by law and ethical guidelines to inform patients of “material” medical information. “There is no legitimate reason that women have not been informed of this information,” she noted.
After Leighton’s diagnosis, she wanted to ensure that other women didn’t suffer the same misfortune of all-too-late tumor discovery on account of dense breast tissue. She gave media interviews, lobbied in Albany and starred in “Happygram,” all the while undergoing chemotherapy. She died four months after the Breast Density Information Bill passed in New York.
Similar legislation has been passed in more than 20 states throughout the country, but not without objection. Many well-intentioned radiologists, poorly informed about alternative screening options, feared that telling women the limitations of mammography would cause them to lose faith in it altogether and not get tested. Others argued that the information would make women anxious, and that it wouldn’t be fair for those who couldn’t afford additional testing. And still further arguments against informing women were possibly impacted by financial considerations, Marron added.
“Women aren’t getting the benefit of full notification across the board yet,” Marron said. “I think that has to change through education. That’s the primary reason we made this movie. There’s been so much resistance within the medical community to telling women. Change isn’t going to come from the medical community, it has to come from the patients.”
Ashkenazi women shouldn’t panic, Kolb says, but they need to carefully examine their breast density and alternative screening options: “Anytime you have a preventative tragedy like that, you have to do everything in your power to stop it from happening.”
Madison Margolin is a freelance writer based in New York. She writes frequently for the Village Voice.
HER-2 is an oncogene and a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or overexpression of HER-2 plays an important role in the development and progression of certain aggressive types of breast cancer.
Breast cancer patients often display elevated levels of proteins in plasma or serum, and many of these proteins correlate to active or recurrent disease, metastasis, prognosis, and therapeutic response. Several of these markers have demonstrated clinical utility, particularly in the area of monitoring disease recurrence after or during therapeutic treatment in advanced disease.
The application of immunoassays to monitor levels of breast-cancer-related proteins in blood or serum is complementary to other diagnostics such as imaging and can be a valuable part of the routine management of disease. The availability of a group of easily applied blood tests is a convenient and powerful addition to the arsenal of technologies available to physicians for patient management. Immunoassays are available for serum-borne breast-cancer-related markers such as cancer antigen 15-3 (CA 15-3), carcinoembryonic antigen (CEA), tissue inhibitor of metalloproteinases-1 (TIMP-1), and human epidermal growth factor receptor 2 (HER2)—all of which are discussed in this article.
Clinical Utility Considered by Multiple Studies
While essential for diagnosis and typing, tissue testing during long-term breast cancer management is impractical, costly, and painful for patients. A growing number of studies support serum-based immunoassay testing to monitor drug response, disease progression, and potential for metastases.
In 2012, Tsai et al.1 studied serum levels of HER2 and TIMP-1 in 185 breast cancer patients in Taiwan. They concluded that TIMP-1 was significantly associated with serum HER2-positive status in circulation, as well as poorer disease-free survival—suggesting that monitoring both of these biomarkers may be beneficial.
In the same year, Kontani et al.2 reported that serum HER2 is a useful biomarker not only for detecting breast cancer recurrence but also for predicting tumor responses to trastuzumab. In 2014, Shao et al.3 found that elevated serum HER2 levels were significantly associated with short-term response to trastuzumab treatment. The median progression-free survival was significantly longer in patients with low levels of serum HER2. Furthermore, they found that over time patients with remaining low serum HER2 levels or those who achieved low serum HER2 levels after treatment had significantly longer progression-free survival than those whose levels remained high or converted from low to high.
In 2015, Di Gioia et al.4 reported that they had investigated the combination of CEA, CA 15-3, and serum HER2 in the pretherapeutic serum of 241 patients as biomarkers for prognosis in early breast cancer. Their retrospective analysis confirmed that serum HER-2 and CA15-3 (but not CEA) were independent and better prognostic tools than HER-2 in tissue. However, they concluded that prospective validation is necessary to confirm usefulness in routine clinical practice.
In a review published in 2015, Ravelli et al.5 discussed the advantages, drawbacks, and new insights with respect to measuring circulating breast-cancer-related biomarkers with immunoassays as compared to standard tissue-based biopsies. They suggested that a reliable panel of circulating cancer biomarkers would be helpful for the following tasks:
Screening and diagnostic procedures
Predicting prognosis
Selecting therapeutic options, including experimental ones
Detecting a lack of efficacy of an ongoing therapy and predicting side-effects
Identifying recurrence
In this review, the authors emphasized “classic” markers such as CA 15-3, CEA, and serum HER2. With respect to CA 15-3 and CEA, the authors noted that they might be most useful in combination. “[More] reliable prediction power have been obtained with the combination of the two biomarkers, whereas when measured singularly, both sensitivity and specificity values were drastically decreased,” the authors wrote. “As a result, the association of the two markers may be a useful independent tool in the follow-up of [breast cancer] patients.”
In their discussion of HER2, the researchers pointed out that increased serum HER2 levels have been clearly associated with the tissue HER2 status, the presence and number of metastases, and the levels of CA 15-3 and CEA.
Utility Strongly Suggested by a Large Study
The review cited a 2014 study that reported how 2,862 primary breast cancer patients had been monitored to demonstrate the correlation between serum HER2 and tissue HER2 overexpression.6This study showed that 15% of tissue-HER2-positive patients also had increased serum HER2 levels and that there was a linear correlation with the increased aggressiveness of tumors. Multivariate analysis confirmed that increased serum HER2 is an independent prognostic factor that can be clinically useful, particularly in patients with tissue-HER2-positive tumors.
The authors of the study concluded that measuring serum HER2 could help oncologists monitor patient response to trastuzumab in the absence of tissue HER2. They added that further clinical validation would be worthwhile.
Summary
Basic immunoassay technology has been in place since the 1950s and has become the gold standard for clinical protein measurement due to high sensitivity and selectivity.7 Growing evidence suggests that these reliable, robust tests can provide valuable insights for physicians managing breast cancer treatment.
This post contains a curation of all Early Diagnosis posts on this site as well as a curation of the Early Detection Research Network.
Early Research Detection Network (EDRN)
Welcome to EDRN
The Early Detection Research Network (EDRN), an initiative of the National Cancer Institute (NCI), brings together dozens of institutions to help accelerate the translation of biomarker information into clinical applications and to evaluate new ways of testing cancer in its earliest stages and for cancer risk.
Getting Started…
Check out the EDRN Highlights — a listing of our accomplishments and milestones.
Since its inception in 1999 EDRN has achieved several key milestones, summarized below:
1998 through 2000: Inception and Inauguration of EDRN
2001 to 2003: Meeting the Challenges to Harness and Share Emerging Scientific Knowledge
EDRN Second Report, Translational Research to Identify Early Cancer and Cancer Risk, October 2002, http://edrn.nci.nih.gov/docs.) published.
EDRN joined the Gordon Research Conferences to co-host the New Frontiers in Cancer detection and Diagnosis in 2002.
Guidelines Set for Studies Measuring Biomarker Predictive Power Journal of National Cancer Institute (Vol. 93, No. 14, July 18, 2001).
EDRN Associate Membership Program Initiated: This novel approach to make EDRN inclusive has been extremely successful. EDRN has now more than 120 Associate Members who are significantly contributing to EDRN efforts in biomarker discovery, development and validation.
2003 to 2004: Network Surges Ahead in Real-time
Collaborative Discovery and Validation Projects: More than 100 collaborative projects spanned the various organ sites. These projects are monitored through the EDRN’s electronic System Information System (eSIS).
EDRN Virtual Specimen Bank and Validation Management System Launched: The EDRN Virtual Specimen Bank, also known as ERNE knowledge system, was deployed to 10 institutions in early 2003, allowing a common web-based query to search for available specimens across the EDRN Clinical Epidemiology and Validation Centers https://ginger.fhcrc.org/edrn/imp/GateServlet?pwd. VSIMS was created to allow multiple studies to be administered efficiently by minimizing development time with standardization of information and data management across multiple activities and research sites. This system encompasses all the security features of Food and Drug Administration (FDA)-required auditing systems.
Partnership on the Plasma Proteome Project (PPP) Initiative of the Human Proteome Organization (HUPO): PPP project was initiated to evaluate multiple technology platforms, develop bioinformatic tools and standards for protein identification, and create a database of the plasma proteome. The entire study was published in the August issue of the journal Proteomics August 2005, Volume 4 (4), pp 1045-1450.
2005 to 2008: An Investment in Prevention
In late 2006, EDRN’s Program for Rapid, Independent Diagnostic Evaluation (PRIDE), was established (http://grants.nih.gov/grants/guide/notice-files/NOT-CA-07-003.html ) as an administrative means to assist extramural investigators in successfully conducting cross-laboratory validation of biomarkers. Ten applications have been reviewed and five are being supported.
EDRN underwent external reviews in 2007 and 2008.
The Canary Foundation, Palo Alto, CA signed a Memorandum of Understanding with EDRN, NCI on supporting prostate cancer surveillance network of investigators from seven institutions. The tissue and serum will be collected during a three-year period and will be made available to extramural scientists for discovery and validation research.
The Lustgarten Foundation, N.Y., funded 6 institutions to generate monoclonal antibodies and associated hybridoma cell lines for pancreatic cancer antigens (biomarkers) identified by EDRN and non-EDRN investigators. These resources will be stored at the NCI-Frederick Facility for distribution to extramural investigators.
2009 to 2011: Realizing Investment for Clinical Use
Two biomarker tests approved by FDA and two IVDs pending FDA review.
Six biomarker tests offered by CLIA labs.
One biomarker test approved for clinical use outside the USA
A Curation of Posts on Early Detection of Cancer and Other Early Detection Networks is Included Below
New Generation of Platinated Compounds to Circumvent Resistance
Curator/Writer: Stephen J. Williams, Ph.D.
Resistance to chemotherapeutic drugs continues to be a major hurdle in the treatment of neoplastic disorders, irregardless if the drug is a member of the cytotoxic “older” drugs or the cytostatic “newer” personalized therapies like the tyrosine kinase inhibitors. For the platinatum compounds such as cisplatin and carboplatin, which are mainstays in therapeutic regimens for ovarian and certain head and neck cancers, development of resistance is often regarded as the final blow, as new options for these diseases have been limited.
Although there are many mechanisms by which resistance to platinated compounds may develop the purpose of this posting is not to do an in-depth review of this area except to refer the reader to the book Ovarian Cancer and just to summarize the well accepted mechanisms of cisplatin resistance including:
Decreased cellular cisplatin influx
Increased cellular cisplatin efflux
Increased cellular glutathione and subsequent conjugation, inactivation
Increased glutathione-S-transferase activity (GST) and subsequent inactivation, conjugation
Increased γ-GGT
Increased metallothionenes with subsequent conjugation, inactivation
Increased DNA repair: increased excision repair
DNA damage tolerance: loss of mismatch repair (MMR)
altered cell signaling activities and cell cycle protein expression
Williams, S.J., and Hamilton, T.C. Chemotherapeutic resistance in ovarian cancer. In: S.C. Rubin, and G.P. Sutton (eds.), Ovarian Cancer, pp.34-44. Lippincott, Wilkins, and Williams, New York, 2000.
Also for a great review on clinical platinum resistance by Drs. Maritn, Hamilton and Schilder please see the following Clinical Cancer Research link here.
This curation represents the scientific rationale for the development of a new class of platinated compounds which are meant to circumvent mechanisms of resistance, in this case the loss of mismatch repair (MMR) and increased tolerance to DNA damage.
An early step in the production of cytotoxicity by the important anticancer drug cisplatin and its analog carboplatin is the formation of intra- and inter-strand adducts with tumor cell DNA 1-3. This damage triggers a cascade of events, best characterized by activation of damage-sensing kinases (reviewed in 4), p53 stabilization, and induction of p53-related genes involved in apoptosis and cell cycle arrest, such as bax and the cyclin-dependent kinase inhibitor p21waf1/cip1/sdi1 (p21), respectively 5,6. DNA damage significantly induces p21 in various p53 wild-type tumor cell lines, including ovarian carcinoma cells, and this induction is responsible for the cell cycle arrest at G1/S and G2/M borders, allowing time for repair 7,8. DNA lesions have the ability of to result in an opening of chromatin structure, allowing for transcription factors to enter 56-58. Therefore the anti-tumoral ability of cisplatin and other DNA damaging agents is correlated to their ability to bind to DNA and elicit responses, such as DNA breaks or DNA damage responses which ultimately lead to cell cycle arrest and apoptosis. Therefore either repair of such lesions, the lack of recognition of such lesions, or the cellular tolerance of such lesions can lead to resistance of these agents.
Mechanisms of Cisplatin Sensitivity and Resistance. Red arrows show how a DNA lesion results in chemo-sensitivity while the beige arrow show common mechanisms of resistance including increased repair of the lesion, effects on expression patterns, and increased inactivation of the DNA damaging agent by conjugation reactions
Increased DNA Repair Mechanisms of Platinated Lesion Lead to ChemoResistance
Description of Different Types of Cellular DNA Repair Pathways. Nucleotide Excision Repair is commonly up-regulated in highly cisplatin resistant cells
Loss of Mismatch Repair Can Lead to DNA Damage Tolerance
In the following Cancer Research paper Dr. Vaisman in the lab of Dr. Steve Chaney at North Carolina (and in collaboration with Dr. Tom Hamilton) describe how cisplatin resistance may arise from loss of mismatch repair and how oxaliplatin lesions are not recognized by the mismatch repair system.
Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.
The following are slides I had co-prepared with my mentor Dr. Thomas C. Hamilton, Ph.D. of Fox Chase Cancer Center on DNA Mismatch Repair, Oxaliplatin and Ovarina Cancer.
Multiple Platinum Analogs of Cisplatin (like Oxaliplatin )Had Been Designed to be Sensitive in MMR Deficient Tumors
Please see below video on 2015 Nobel Laureates and their work to elucidate the celluar DNA repair mechanisms.
Clinical genetics expert Kenneth Offit gives an overview of Lynch syndrome, a genetic disorder that can cause colon (HNPCC) and other cancers by defects in the MSH2 DNA mismatch repair gene. (View Video)
References
Johnson, S. W. et al. Relationship between platinum-DNA adduct formation, removal, and cytotoxicity in cisplatin sensitive and resistant human ovarian cancer cells. Cancer Res54, 5911-5916 (1994).
Eastman, A. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology and Therapeutics34, 155-166 (1987).
Zhen, W. et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Molecular and Cellular Biology12, 3689-3698 (1992).
Durocher, D. & Jackson, S. P. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol13, 225-231 (2001).
el-Deiry, W. S. p21/p53, cellular growth control and genomic integrity. Curr Top Microbiol Immunol227, 121-37 (1998).
Ewen, M. E. & Miller, S. J. p53 and translational control. Biochim Biophys Acta1242, 181-4 (1996).
Gartel, A. L., Serfas, M. S. & Tyner, A. L. p21–negative regulator of the cell cycle. Proc Soc Exp Biol Med213, 138-49 (1996).
Chang, B. D. et al. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene19, 2165-70 (2000).
Davies, N. P., Hardman, L. C. & Murray, V. The effect of chromatin structure on cisplatin damage in intact human cells. Nucleic Acids Res28, 2954-2958 (2000).
Vichi, P. et al. Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP. Embo J16, 7444-7456 (1997).
Xiao, G. et al. A DNA damage signal is required for p53 to activate gadd45. Cancer Res60, 1711-9 (2000).
Other articles in this Open Access Journal on ChemoResistance Include:
The cancer stem-cell hypothesis proposes the existence of a subset of cells within a heterogeneous tumor cell population that have stem-cell like properties [1], and may be essential for the progression and metastases of epithelial malignancies, by providing a reservoir of cells that self-renew and differentiate into the bulk of the tumor [2]. The stem-cell hypothesis implies that similar genetic regulatory pathways might define critical stem-cell like functions, such as self-renewal and pluripotency, in both normal and cancer stem-cells. Indeed, cancer stem-cells have been identified in many tumor types, such as breast [3], pancreas [4] and ovarian [5], based on screening with cellular markers typically found in normal stem-cells such as CD44, ALDH1, and CD133 (reviewed in [2]). A number of studies have suggested that the expression of these stem-cell markers is correlated with poor prognosis [6-9]. The ability to identify and isolate these populations may have a significant impact on design of individualized therapies.
Great general posts and good review on this site about Cancer Stem Cells, their markers, and ability to target them with chemotherapy can be seen here.
However, there has been growing acknowledgement of the ability of cancer stem cell populations to resist the cytotoxic effects of most chemotherapeutic agents, including cisplatin, topoisomerase inhibitors, DNA damaging agents, and even tyrosine kinase inhibitors (TKI). Indeed, some feel that intrinsic resistance to cytotoxic drugs may be a biological feature of cancer stem cells.
Definitions:
Acquired resistance: a resistance to a particular drug which results following continued exposure to said drug. Can take days (in cases of some TKIs) or months to develop. Acquired resistant cells lines are developed by exposure to increasing drug concentration over a time period (either intermittent exposure or continuous exposure)
Intrinsic resistance: a pre-existing resistance usually termed refractory where cancer cells THAT HAVE NOT BEEN EXPOSED to drug, do not respond to initial drug exposure. Can be seen experimentally in panels of unrelated cancer cells lines isolated from untreated patients which show no cytotoxicity to drug exposure in vitro.
Below is one of the first reports which described the drug resistant phenotype of cancer stem cells in an in vivo (mouse) model of breast cancer with videos.
Cancer Res. 2008 May 1;68(9):3243-50. doi: 10.1158/0008-5472.CAN-07-5480.
The majority of BRCA1-associated breast cancers are basal cell-like, which is associated with a poor outcome. Using a spontaneous mouse mammary tumor model, we show that platinum compounds, which generate DNA breaks during the repair process, are more effective than doxorubicin in Brca1/p53-mutated tumors. At 0.5 mg/kg of daily cisplatin treatment, 80% primary tumors (n = 8) show complete pathologic response. At greater dosages, 100% show complete response (n = 19). However, after 2 to 3 months of complete remission following platinum treatment, tumors relapse and become refractory to successive rounds of treatment. Approximately 3.8% to 8.0% (mean, 5.9%) of tumor cells express the normal mammary stem cell markers, CD29(hi)24(med), and these cells are tumorigenic, whereas CD29(med)24(-/lo) and CD29(med)24(hi) cells have diminished tumorigenicity or are nontumorigenic, respectively. In partially platinum-responsive primary transplants, 6.6% to 11.0% (mean, 8.8%) tumor cells are CD29(hi)24(med); these populations significantly increase to 16.5% to 29.2% (mean, 22.8%; P < 0.05) in platinum-refractory secondary tumor transplants. Further, refractory tumor cells have greater colony-forming ability than the primary transplant-derived cells in the presence of cisplatin. Expression of a normal stem cell marker, Nanog, is decreased in the CD29(hi)24(med) populations in the secondary transplants. Top2A expression is also down-regulated in secondary drug-resistant tumor populations and, in one case, was accompanied by genomic deletion of Top2A. These studies identify distinct cancer cell populations for therapeutic targeting in breast cancer and implicate clonal evolution and expansion of cancer stem-like cells as a potential cause of chemoresistance.
Please Watch Videos
Below is a curation of talks and abstracts from the 2015 Annual AACR Meeting in Philadelphia, PA.
The Talk by Dr. Cheresh is an example of this school of thought; that inducing cancer cell stemness can result in development of drug resistance, in this case to a TKI. (For a press release on this finding see here.)
Induction of cancer stemness and drug resistance by EGFR blockade
Presentation Time:
Tuesday, Apr 21, 2015, 12:00 PM -12:15 PM
Abstract Body:
Tumor drug resistance is often accompanied by genetic and biological changes in the tumor cell population reflecting the acquisition of a stem-like state. However, it is not clear whether cancer therapies select for the growth of drug resistance cancer stem cells and/or directly induce the reprograming of tumor cells to a cancer stem-like, drug resistance state. We provide evidence that breast, pancreas and lung carcinomas in the presence of prolonged exposure to EGFR inhibitors undergo an epigenetic reprogramming resulting in a drug resistant stem-like tumor population expressing the cell surface marker CD61 (b3 integrin). In fact, CD61 in the context of KRAS, is necessary and sufficient to account for drug resistance, tumor initiation, self-renewal and expression of the pluripotent genes Oct 4 and Nanog. Once expressed, CD61 in the unligated state recruits KRAS to the plasma membrane leading to the activation of RalB, TBK1 and c-Rel driving both stemness and EGFR inhibitor resistance. Pharmacological targeting this pathway with drugs such as bortezomib or revlimid not only reverses stemness but resensitizes these epithelial tumors to EGFR inhibition. This epigenetic pathway can also be initiated by range of cellular stresses found within the tumor microenvironment such as hypoxia, nutrient deprivation, low pH, and oxidative stress. In normal tissues CD61 is induced during tissue remodeling and repair. For example, CD61 was found to be critical for mammary gland remodeling during pregnancy and as a mediator of pathological neovascularization. Together these findings reveal a stress-induced epigenetic pathway characterized by the upregulation of CD61 that promotes the remodeling of normal tissues but in tumors contributes to EGFR inhibitor resistance and tumor progression.
Molecular and Cellular Biology – Poster Presentations – Proffered Abstracts – Poster Presentations – Cell Death Mechanisms: Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines
Aoi Kuroda,
Keiko Ohgino,
Hiroyuki Yasuda,
Junko Hamamoto,
Daisuke Arai,
Kota Ishioka,
Tetsuo Tani,
Shigenari Nukaga,
Ichiro Kawada,
Katsuhiko Naoki,
Kenzo Soejima,
and Tomoko Betsuyaku
Cancer Res August 1, 2015 75:4; doi:10.1158/1538-7445.AM2015-4
Molecular and Cellular Biology – Poster Presentations – Proffered Abstracts – Poster Presentations – Cell Death Mechanisms: Abstract 6: Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199)
Sha Jin,
Paul Tapang,
Donald J. Osterling,
Wenqing Gao,
Daniel H. Albert,
Andrew J. Souers,
Joel D. Leverson,
Darren C. Phillips,
and Jun Chen
Cancer Res August 1, 2015 75:6; doi:10.1158/1538-7445.AM2015-6
Molecular and Cellular Biology – Poster Presentations – Proffered Abstracts – Poster Presentations – Cell Death Mechanisms: Abstract 24: The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage
Nurmaa Khund Dashzeveg and
Kiyotsugu Yoshida
Cancer Res August 1, 2015 75:24; doi:10.1158/1538-7445.AM2015-24
Molecular and Cellular Biology – Poster Presentations – Proffered Abstracts – Poster Presentations – Cell Signaling in Cancer 1: Abstract 48: Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells
Tomoaki Ohtsuka,
Masakiyo Sakaguchi,
Katsuyoshi Takata,
Shinsuke Hashida,
Mototsugu Watanabe,
Ken Suzawa,
Yuho Maki,
Hiromasa Yamamoto,
Junichi Soh,
Hiroaki Asano,
Kazunori Tsukuda,
Shinichiro Miyoshi,
and Shinichi Toyooka
Cancer Res August 1, 2015 75:48; doi:10.1158/1538-7445.AM2015-48
Abstract 1 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Death Mechanisms
Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Rationale:
ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms.
Methods:
The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA.
Results:
By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity.
Conclusion:
We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels.
Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2015-4
Abstract 2 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Death Mechanisms
Abstract 6: Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199)
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
The BCL-2-selective inhibitor venetoclax (ABT-199) binds with high affinity to the BH3-binding groove of BCL-2, thereby competing for binding with the BH3-only protein BIM (Souers et al., 2013). Venetoclax is currently being evaluated in clinical trials for CLL, AML, multiple myeloma and NHL. To facilitate these studies, we developed and validated a 384-well electrochemiluminescent ELISA (MSD, Gaithersburg, MD,USA) that quantifies expression of BCL-2, BCL-XL, and MCL-1protein alone or in complex with BIM. We subsequently quantified expression of BCL-2 and BCL-2:BIM complexes in 16 hematologic tumor cell lines. We found the EC50 of venetoclax in these tumor cell lines to correlate strongly with baseline BCL-2:BIM complex levels. This correlation was superior to the correlation between venetoclax EC50 and absolute BCL-2 expression. We also applied the assay to measure disruption of BCL-2:BIM complexes in vivo. Treatment of the Non-Hodgkin’s Lymphoma (NHL) xenograft model SU-DHL-4 with a BCL-2-selective inhibitor resulted in disruption of tumor BCL-2:BIM complexes that aligned with serum and tumor concentrations of inhibitor. Collectively, these data demonstrate that quantifying BCL-2:BIM complexes offers an accurate means of assessing target engagement by venetoclax and, potentially, predicting its efficacy. The utility of this assay is currently being assessed in clinical trials.
Citation Format: Sha Jin, Paul Tapang, Donald J. Osterling, Wenqing Gao, Daniel H. Albert, Andrew J. Souers, Joel D. Leverson, Darren C. Phillips, Jun Chen. Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 6. doi:10.1158/1538-7445.AM2015-6
Abstract 3 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Death Mechanisms
Abstract 19: Antitumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Survivin, which is highly expressed and promotes cell survival in diffuse malignant peritoneal mesothelioma (DMPM), exclusively relies on the nuclear exportin 1 (XPO1/CRM1) to be released in the cytoplasm and perform its anti-apoptotic function. Here, we explored the efficacy of selective inhibitors of nuclear export (SINEs) in patient-derived DMPM preclinical models. Exposure to individual SINE (KPT-251, KPT-276, KPT-330) was able to induce a time- and dose-dependent inhibition of the growth of two DMPM cell lines without affecting normal cell proliferation. Such a cell growth inhibition was preceded by a decline in the nuclear XPO1/CRM1 levels and an increase in the nuclear accumulation of its cargo proteins p53 and p21, which led to a cell cycle arrest at G1-phase. Our results also indicated that survivin is an essential component of the downstream signaling pathway of XPO1/CRM1 inhibition in DMPM cells. In fact, in both cell lines, exposure to SINEs led to a time-dependent reduction of cytoplasmic survivin levels and, after an initial survivin nuclear accumulation, also to a progressive decrease in the nuclear protein abundance, through the ubiquitin-proteasomal degradation pathway, leading to the complete depletion of total survivin levels. In both DMPM cell models, according to survivin anti-apoptotic activity, drug-induced reduction of cytoplasmic survivin levels correlated with the onset of caspase-dependent apoptosis. We further observed that SINEs can be combined with other survivin inhibitors, such as the survivin suppressant YM155 to achieve enhanced growth inhibition in DMPM cells. Initial in vivo experiments with orally administered KPT-251, KPT-276 and the orally available, clinical stage KPT-330 (selinexor) indicated that each compound was able to significantly reduce the growth of early-stage subcutaneous DMPM xenografts. Interestingly, additional experiments carry out with selinexor demonstrated that the compound was also able to inhibit the growth of late-stage subcutaneous DMPM xenografts in nude mice. Most importantly, oral administration of selinexor to SCID mice reduced the growth of orthotopic DMPM xenografts, which properly recapitulate the dissemination pattern in the peritoneal cavity of human DMPM and, for this reason, represent a valuable model for investigating novel therapeutic approaches for the disease. Consistent with an important role of survivin as a determinant of anti-cancer activity of SINE compounds, a reduction of the protein expression was observed in tumor specimens obtained from selinexor treated mice. Overall, our results (i) demonstrate a marked efficacy of SINEs in DMPM preclinical models, which is, at least in part, dependent on the interference with survivin intracellular distribution and function, and (ii) suggest SINE-mediated XPO1/CRM1 inhibition as a novel therapeutic option for the disease.
Citation Format: Nadia Zaffaroni, Michelandrea De Cesare, Denis Cominetti, Valentina Doldi, Alessia Lopergolo, Marcello Deraco, Paolo Gandellini, Yosef Landesman, Sharon Friedlander, Michael G. Kauffman, Sharon Shacham, Marzia Pennati. Antitumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2015-19
Abstract 4 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Death Mechanisms
Abstract 24: The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Tumor suppressor p53 plays a pivotal role in cell cycle arrest, DNA repair, and apoptosis in response to DNA damage. Promoter selectivity of p53 depends mainly on post-translational modification. Notably, the apoptotic function of p53 is related to its phosphorylation at serine-46 (ser46) to promote pro-apoptotic genes. However, little is known about the pro-apoptotic genes induced by Ser46 phosphorylation. Our research achieved to investigate the pro-apoptotic genes induced by p53 in a phospho-ser46-specific manner using microarray and ChIP sequencing in human cancer cell lines. As a result, palmdelphin (PALMD), an isoform of paralemmin protein, was strongly elicited from the phosphorylation of ser46. The mRNA and protein expression of PALMD increased only in wild type p53 transfected cells, but not in ser46-mutated cells. Importantly, PALMD moved to the nucleus in response to DNA damage and the apoptotic function of PALMD was tightly exerted with localization into nucleus. Interestingly, down-regulation of PALMD by siRNA resulted in necroptosis-like cell death through ATP depletion. Moreover, we found vimentin as a PALMD interacting protein and the depletion of vimentin increased PALMD level to accelerate apoptosis. These results demonstrate that p53 regulates cell death fate (apoptosis or necroptosis-like cell death) through promoting PALMD expression in a phospho-ser46-specific manner in response to DNA damage.
Citation Format: Nurmaa Khund Dashzeveg, Kiyotsugu Yoshida. The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2015-24
Abstract 5 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Signaling in Cancer 1
Abstract 48: Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Human epidermal growth factor receptor 2 (HER2) is a member of epidermal growth factor receptor (EGFR) family. Previous studies have revealed that many kinds of malignant tumors have genetic mutations or amplification of HER2, indicating that HER2 alterations are oncogenic. Many kinds of HER2 targeted therapies are effective to HER2 positive tumors, but those treated tumors often get resistance to drugs. Thus, to elucidate HER2 related pathway in cancer biology is important to develop new therapeutic strategy for cancers.
Recently, we newly identified a protein X (a temporary name) as a novel binding protein to HER2 with immunoprecipitation and following LC-Ms/Ms analysis. The protein generally expressed in lung and breast cancers at remarkable level.
We constructed plasmid vectors carrying wild type HER2 and gene X. These vectors were simultaneously introduced to HEK293T cells to examine the binding ability of protein X and HER2 as well as the effect of gene X on HER2-mediated signal-transduction pathway. The approach clearly showed that the expression of gene X, resulted in phosphorylation of HER2 and subsequent activation of oncogenic effector molecules.
We next constructed several kinds of gene X-truncated variants and subjected to the binding assay to look for the binding domain of gene X to HER2. The analysis showed that N-terminal head domain of gene X was essential for the HER2 binding. This domain has an ability to induce HER2 phosphorylation and subsequent activation of the effector kinase, ERK.
In conclusion, we found that gene X is a novel binding protein to HER2 and has a role in HER2 activation.
Citation Format: Tomoaki Ohtsuka, Masakiyo Sakaguchi, Katsuyoshi Takata, Shinsuke Hashida, Mototsugu Watanabe, Ken Suzawa, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Shinichi Toyooka. Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 48. doi:10.1158/1538-7445.AM2015-48
Abstract 6 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Signaling in Cancer 1
Abstract 54: Ezrin enhances signaling and nuclear translocation of the epidermal growth factor receptor in non-small cell lung cancer cells
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
The cytoskeletal cross linker protein ezrin is a member of the ezrin-radixin-moesin (ERM) family and plays important roles not only in cell motility, cell adhesion, and apoptosis, but also in various cell-signaling pathways. Ezrin interacts with EGFR in the cell membrane and involves in cell motility events, but little is known about the effects of this interaction on the EGFR signaling pathway. We investigated the role of Ezrin in EGFR signaling and nuclear trafficking in non-small cell lung cancer (NSCLC) cell lines. The ligand induced interaction between Ezrin and EGFR was evaluated by immunoprecipitation (IP) and immunofluorescence (IF) in H292 and A549 cells. Ezrin levels were reduced using siRNA in these two cell lines. Downstream signaling protein phosphorylation and nuclear localization of EGFR were detected after EGF treatment. Expressions of nuclear EGFR target genes were evaluated by qPCR. Endogenous Ezrin was found in a complex with EGFR in IP and IF. When Ezrin protein expression was inhibited, phosphorylation levels of EGFR at Y1068, Y1101 and Y845 were reduced as well as phosphorylation levels of downstream signaling pathway proteins ERK and STAT3. Cell fractionation revealed that EGFR nuclear translocation after EGF treatment significantly reduced in Ezrin-knockdown cells. Further, mRNA levels of EGFR target genes AuroraK-A, COX2, Cyclin D1 and iNOS were decreased in Ezrin-knockdown A549 cells. Small molecule ezrin inhibitors showed strong synergy with EGFR inhibitors in cytotoxicity assays. These results suggest that Ezrin has a role as an enhancer in the EGFR pathway and targeting ezrin may potentiate anti-EGFR based therapies in NSCLC.
Citation Format: Yasemin Saygideger Kont, Haydar Celik, Hayriye V. Erkizan, Tsion Minas, Jenny Han, Jeffrey Toretsky, Aykut Uren. Ezrin enhances signaling and nuclear translocation of the epidermal growth factor receptor in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2015-54
Abstract 7 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Cell Signaling in Cancer 1
Abstract 57: Substrates of protein kinase C drive cell rac1-dependent motility
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
This laboratory has identified and/or characterized substrates of PKC that upon phosphorylation give rise to motility, an aspect of metastasis. By use of the traceable kinase method, we discovered that alpha-tubulin and Cdc42 effector protein-4 (CEP4) are PKC substrates. Phosphorylation of alpha-tubulin stimulates its incorporation into microtubules (MTs), consequently increasing the stability and prolonged growth of MTs and leading to the activation of the small GTPase Rac1. CEP4 undergoes phosphorylation by PKC that results in its release from Cdc42, whereupon CEP4 binds a guanine nucleotide exchange factor (GEF) that in turn activates Rac1 GTPase. These results imply that Rac1 acts as a node in pathways driven by phosphorylated PKC substrates. Since translocation of IQGAP to the membrane is known to be promoted by Rac1, a role is explored in non-transformed human MCF-10A cells that express a specific phospho-mimetic mutant substrate. In addition, the phospho-mimetic mutant for each substrate expressed in human metastatic MDA-MB-231 cells produces different morphologies in 3-D growth assays. This research is being supported by NIH CA125632.
Citation Format: Susan A. Rotenberg, Xin Zhao, Shatarupa De. Substrates of protein kinase C drive cell rac1-dependent motility. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 57. doi:10.1158/1538-7445.AM2015-57
Abstract 8 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Deregulation of Gene Expression in Prostate Cancer and Sarcoma
Abstract 88: The Nkx3.1 homeobox gene maintains prostatic identity while its loss leads to prostate cancer initiation
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Background
Maintenance of epithelial cell identity is tightly coordinated by tissue-specific gene expression programs, which are often deregulated during tumorigenesis. The homeodomain-containing transcription factor, Nkx3.1, is a key regulator of normal prostatic development and is frequently lost at early stages of prostate cancer initiation. In this study, we aim to elucidate detailed mechanisms governing Nkx3.1-driven maintenance of prostate identity and how deregulation of such can lead to prostate tumorigenesis.
Models and Methods
We evaluated the consequences of Nkx3.1 loss or gain of function in vivo using genetically-engineered mouse models and cell-recombination assays. RNA sequencing was performed to generate gene expression profiles, which were analyzed using Gene Set Enrichment analysis (GSEA), and validated by quantitative real-time PCR. In parallel, protein expression was assessed by immunofluorescence and western blot. Immunoprecipitation (IP) and chromatin-immunoprecipitation (ChIP) assays were performed using RWPE1 prostate epithelial cells.
Results
Here, we show that loss of function of Nkx3.1 leads to the progressive down-regulation of a prostate-specific gene expression program and to aberrant expression of genes that are not typically expressed in the prostate epithelium. Conversely, gain of function of Nkx3.1 in non-prostatic epithelium leads to the acquisition of a prostate-like morphology and expression of prostate-related genes. Our findings indicate that the underlying mechanism by which Nkx3.1 promotes prostatic identity is via epigenetic regulation of gene expression. In particular, we show that Nkx3.1 interacts with the histone methyl-transferase complex G9a/Glp. Finally, we demonstrate that this interaction is necessary for maintenance of prostate identity in vivo and that Nkx3.1 and G9a cooperate to control expression of genes that coordinate prostatic epithelial integrity.
Conclusions
Our results suggest that Nkx3.1 promotes prostatic identity by interacting with histone modifying enzymes to coordinate the expression of prostate-specific genes and that the loss of this function results in a failure to maintain prostate identity associated with early stages of prostate tumorigenesis.
Citation Format: Clémentine Le Magnen, Aditya Dutta, Cory Abate-Shen. The Nkx3.1 homeobox gene maintains prostatic identity while its loss leads to prostate cancer initiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2015-88
Abstract 9 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Deregulation of Gene Expression in Prostate Cancer and Sarcoma
Abstract 90: K63-linked JARID1B ubiquitination by TRAF6 contributes to aberrant elevation of JARID1B in prostate cancer
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Aberrant elevation of JARID1B and histone H3 Lys4 trimethylations (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulations of JARID1B and H3K4me3 through epigenetic modifications still remain poorly understood. In this study we performed immunohistochemistry staining, immunofluorescence imaging, immunoprecipitation, shRNA and Western blotting analysis in mouse embryonic fibroblasts (MEFs), mouse models, and cultured human prostate cancer cells. As a result, we discovered that SKP2 modulates JARID1B and H3K4me3 levels in vitro in PTEN null prostate cancer cells and in vivo in Pten/Trp53 mouse models. We demonstrated that levels of SKP2, JARID1B and H3K4me3 are strikingly elevated in vitro and in vivo when both PTEN and P53 are inactivated. Importantly, SKP2 inactivation resulted in a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, JARID1B is ubiquitinated by E3 ligase TRAF6 through the K63-linkage in prostate cancer cells. Interestingly, SKP2 contributes to JARID1B ubiquitination machinery as a non-E3 ligase regulator by decreasing TRAF6-mediated ubiquitination of JARID1B. SKP2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the aberrant levels of SKP2, JARID1B, and H3K4me3 are associated with malignant features of castration-resistant prostate cancer (CRPC) in mice. Overall, our findings reveal a novel network of SKP2- JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control.
Citation Format: Wenfu Lu, Shenji Liu, Bo Li, Yingqiu Xie, Christine Adhiambo, Qing Yang, Billy R. Ballard, Keiichi I. Nakayama, Robert J. Matusik, Zhenbang Chen. K63-linked JARID1B ubiquitination by TRAF6 contributes to aberrant elevation of JARID1B in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 90. doi:10.1158/1538-7445.AM2015-90
Abstract 10 of 10Molecular and Cellular Biology / Poster Presentations – Proffered Abstracts / Poster Presentations – Histone Methylation and Acetylation
Abstract 97: CARM1 preferentially methylates H3R17 over H3R26 through a random kinetic mechanism
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
CARM1 (PRMT4) is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression and the DNA damage response. Overexpression of CARM1 has been implicated in breast, prostate, and colorectal cancers. Since CARM1 appears to be a good target for the development of therapies against these cancers, we studied the substrate specificity and kinetic mechanism of the full-length human enzyme. CARM1 has been shown to methylate both residues R17 and R26 of histone H3. Substrate specificity was examined by testing CARM1 activity with several H3-based peptide substrates using a radiometric assay. Comparison of kcat/KM values reveal that methylation of H3R17 is preferred over H3R26. An R17/R26K peptide produced 8-fold greater kcat/KM value compared to the corresponding R17K/R26 peptide. These effects are KM-driven as kcat values remain relatively constant for the peptides tested. Shortening the peptide at the C-terminus by 5 amino acid residues greatly reduced the specificity (16-24-fold), demonstrating the contribution of distal residues to substrate binding. In contrast, adding residues to the N-terminus of the shortened peptide had a negative effect on activity. CARM1 displays little preference for monomethylated over unmethylated H3R17 (2-5-fold by kcat/KM) suggesting that it operates through a distributive mechanism. Previous crystallographic studies with mouse CARM1 showed that part of the substrate binding groove was formed by cofactor binding, thereby suggesting an ordered kinetic mechanism (Yue et al., EMBO J., 2007). Our results from dead-end and product inhibition studies performed with human CARM1, however, are consistent with a random kinetic mechanism. SAH and sinefungin demonstrate competitive inhibition with respect to SAM and produced noncompetitive inhibition patterns with respect to peptide. Both dimethylated R17 product peptide and dead-end R17K peptide exhibited noncompetitive inhibition patterns with respect to SAM. Furthermore, binding of SAM and peptide substrates were shown to be independent of each other in initial velocity experiments where both substrates were varied. Together, these results elucidate the kinetic mechanism of CARM1 and highlight elements important for binding affinity.
Citation Format: Suzanne L. Jacques, Katrina P. Aquino, Jodi Gureasko, P Ann Boriack-Sjodin, Robert A. Copeland, Thomas V. Riera. CARM1 preferentially methylates H3R17 over H3R26 through a random kinetic mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 97. doi:10.1158/1538-7445.AM2015-97
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Additional Articles on this Open Access Journal on Cancer Stem Cells Include
Cyclin-dependent kinases (CDKs), in complex with their cyclin partners, modulate the transition through phases of the cell division cycle. Cyclin D–CDK complexes are important in cancer progression, especially for certain types of breast cancer. Fassl et al. discuss advances in understanding the biology of cyclin D–CDK complexes that have led to new concepts about how drugs that target these complexes induce cancer cell cytostasis and suggest possible combinations to widen the types of cancer that can be treated. They also discuss progress in overcoming resistance to cyclin D–CDK inhibitors and their possible application to diseases beyond cancer. —GKA
Structured Abstract
BACKGROUND
Cyclins and cyclin-dependent kinases (CDKs) drive cell division. Of particular importance to the cancer field are D-cyclins, which activate CDK4 and CDK6. In normal cells, the activity of cyclin D–CDK4/6 is controlled by the extracellular pro-proliferative or inhibitory signals. By contrast, in many cancers, cyclin D–CDK4/6 kinases are hyperactivated and become independent of mitogenic stimulation, thereby driving uncontrolled tumor cell proliferation. Mouse genetic experiments established that cyclin D–CDK4/6 kinases are essential for growth of many tumor types, and they represent potential therapeutic targets. Genetic and cell culture studies documented the dependence of breast cancer cells on CDK4/6. Chemical CDK4/6 inhibitors were synthesized and tested in preclinical studies. Introduction of these compounds to the clinic represented a breakthrough in breast cancer treatment and will likely have a major impact on the treatment of many other tumor types.
ADVANCES
Small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) showed impressive results in clinical trials for patients with hormone receptor–positive breast cancers. Addition of CDK4/6 inhibitors to standard endocrine therapy substantially extended median progression-free survival and prolonged median overall survival. Consequently, all three CDK4/6 inhibitors have been approved for treatment of women with advanced or metastatic hormone receptor–positive breast cancers. In the past few years, the renewed interest in CDK4/6 biology has yielded several surprising discoveries. The emerging concept is that CDK4/6 kinases regulate a much wider set of cellular functions than anticipated. Consequently, CDK4/6 inhibitors, beyond inhibiting tumor cell proliferation, affect tumor cells and the tumor environment through mechanisms that are only beginning to be elucidated. For example, inhibition of CDK4/6 affects antitumor immunity acting both on tumor cells and on the host immune system. CDK4/6 inhibitors were shown to enhance the efficacy of immune checkpoint blockade in preclinical mouse cancer models. These new concepts are now being tested in clinical trials.
OUTLOOK
Palbociclib, ribociclib, and abemaciclib are being tested in more than 300 clinical trials for more than 50 tumor types. These trials evaluate CDK4/6 inhibitors in combination with a wide range of therapeutic compounds that target other cancer-relevant pathways. Several other combination treatments were shown to be efficacious in preclinical studies and will enter clinical trials soon. Another CDK4/6 inhibitor, trilaciclib, is being tested for its ability to shield normal cells of the host from cytotoxic effects of chemotherapy. New CDK4/6 inhibitors have been developed and are being assessed in preclinical and clinical trials. The major impediment in the therapeutic use of CDK4/6 inhibitors is that patients who initially respond to treatment often develop resistance and eventually succumb to the disease. Moreover, a substantial fraction of tumors show preexisting, intrinsic resistance to CDK4/6 inhibitors. One of the main challenges will be to elucidate the full range of resistance mechanisms. Even with the current, limited knowledge, one can envisage the principles of new, improved approaches to overcome known resistance mechanisms. Another largely unexplored area for future study is the possible involvement of CDK4/6 in other pathologic states beyond cancer. This will be the subject of intense studies, and it may extend the utility of CDK4/6 inhibitors to the treatment of other diseases.
Targeting cyclin D–CDK4/6 for cancer treatment.
D-cyclins (CycD) activate CDK4 and CDK6 in G1 phase of the cell cycle and promote cell cycle progression by phosphorylating the retinoblastoma protein RB1. RB1 inhibits E2F transcription factors; phosphorylation of RB1 activates E2F-driven transcription. In many cancers, CycD-CDK4/6 is constitutively activated and drives uncontrolled cell proliferation. The development of small-molecule CDK4/6 inhibitors provided a therapeutic tool to repress constitutive CycD-CDK4/6 activity and to inhibit cancer cell proliferation. As with several targeted therapies, tumors eventually develop resistance and resume cell proliferation despite CDK4/6 inhibition. New combination treatments, involving CDK4/6 inhibitors plus inhibition of other pathways, are being tested in the clinic to delay or overcome the resistance.
Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin D–CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor–positive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin D–CDK4/6 inhibitors in cancer treatment.
Cyclin D1, the activator of CDK4 and CDK6, was discovered in the early 1990s (1, 2). The role of cyclin D1 in oncogenesis was already evident at the time of its cloning, as it was also identified as the protein product of the PRAD1 oncogene, which is rearranged and overexpressed in parathyroid adenomas (3), and of the BCL1 oncogene, which is rearranged in B-lymphocytic malignancies (4). Subsequently, the remaining two D-type cyclins, D2 and D3, were discovered on the basis of their homology to cyclin D1 (1).
Cyclins serve as regulatory subunits of cyclin-dependent kinases (CDKs) (5). Shortly after the discovery of D-cyclins, CDK4 and CDK6 were identified as their kinase partners (6). Mouse gene knockout studies revealed that CDK4 and CDK6 play redundant roles in development, and combined ablation of CDK4 and CDK6 was found to result in embryonic lethality (7). The essentially identical phenotype was seen in cyclin D–knockout mice, thereby confirming the role of D-cyclins as CDK4/6 activators in vivo (8). Surprisingly, these analyses revealed that many normal nontransformed mammalian cell types can proliferate without any cyclin D–CDK4/6 activity (7, 8).
CDK4 and CDK6 are expressed at constant levels throughout the cell cycle. By contrast, D-cyclins are labile proteins that are transcriptionally induced upon stimulation of cells with growth factors. For this reason, D-cyclins are regarded as links between the cellular environment and the cell cycle machinery (6).
Cell cycle inhibitors play an important role in regulating the activity of cyclin D–CDK4/6 (Fig. 1). The INK inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) bind to CDK4 or CDK6 and prevent their interaction with D-type cyclins, thereby inhibiting cyclin D–CDK4/6 kinase activity. By contrast, KIP/CIP inhibitors (p27KIP1, p57KIP2, p21CIP1), which inhibit the activity of CDK2-containing complexes, serve as assembly factors for cyclin D–CDK4/6 (6, 9). This was demonstrated by the observation that mouse fibroblasts devoid of p27KIP1 and p21CIP1 fail to assemble cyclin D–CDK4/6 complexes (10).
Fig. 1. Molecular events governing progression through the G1 phase of the cell cycle.
The mammalian cell cycle can be divided into G1, S (DNA synthesis), G2, and M (mitosis) phases. During G1 phase, cyclin D (CycD)–CDK4/6 kinases together with cyclin E (CycE)–CDK2 phosphorylate the retinoblastoma protein RB1. This activates the E2F transcriptional program and allows entry of cells into S phase. Members of the INK family of inhibitors (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) inhibit cyclin D–CDK4/6; KIP/CIP proteins (p21CIP1, p27KIP1, and p57KIP2) inhibit cyclin E–CDK2. Cyclin D–CDK4/6 complexes use p27KIP1 and p21CIP1 as “assembly factors” and sequester them away from cyclin E–CDK2, thereby activating CDK2. Proteins that are frequently lost or down-regulated in cancers are marked with green arrows, overexpressed proteins with red arrows.
p27KIP1 can bind cyclin D–CDK4/6 in an inhibitory or noninhibitory mode, depending on p27KIP1 phosphorylation status. Cyclin D–p27KIP1-CDK4/6 complexes are catalytically inactive unless p27KIP1 is phosphorylated on Tyr88 and Tyr89 (11). Two molecular mechanisms may explain this switch. First, Tyr88/Tyr89 phosphorylation may dislodge the helix of p27KIP1 from the CDK active site and allow adenosine triphosphate (ATP) binding (12). Second, the presence of tyrosine-unphosphorylated p27KIP1 within the cyclin D–CDK4 complex prevents the activating phosphorylation of CDK4’s T-loop by the CDK-activating kinase (CAK) (12). Brk has been identified as a physiological kinase of p27KIP1 (13); Abl and Lyn can phosphorylate p27KIP1 in vitro, but their in vivo importance remains unclear (11, 14).
The activity of cyclin D–CDK4/6 is also regulated by proteolysis. Cyclin D1 is an unstable protein with a half-life of less than 30 min. At the end of G1 phase, cyclin D1 is phosphorylated at Thr286 by GSK3β (15). This facilitates association of cyclin D1 with the nuclear exportin CRM1 and promotes export of cyclin D1 from the nucleus to the cytoplasm (16). Subsequently, phosphorylated cyclin D1 becomes polyubiquitinated by E3 ubiquitin ligases, thereby targeting it for proteasomal degradation. Several substrate receptors of E3 ubiquitin ligases have been implicated in recognizing phosphorylated cyclin D1, including F-box proteins FBXO4 (along with αB crystallin), FBXO31, FBXW8, β-TrCP1/2, and SKP2 (17). The anaphase-promoting complex/cyclosome (APC/C) was also proposed to target cyclin D1 while F-box proteins FBXL2 and FBXL8 target cyclins D2 and D3 (17, 18). Surprisingly, the level and stability of cyclin D1 was unaffected by depletion of several of these proteins, indicating that some other E3 plays a rate-limiting role in cyclin D1 degradation (19). Indeed, recent studies reported that D-cyclins are ubiquitinated and targeted for proteasomal degradation by the E3 ubiquitin ligase CRL4, which uses AMBRA1 protein as its substrate receptor (20–22).
Cyclin D–CDK4/6 in cancer
Genomic aberrations of the cyclin D1 gene (CCND1) represent frequent events in different tumor types. The t(11;14)(q13;q32) translocation juxtaposing CCND1 with the immunoglobulin heavy-chain (IGH) locus represents the characteristic feature of mantle-cell lymphoma and is frequently observed in multiple myeloma or plasma cell leukemia (23, 24). Amplification of CCND1 is seen in many other malignancies—for example, in 13 to 20% of breast cancers (23, 24), more than 40% of head and neck squamous cell carcinomas, and more than 30% of esophageal squamous cell carcinomas (23). A higher proportion of cancers (e.g., up to 50% of mammary carcinomas) overexpress cyclin D1 protein (24). Also, cyclins D2 and D3, CDK4, and CDK6 are overexpressed in various tumor types (5, 9). Cyclin D–CDK4/6 can also be hyperactivated through other mechanisms such as deletion or inactivation of INK inhibitors, most frequently p16INK4A (5, 9, 23). Altogether, a very large number of human tumors contain lesions that hyperactivate cyclin D–CDK4/6 (5).
An oncogenic role for cyclin D–CDK4/6 has been supported by mouse cancer models. For example, targeted overexpression of cyclin D1 in mammary glands of transgenic mice led to the development of mammary carcinomas (25). Also, overexpression of cyclin D2, D3, or CDK4, or loss of p16INK4a resulted in tumor formation (9).
Conversely, genetic ablation of D-cyclins, CDK4, or CDK6 decreased tumor sensitivity (9). For instance, Ccnd1– or Cdk4-null mice, or knock-in mice expressing kinase-inactive cyclin D1–CDK4/6, were resistant to develop human epidermal growth factor receptor 2 (HER2)–driven mammary carcinomas (26–29). An acute, global shutdown of cyclin D1 in mice bearing HER2-driven tumors arrested tumor growth and triggered tumor-specific senescence while having no obvious impact on normal tissues (30). Likewise, an acute ablation of CDK4 arrested tumor cell proliferation and triggered tumor cell senescence in a KRAS-driven non–small-cell lung cancer (NSCLC) mouse model (31). These observations indicated that CDK4 and CDK6 might represent excellent therapeutic targets in cancer treatment.
CDK4/6 functions in cell proliferation and oncogenesis
The best-documented function of cyclin D–CDK4/6 in driving cell proliferation is phosphorylation of the retinoblastoma protein, RB1, and RB-like proteins, RBL1 and RBL2 (5, 6) (Fig. 1). Unphosphorylated RB1 binds and inactivates or represses E2F transcription factors. According to the prevailing model, phosphorylation of RB1 by cyclin D–CDK4/6 partially inactivates RB1, leading to release of E2Fs and up-regulation of E2F-transcriptional targets, including cyclin E. Cyclin E forms a complex with its kinase partner, CDK2, and completes full RB1 phosphorylation, leading to activation of the E2F transcriptional program and facilitating S-phase entry (5, 6). In normal, nontransformed cells, the activity of cyclin D–CDK4/6 is tightly regulated by the extracellular mitogenic milieu. This links inactivation of RB1 with mitogenic signals. In cancer cells carrying activating lesions in cyclin D–CDK4/6, the kinase is constitutively active, thereby decoupling cell division from proliferative and inhibitory signals (5).
This model has been questioned by the demonstration that RB1 exists in a monophosphorylated state throughout G1 phase and becomes inactivated in late G1 by cyclin E–CDK2, which “hyperphosphorylates” RB1 on multiple residues (32). However, recent single-cell analyses revealed that cyclin D–CDK4/6 activity is required for the hyperphosphorylation of RB1 throughout G1, whereas cyclin E/A–CDK maintains RB1 hyperphosphorylation in S phase (33). Moreover, phosphorylation of RB1 by cyclin D–CDK4/6 was shown to be required for normal cell cycle progression (34).
In addition to this kinase-dependent mechanism, up-regulation of D-cyclin expression and formation of cyclin D–CDK4/6 complexes lead to redistribution of KIP/CIP inhibitors from cyclin E–CDK2 complexes (which are inhibited by these proteins) to cyclin D–CDK4/6 (which use them as assembly factors), thereby activating the kinase activity of cyclin E–CDK2 (6). Cyclin E–CDK2 in turn phosphorylates RB1 and other cellular proteins and promotes cell cycle progression.
Cyclin D1–CDK4/6 directly phosphorylates, stabilizes, and activates the transcription factor FOXM1. This promotes cell cycle progression and protects cancer cells from entering senescence (35). Cyclin D–CDK4 also phosphorylates and inactivates SMAD3, which mediates transforming growth factor–β (TGF-β) antiproliferative response. CDK4/6-dependent phosphorylation of SMAD3 inhibits its transcriptional activity and disables the ability of TGF-β to induce cell cycle arrest (36). FZR1/CDH1, an adaptor protein of the APC complex, is another phosphorylation substrate of CDK4. Depletion of CDH1 in human cancer cells partially rescued the proliferative block upon CDK4/6 inhibition, and it cooperated with RB1 depletion in restoring full proliferation (37).
Cyclin D–CDK4/6 also phosphorylates and inactivates TSC2, a negative regulator of mTORC1, thereby resulting in mTORC1 activation. Conversely, inhibition of CDK4/6 led to decreased mTORC1 activity and reduced protein synthesis in cells representing different human tumor types. It was proposed that through TSC2 phosphorylation, activation of cyclin D–CDK4/6 couples cell growth with cell division (38). Consistent with this, the antiproliferative effect of CDK4/6 inhibition was reduced in cells lacking TSC2 (38).
MEP50, a co-regulatory factor of protein arginine-methyltransferase 5 (PRMT5), is phosphorylated by cyclin D1–CDK4. Through this mechanism, cyclin D1–CDK4/6 increases the catalytic activity of PRMT5/MEP50 (39). It was proposed that deregulation of cyclin D1–CDK4 kinase in tumor cells, by increasing PRMT5/MEP50 activity, reduces the expression of CUL4, a component of the E3 ubiquitin-ligase complex, and stabilizes CUL4 targets such as CDT1 (39). In addition, by stimulating PRMT5/MEP50-dependent arginine methylation of p53, cyclin D–CDK4/6 suppresses the expression of key antiproliferative and pro-apoptotic p53 target genes (40). Another study proposed that PRMT5 regulates splicing of the transcript encoding MDM4, a negative regulator of p53. CDK4/6 inhibition reduced PRMT5 activity and altered the pre-mRNA splicing of MDM4, leading to decreased levels of MDM4 protein and resulting in p53 activation. This, in turn, up-regulated the expression of a p53 target, p21CIP1, that blocks cell cycle progression (41).
During oncogenic transformation of hematopoietic cells, chromatin-bound CDK6 phosphorylates the transcription factors NFY and SP1 and induces the expression of p53 antagonists such as PRMT5, PPM1D, and MDM4 (42). Also, in acute myeloid leukemia cells expressing constitutively activated FLT3, CDK6 binds the promoter region of the FLT3 gene as well as the promoter of PIM1 pro-oncogenic kinase and stimulates their expression. Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased FLT3 and PIM1 expression and triggered cell cycle arrest and apoptosis (43). The relevance of these various mechanisms in the context of human tumors is unclear and requires further study.
Mechanism of action of CDK4/6 inhibitors
Three small-molecule CDK4/6 inhibitors have been extensively characterized in preclinical studies: palbociclib and ribociclib, which are highly specific CDK4/6 inhibitors, and abemaciclib, which inhibits CDK4/6 and other kinases (Table 1). It has been assumed that these compounds act in vivo by directly inhibiting cyclin D–CDK4/6 (9). This simple model has been recently questioned by observations that palbociclib inhibits only cyclin D–CDK4/6 dimers, but not trimeric cyclin D–CDK4/6-p27KIP1 (44). However, it is unlikely that substantial amounts of cyclin D–CDK4 dimers ever exist in cells, because nearly all cyclin D–CDK4 in vivo is thought to be complexed with KIP/CIP proteins (11, 14, 44). Palbociclib also binds monomeric CDK4 (44). Surprisingly, treatment of cancer cells with palbociclib for 48 hours failed to inhibit CDK4 kinase, despite cell cycle arrest, but it inhibited CDK2 (44). Hence, palbociclib might prevent the formation of active CDK4-containing complexes (through binding to CDK4) and indirectly inhibit CDK2 by liberating KIP/CIP inhibitors. This model needs to be reconciled with several observations. First, treatment of cells with CDK4/6 inhibitors results in a rapid decrease of RB1 phosphorylation on cyclin D–CDK4/6-dependent sites, indicating an acute inhibition of CDK4/6 (45–47). Moreover, CDK4/6 immunoprecipitated from cells can be inhibited by palbociclib (48) and p21CIP-associated cyclin CDK4/6 kinase is also inhibited by treatment of cells with palbociclib (49). Lastly, CDK2 is dispensable for proliferation of several cancer cell lines (50, 51), hence the indirect inhibition of CDK2 alone is unlikely to be responsible for cell cycle arrest.
Name of compound
IC50
Other known targets
Stage of clinical development
Palbociclib (PD-0332991)
D1-CDK4, 11 nM;
D2-CDK6, 15 nM;
D3-CDK4, 9 nM
FDA-approved for HR+/HER2– advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Ribociclib (LEE011)
D1-CDK4, 10 nM;
D3-CDK6, 39 nM
FDA-approved for HR+/HER2– advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Abemaciclib (LY2835219)
D1-CDK4, 0.6 to 2 nM;
D3-CDK6, 8 nM
Cyclin T1–CDK9, PIM1, HIPK2, CDKL5,
CAMK2A, CAMK2D, CAMK2G,
GSK3α/β, and (at higher doses)
cyclin E/A–CDK2 and cyclin B–CDK1
FDA-approved for early (adjuvant) and
advanced HR+/HER2– breast cancer in
combination with endocrine therapy;
FDA-approved as monotherapy in advanced
HR+/HER2– breast cancer; phase 2/3 trials
for several other tumor types
Trilaciclib (G1T28)
D1-CDK4, 1 nM;
D3-CDK6, 4 nM
FDA-approved for small-cell lung cancer
to reduce chemotherapy-induced bone
marrow suppression; phase 2/3 trials
for other solid tumors
Lerociclib (G1T38)
D1-CDK4, 1 nM;
D3-CDK6, 2 nM
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and EGFR-mutant
non–small-cell lung cancer
SHR6390
CDK4, 12 nM;
CDK6, 10 nM
Phase 1/2/3 trials for HR+/HER2– advanced
breast cancer and other solid tumors
PF-06873600
CDK4, 0.13 nM (Ki),
CDK6, 0.16 nM (Ki)
CDK2, 0.09 nM (Ki)
Phase 2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
FCN-437
D1-CDK4, 3.3 nM;
D3-CDK6, 13.7 nM
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
Birociclib (XZP-3287)
Not reported
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
HS-10342
Not reported
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
This table lists major inhibitors of CDK4 and CDK6, half-maximal inhibitory concentration (IC50) for different cyclin D–CDK4/6 complexes (if known), other known targets, and the stage of clinical development. Ki, inhibitory constant.
Palbociclib, ribociclib, and abemaciclib were shown to block binding of CDK4 and CDK6 to CDC37, the kinase-targeting subunit of HSP90, thereby preventing access of CDK4/6 to the HSP90-chaperone system (52). Because the HSP90-CDC37 complex stabilizes several kinases (53), these observations suggest that CDK4/6 inhibitors, by disrupting the interaction between CDC37 and CDK4 or CDK6, might promote degradation of CDK4 and CDK6. However, depletion of CDK4/6 is typically not observed upon treatment with CDK4/6 inhibitors (54). More studies are needed to resolve these conflicting reports and to establish how CDK4/6 inhibitors affect the cell cycle machinery in cancer cells.
Validation of CDK4/6 inhibitors as anticancer agents
Consistent with the notion that RB1 represents the major rate-limiting substrate of cyclin D–CDK4/6 in cell cycle progression (55–57), palbociclib, ribociclib, and abemaciclib were shown to block proliferation of several RB1-positive cancer cell lines, but not cell lines that have lost RB1 expression (46, 58, 59). Breast cancer cell lines representing the luminal, estrogen receptor–positive (ER+) subtype were shown to be most susceptible to cell proliferation arrest upon palbociclib treatment (45). Palbociclib, ribociclib, abemaciclib, and another CDK4/6 inhibitor, lerociclib, were demonstrated to display potent antitumor activity in xenografts of several tumor types, including breast cancers (46, 60–62). Palbociclib and abemaciclib cross the blood-brain barrier and inhibit growth of intracranial glioblastoma (GBM) xenografts, with abemaciclib being more efficient in reaching the brain (63, 64). Recently, additional CDK4/6 inhibitors were shown to exert therapeutic effects in mouse xenograft models of various cancer types, including SHR6390 (65), FCN-437 (66), and compound 11 (67); the latter two were reported to cross the blood-brain barrier. In most in vivo studies, the therapeutic effect was dependent on expression of intact RB1 protein in tumor cells (46, 63). However, antitumor effects of palbociclib were also reported in bladder cancer xenografts independently of RB1 status; this was attributed to decreased phosphorylation of FOXM1 (68).
Tumor cell senescence upon CDK4/6 inhibition
In addition to blocking cell proliferation, inhibition of CDK4/6 can also trigger tumor cell senescence (63), which depends on RB1 and FOXM1 (35, 54). The role of RB1 in enforcing cellular senescence is well established (69). In addition, cyclin D–CDK4/6 phosphorylates and activates FOXM1, which has anti-senescence activity (35, 70). Senescence represents a preferred therapeutic outcome to cell cycle arrest, as it may lead to a durable inhibition of tumor growth.
It is not clear what determines the extent of senescence upon treatment of cancer cells with CDK4/6 inhibitors. A recent study showed that inhibition of CDK4/6 leads to an RB1-dependent increase in reactive oxygen species (ROS) levels, resulting in activation of autophagy, which mitigates the senescence of breast cancer cells in vitro and in vivo (71). Co-treatment with palbociclib plus autophagy inhibitors strongly augmented the ability of CDK4/6 inhibitors to induce tumor cell senescence and led to sustained inhibition of cancer cell proliferation in vitro and of xenograft growth in vivo (71). Decreased mTOR signaling after long-term CDK4/6 inhibition was shown to be essential for the induction of senescence in melanoma cells, and activation of mTORC1 overrode palbociclib-induced senescence (72). Others postulated that expression of the chromatin-remodeling enzyme ATRX and degradation of MDM2 determines the choice between quiescence and senescence upon CDK4/6 inhibition (73). Inhibition of CDK4 causes dissociation of the deubiquitinase HAUSP/USP7 from MDM2, thereby driving autoubiquitination and proteolytic degradation of MDM2, which in turn promotes senescence. This mechanism requires ATRX, which suggests that expression of ATRX can be used to predict the senescence response (73). Two additional proteins that play a role in this process are PDLIM7 and type II cadherin CDH18. Expression of CDH18 correlated with a sustained response to palbociclib in a phase 2 trial for patients with liposarcoma (74).
Markers predicting response to CDK4/6 inhibition
Only tumors with intact RB1 respond to CDK4/6 inhibitor treatment by undergoing cell cycle arrest or senescence (9, 58). In addition, “D-cyclin activating features” (CCND1 translocation, CCND2 or CCND3 amplification, loss of the CCND1-3 3′-untranslated region, and deletion of FBXO31 encoding an F-box protein implicated in cyclin D1 degradation) were shown to confer a strong response to abemaciclib in cancer cell lines (58). Moreover, co-deletion of CDKN2A and CDKN2C (encoding p16INK4A/p19ARF and p18INK4C, respectively) confers palbociclib sensitivity in glioblastoma (75). Thr172 phosphorylation of CDK4 and Tyr88 phosphorylation of p27KIP1 (both associated with active cyclin D–CDK4) correlate with sensitivity of breast cancer cell lines or tumor explants to palbociclib (76, 77). Surprisingly, in PALOMA-1, PALOMA-2, and PALOMA-3 trials (78–80), and in another independent large-scale study (81), CCND1 gene amplification or elevated levels of cyclin D1 mRNA or protein were not predictive of palbociclib efficacy. Conversely, overexpression of CDK4, CDK6, or cyclin E1 is associated with resistance of tumors to CDK4/6 inhibitors (see below).
Synergy of CDK4/6 inhibitors with other compounds
Several preclinical studies have documented the additive or synergistic effects of combining CDK4/6 inhibitors with inhibitors of the receptor tyrosine kinases as well as phosphoinositide 3-kinase (PI3K), RAF, or MEK (Table 2). This synergism might be because these pathways impinge on the cell cycle machinery through cyclin D–CDK4/6 (82–86). In some cases, the effect was seen in the presence of specific genetic lesions, such as EGFR, BRAFV600E, KRAS, and PIK3CA mutations (59, 87–89) (Table 2). When comparing different dosing regimens, continuous treatment with a MEK inhibitor with intermittent palbociclib resulted in more complete tumor responses than other combination schedules (90). Treatment with CDK4/6 inhibitors sensitized cancer cells to ionizing radiation (63) or cisplatin (68). The synergism with platinum-based chemotherapy was attributed to the observation that upon this treatment, CDK6 phosphorylates and stabilizes the FOXO3 transcription factor, thereby promoting tumor cell survival. Consequently, inhibition of CDK6 increases platinum sensitivity by enhancing tumor cell death (91).
In several instances, co-treatment with CDK4/6 inhibitors prevented the development of resistance to other compounds or inhibited the proliferation of resistant tumor cells. Co-treatment of melanoma patient-derived xenografts (PDXs) with ribociclib plus the RAF inhibitor encorafenib delayed or prevented development of encorafenib resistance (92). PDXs that acquired encorafenib resistance remained sensitive to the combination of encorafenib plus ribociclib (59). Treatment of BRAFV600E-mutant melanoma xenografts with palbociclib plus the BRAFV600E inhibitor PLX4720 prevented development of resistance (89). BRAFV600E-mutant melanoma cell lines that acquired resistance to the BRAFV600E inhibitor vemurafenib remained sensitive to palbociclib or abemaciclib, and xenografts underwent senescence and tumor regression upon CDK4/6 inhibition (72, 93). Treatment of ALK-mutant, ALK kinase inhibitor–resistant neuroblastoma xenografts with palbociclib restored the sensitivity to these compounds (94). A combination of PI3K and CDK4/6 inhibitors overcame the intrinsic and acquired resistance of breast cancers to PI3K inhibitors and resulted in regression of PIK3CA-mutant xenografts (88).
Up-regulation of cyclin D1 expression was shown to mediate acquired resistance of HER2+ tumors to anti-HER2 therapies in a mouse breast cancer model (95). Treatment of mice bearing trastuzumab-resistant tumors or PDXs of resistant HER2+ mammary carcinomas with abemaciclib restored the sensitivity of tumors to HER2 inhibitors and inhibited tumor cell proliferation. Moreover, in the case of treatment-naïve tumors, co-administration of abemaciclib significantly delayed the development of resistance to anti-HER2 therapies (95).
Several anticancer treatments, such as chemotherapy, target dividing cells. Because CDK4/6 inhibitors block tumor cell proliferation, they might impede the effects of chemotherapy. Indeed, several reports have documented that co-administration of CDK4/6 inhibitors antagonized the antitumor effects of compounds that act during S phase (doxorubicin, gemcitabine, methotrexate, mercaptopurine) or mitosis (taxanes) (96, 97). However, some authors reported synergistic effects (98, 99), although the molecular underpinnings are unclear.
A recent report documented that administration of CDK4/6 inhibitors prior to taxanes inhibited tumor cell proliferation and impeded the effect of taxanes (100). By contrast, administration of taxanes first (or other chemotherapeutic compounds that act on mitotic cells or cells undergoing DNA synthesis), followed by CDK4/6 inhibitors, had a strong synergistic effect. The authors showed that by repressing the E2F-dependent transcriptional program, CDK4/6 inhibitors impaired the expression of genes required for DNA-damage repair via homologous recombination. Because treatment of cancer cells with chemotherapy triggers DNA damage, the impairment of DNA-damage repair induced cytotoxicity, thereby explaining the synergistic effect (100).
Cells with impaired homologous recombination rely on poly-(ADP-ribose) polymerase (PARP) for double-stranded DNA-damage repair, which renders them sensitive to PARP inhibition. Indeed, a strong synergistic effect has been demonstrated between CDK4/6 inhibitors and PARP inhibitors in PDX-derived cell lines (100). Such synergy was also reported for ovarian cancer cells (101). Another study found that inhibition of CDK4/6 resulted in down-regulation of PARP levels (102).
Protection against chemotherapy-induced toxicity
Administration of palbociclib to mice induced reversible quiescence in hematopoietic stem/progenitor cells (HSPCs). This effect protected mice from myelosuppression after total-body irradiation. Moreover, treatment of tumor-bearing mice with CDK4/6 inhibitors together with irradiation mitigated radiation-induced toxicity without compromising the therapeutic effect (103). Co-administration of a CDK4/6 inhibitor, trilaciclib, with cytotoxic chemotherapy (5-FU, etoposide) protected animals from chemotherapy-induced exhaustion of HSPCs, myelosuppression, and apoptosis of bone marrow (60, 104). These observations led to phase 2 clinical trial, which evaluated the effects of trilaciclib administered prior to etoposide and carboplatin for treatment of small-cell lung cancer. Trilaciclib improved myelopreservation while having no adverse effect on antitumor efficacy (105). A similar phase 2 clinical trial investigating trilaciclib in combination with gemcitabine and carboplatin chemotherapy in patients with metastatic triple-negative breast cancer (TNBC) did not observe a significant difference in myelosuppression. However, this study demonstrated an overall survival benefit of the combination therapy (106, 107).
Metabolic function of CDK4/6 in cancer cells
The role of CDK4/6 in tumor metabolism is only starting to be appreciated (Fig. 2A). Treatment of pancreatic cancer cells with CDK4/6 inhibitors was shown to induce tumor cell metabolic reprogramming (108). CDK4/6 inhibition increased the numbers of mitochondria and lysosomes, activated mTOR, and increased the rate of oxidative phosphorylation, likely through an RB1-dependent mechanism (108). Combined inhibition of CDK4/6 and mTOR strongly suppressed tumor cell proliferation (108). Moreover, CDK4/6 can phosphorylate and inactivate TFEB, the master regulator of lysosomogenesis, and through this mechanism reduce lysosomal numbers. Conversely, CDK4/6 inhibition activated TFEB and increased the number of lysosomes (109). Another mechanism linking CDK4/6 and lysosomes was provided by the observation that treatment of TNBC cells with CDK4/6 inhibitors decreased mTORC1 activity and impaired the recruitment of mTORC1 to lysosomes (110). Consistent with the idea that mTORC1 inhibits lysosomal biogenesis, CDK4/6 inhibition increased the number of lysosomes in tumor cells. Because an increased lysosomal biomass underlies some cases of CDK4/6 inhibitor resistance (see below) (111), stimulation of lysosomogenesis by CDK4/6 inhibitors might limit their clinical efficacy by inducing resistance.
Fig. 2. CDK4 and CDK6: More than cell cycle kinases.
Although the role of CDK4 and CDK6 in cell cycle progression has been well documented, both kinases regulate several other functions that are only now starting to be unraveled. (A) Inhibition of CDK4/6 (CDK4/6i) affects lysosome and mitochondrial numbers as well as oxidative phosphorylation. Cyclin D3–CDK6 phosphorylates glycolytic enzymes 6-phosphofructokinase (PFKP) and pyruvate kinase M2 (PKM2), thereby controlling ROS levels via the pentose phosphate (PPP) and serine synthesis pathways. (B) Inhibition of CDK4/6 affects antitumor immunity, acting both within cancer cells and on the immune system of the host. In tumor cells, inhibition of CDK4/6 impedes expression of an E2F target, DNA methyltransferase (DNMT). DNMT inhibition reduces methylation of endogenous retroviral genes (ERV) and increases intracellular levels of double-stranded RNA (dsRNA) (114). In effector T cells, inhibition of CDK4/6 stimulates NFAT transcriptional activity and enhances secretion of IFN-γ and interleukin 2 (IL-2) (115).
Lastly, CDK4/6 inhibition impaired lysosomal function and the autophagic flux in cancer cells. It was argued that this lysosomal dysfunction was responsible for the senescent phenotype in CDK4/6 inhibitor–treated cells (110). Because lysosomes are essential for autophagy, the authors co-treated TNBC xenografts with abemaciclib plus an AMPK activator, A769662 (which induces autophagy), and found that this led to cancer cell death and subsequent regression of tumors (110).
Cyclin D3–CDK6 phosphorylates and inhibits two rate-limiting glycolytic enzymes, 6-phosphofructokinase and pyruvate kinase M2. This redirects glycolytic intermediates into the pentose phosphate pathway (PPP) and serine synthesis pathway. Through this mechanism, cyclin D3–CDK6 promotes the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) and helps to neutralize ROS (112). Treatment of tumors expressing high levels of cyclin D3–CDK6 (such as leukemias) with CDK4/6 inhibitors reduced the PPP- and serine-synthesis pathway flow, thereby depleting the antioxidants NADPH and GSH. This increased ROS levels and triggered tumor cell apoptosis (112).
Another link between cyclin D–CDK4/6 in metabolism and cancer was provided by the observation that livers of obese/diabetic mice up-regulate cyclin D1 expression (113). Treatment of these mice with an antidiabetic compound, metformin, reduced liver cyclin D1 levels and largely protected mice against development of hepatocellular carcinoma. Also, genetic ablation of cyclin D1 protected obese/diabetic mice from liver cancer, and administration of palbociclib inhibited liver cancer progression. These treatments had no effect on tumors in nonobese animals (113). These observations raise the possibility of using antidiabetic compounds with CDK4/6 inhibitors for treatment of liver cancers in obese patients.
CDK4/6 inhibitors and antitumor immune responses
Several recent reports have started to unravel how inhibition of CDK4/6 influences antitumor immune responses, acting both on tumor cells as well as on the tumor immune environment (Fig. 2B). Treatment of breast cancer–bearing mice or breast cancer cells with abemaciclib activated expression of endogenous retroviral elements in tumor cells, thereby increasing the levels of double-stranded RNA. This, in turn, stimulated production of type III interferons and increased presentation of tumor antigens. Hence, CDK4/6 inhibitors, by inducing viral gene expression, trigger antiviral immune responses that help to eliminate the tumor (114).
Inhibition of CDK4/6 also affects the immune system by impeding the proliferation of CD4+FOXP3+ regulatory T cells (Tregs), which normally inhibit the antitumor response. Because cytotoxic CD8+ T cells are less affected by CDK4/6 inhibition, abemaciclib treatment decreases the Treg/CD8+ ratio of intratumoral T cells and facilitates tumor cell killing by cytotoxic CD8+ T cells (114).
Inhibition of CDK4/6 also resulted in activation of T cells through derepression of NFAT signaling. NFAT4 (and possibly other NFATs) are phosphorylated by cyclin D3–CDK6 (115). Inhibition of CDK4/6 decreased phosphorylation of NFATs, resulting in their nuclear translocation and enhanced transcriptional activity. This caused up-regulation of NFAT targets, resulting in T cell activation, which enhanced the antitumor immune response. In addition, CDK4/6 inhibitors increased the infiltration of effector T cells into tumors, likely because of elevated levels of chemokines CXCL9 and CXCL10 after CDK4/6 inhibitor treatment (115). Abemaciclib treatment also induced inflammatory and activated T cell phenotypes in tumors and up-regulated the expression of immune checkpoint proteins CD137, PD-L1, and TIM-3 on CD4+ and CD8+ cells (116).
CDK4/6 inhibition also caused up-regulation of PD-L1 protein expression in tumor cells (117, 118). This effect was shown to be independent of RB1 status in the tumor. Mechanistically, CDK4/6 phosphorylates and stabilizes SPOP, which promotes PD-L1 polyubiquitination and degradation (118). Cyclin D–CDK4 also represses expression of PD-L1 through RB1. Specifically, cyclin D–CDK4/6-mediated phosphorylation of RB1 on S249/T252 promotes binding of RB1 to NF-κB protein p65, and this represses the expression of a subset NF-κB–regulated genes, including PD-L1 (119).
These observations prompted tests of the efficacy of combining CDK4/6 inhibitors with antibodies that elicit immune checkpoint blockade. Indeed, treatment of mice bearing autochthonous breast cancers, or cancer allografts, with CDK4/6 inhibitors together with anti-PD-1/PD-L1 antibodies enhanced the efficacy of immune checkpoint blockade and led to complete tumor regression in a high proportion of animals (114, 115, 118). Conversely, activation of the cyclin D–CDK4 pathway by genomic lesions in human melanomas correlated with resistance to anti–PD-1 therapy (117).
Some authors did not observe synergy when abemaciclib was administered concurrently with immune checkpoint inhibitors in allograft tumor models (116, 120). However, a strong synergistic antitumor effect was detected when abemaciclib was administered first (and continued) and anti–PD-L1 antibody was administered later. The combined treatment induced immunological memory, as mice that underwent tumor regression were resistant to rechallenge with the same tumor (116). Abemaciclib plus anti–PD-L1 treatment increased infiltration of CD4+ and CD8+ T cells into tumors, and increased the expression of major histocompatibility complex class I (MHC-I) and MHC-II on tumor cells and on macrophages and MHC-I on dendritic cells (116). In the case of anti–CTLA-4 plus anti–PD-1 treatment in melanoma allograft model, the synergistic effect was observed when immune checkpoint inhibitor treatment was started first, followed by abemaciclib (120).
The synergistic antitumor effect of PI3K and CDK4/6 inhibitors in TNBC is mediated, in part, by enhancement of tumor immunogenicity (121). Combined treatment of TNBC cells with ribociclib plus the PI3K inhibitor apelisib synergistically up-regulated the expression of immune-related pathways in tumor cells, including proteins involved in antigen presentation. Co-treatment of tumor-bearing mice also decreased proliferation of CD4+FOXP3+ Treg cells, increased activation of intratumoral CD4+ and CD8+ T cells, increased the frequency of tumor-infiltrating NKT cells, and decreased the numbers of intratumoral immunosuppressive myeloid-derived suppressor cells. Moreover, combined treatment strongly augmented the response to immune checkpoint therapy with PD-1 and CTLA-4 antibodies (121).
Single-cell RNA sequencing of human melanomas identified an immune resistance program expressed by tumor cells that correlates with T cell exclusion from the tumor mass and immune evasion by tumor cells. The program can predict the response of tumors to immune checkpoint inhibitors. Treatment of human melanoma cells with abemaciclib repressed this program in an RB1-dependent fashion (120).
Together, these findings indicate that CDK4/6 inhibitors may convert immunologically “cold” tumors into “hot” ones. The most pressing issue is to validate these findings in a clinical setting. The utility of combining CDK4/6 inhibitors with PD-1 or PD-L1 antibodies is currently being evaluated in several clinical trials. Note that the effects of CDK4/6 inhibition on the immune system of the host are independent of tumor cell RB1 status, raising the possibility of using CDK4/6 inhibitors to also boost the immune response against RB1-negative tumors.
CDK4/6 inhibitors in clinical trials
Table 3 summarizes major clinical trials with CDK4/6 inhibitors. Given early preclinical data indicating that breast cancers—in particular, the hormone receptor–positive ones—are very sensitive to CDK4/6 inhibition (as discussed above), many clinical trials have focused on this cancer type. Most studies have evaluated CDK4/6 inhibitors administered together with anti-estrogens (the aromatase inhibitors letrozole or anastrozole, or the estrogen receptor antagonist fulvestrant) for treatment of advanced/metastatic HR+/HER2– breast cancers in postmenopausal women. Addition of CDK4/6 inhibitors significantly extended median progression-free survival (78, 122–130) and prolonged median overall survival (131–134). Moreover, abemaciclib has shown clinical activity when administered as a single agent (135). Consequently, palbociclib, ribociclib, and abemaciclib have been approved by the US Food and Drug Administration (FDA) for treatment of patients with advanced/metastatic HR+/HER2– breast cancer (Box 1). A recent phase 3 clinical trial, MonarchE, evaluated abemaciclib plus standard endocrine therapy in treatment of patients with early-stage, high-risk, lymph node–positive HR+/HER2– breast cancer. Addition of abemaciclib reduced the risk of breast cancer recurrence (136). This is in contrast to the similar PALLAS study reported this year, which found no benefit of adding palbociclib to endocrine therapy for women with early-stage breast cancer (137). Analysis of patient populations in these two trials may help to explain the different outcomes. It is also possible that the favorable outcome of the MonarchE study reflects a broader spectrum of kinases inhibited by abemaciclib. The utility of CDK4/6 inhibitors in early-stage breast cancer remains unclear and is being addressed in ongoing clinical trials (PALLAS, PENELOPE-B, EarLEE-1, MonarchE) (138).
CDK4/6 inhibitor
Trial name
Trial details
Treatment
Patients
Outcome
Ref.
Other outcomes
Palbociclib
PALOMA-1
Randomized
phase 2
Aromatase inhibitor
letrozole alone
(standard of care)
versus letrozole
plus palbociclib
Postmenopausal women
with advanced ER+/HER2–
breast cancer who had
not received any systemic
treatment for their
advanced disease
Addition of palbociclib markedly
increased median PFS from
10.2 months in the
letrozole group to
20.2 months in the
palbociclib plus
letrozole group
On the basis of this result, palbociclib
received a “Breakthrough Therapy”
designation status from FDA and was
granted accelerated approval, in
combination with letrozole, for the
treatment of ER+/HER2– metastatic
breast cancer
Palbociclib
PALOMA-2
Double-blind
phase 3
Palbociclib plus
letrozole as first-
line therapy
Postmenopausal women
with ER+/HER2–
breast cancer
Addition of palbociclib strongly
increased median PFS:
14.5 months in the placebo-
letrozole group versus
24.8 months in the
palbociclib-letrozole group
Palbociclib was equally efficacious in
patients with luminal A and B breast
cancers, and there was no single
biomarker associated with the lack of
clinical benefit, except for RB1 loss;
CDK4 amplification was associated
with endocrine resistance, but this
was mitigated by addition of
palbociclib; tumors with high levels
of FGFR2 and ERBB3 mRNA
displayed greater PFS gain
after addition of palbociclib (79)
Palbociclib
PALOMA-3
Randomized
phase 3
Estrogen receptor
antagonist
fulvestrant plus
placebo versus
fulvestrant plus
palbociclib
Women with HR+/HER2–
metastatic breast cancer
that had progressed on
previous endocrine therapy
The study demonstrated a
substantial prolongation
of median PFS in the palbociclib-
treated group: 4.6 months in the
placebo plus fulvestrant group
versus 9.5 months in the
palbociclib plus fulvestrant
group; addition of palbociclib
also extended median overall
survival from 28.0 months
(placebo-fulvestrant) to
34.9 months (palbociclib-
fulvestrant); estimated rate
of survival at 3 years was
41% versus 50%, respectively
Palbociclib in
patients with
early breast
cancer at high
risk of recurrence
Ongoing
Ribociclib
MONA
LEESA-2
Randomized
phase 3
Ribociclib plus
letrozole versus
placebo plus
letrozole
First-line treatment for
postmenopausal women
with HR+/HER2– recurrent
or metastatic breast
cancer who had not
received previous
systemic therapy for
advanced disease
At 18 months, PFS
was 42.2% in the
placebo-letrozole
group and 63.0%
in the ribociclib-
letrozole group
Patients with advanced
(metastatic or recurrent)
HR+/HER2– breast cancer
who have either received no
treatment for the advanced
disease or previously
received a single line of
endocrine therapy for the
advanced disease
Addition of ribociclib significantly
extended median PFS, from
12.8 months (placebo-fulvestrant)
to 20.5 months (ribociclib-
fulvestrant); overall survival at
42 months was also extended
from 45.9% (placebo-fulvestrant)
to 57.8% (ribociclib-fulvestrant)
Ribociclib versus
placebo together
with an anti-
estrogen tamoxifen
or an aromatase
inhibitor (letrozole
or anastrozole)
Premenopausal and
perimenopausal women
with HR+/HER2– advanced
breast cancer who had not
received previous treatment
with CDK4/6 inhibitors
Ribociclib significantly increased
median PFS from 13.0 months in
the placebo-endocrine therapy
group to 23.8 months in the
ribociclib-endocrine therapy
group; overall survival was also
strongly prolonged in the ribociclib
group (estimated overall survival
at 42 months was 46.0% for the
placebo group and 70.2% in the
ribociclib group)
Ribociclib in the
treatment of early-
stage, high-risk
HR+/HER2–
breast cancers
Ongoing
Abemaciclib
MONARCH 1
Phase 2 trial
Abemaciclib as a
single agent
Women with HR+/HER2–
metastatic breast cancer
who had progressed on or
after prior endocrine therapy
and had 1 or 2 chemotherapy
regimens in the metastatic
setting
Abemaciclib exhibited promising activity
in these heavily pretreated patients
with poor prognosis; median
PFS was 6.0 months and overall
survival 17.7 months
The most common adverse events
were diarrhea, fatigue, and
nausea (136)
Abemaciclib
MONARCH 2
Double-blind
phase 3
Abemaciclib in
combination
with fulvestrant
Women with HR+/HER2– breast
cancer who had progressed
while receiving endocrine
therapy, or while receiving
first-line endocrine therapy for
metastatic disease
Addition of abemaciclib significantly
increased PFS from 9.3 months in
the placebo-fulvestrant to 16.4 in
the abemaciclib-fulvestrant group;
median overall survival was also
extended from 37.3 months
to 46.7 months
Abemaciclib plus
an aromatase
inhibitor
(anastrozole
or letrozole)
Postmenopausal women
with advanced HR+/HER2–
breast cancer who had
no prior systemic therapy
in the advanced setting
Addition of abemaciclib prolonged
PFS from 14.8 months (in
the placebo-aromatase
inhibitor group) to 28.2 months
(abemaciclib-aromatase
inhibitor group)
Patients with HR+/HER2–
lymph node–positive,
high-risk early
breast cancer
Preliminary analysis indicates that
addition of abemaciclib resulted
in a significant improvement of
invasive disease-free survival
and of distant relapse-
free survival
Chemotherapy alone
(gemcitabine and
carboplatin),
versus concurrent
administration of
trilaciclib plus
chemotherapy,
versus
administration of
trilaciclib prior to
chemotherapy
(to mitigate the
cytotoxic effect of
chemotherapy on
bone marrow)
Patients with recurrent or
metastatic triple-negative
breast cancer who had no
more than two previous
lines of chemotherapy
Addition of trilaciclib did not offer
detectable myeloprotection, but
resulted in increased overall
survival (from 12.8 months in the
chemotherapy-only group to
20.1 months in the concurrent
trilaciclib and chemotherapy
group and 17.8 months in trilaciclib
before chemotherapy group)
Approved by FDA in 2016, in combination with fulvestrant for the treatment of hormone receptor–positive, HER2-negative (HR+/HER2–) advanced or metastatic breast cancer in women with disease progression following endocrine therapy. Approved in 2017 for the treatment of HR+/HER2– advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine-based therapy in postmenopausal women.
Palbociclib is administered at a dose of 125 mg (given orally) daily for 3 weeks followed by 1 week off, or 200 mg daily for 2 weeks followed by 1 week off. The rate-limiting toxicities are neutropenia, thrombocytopenia, and anemia.
Ribociclib
Approved by FDA in 2017, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with HR+/HER2– advanced or metastatic breast cancer. In 2018, the FDA expanded the indication for ribociclib in combination with an aromatase inhibitor for pre/perimenopausal women with HR+/HER2– advanced or metastatic breast cancer, as initial endocrine-based therapy. FDA also approved ribociclib in combination with fulvestrant for postmenopausal women with HR+/HER2– advanced or metastatic breast cancer, as initial endocrine-based therapy or following disease progression on endocrine therapy.
Ribociclib is administered at a dose of 600 mg (given orally) daily for 3 weeks followed by 1 week off. The main toxicities are neutropenia and thrombocytopenia.
Abemaciclib
Approved by FDA in 2017, in combination with fulvestrant for women with HR+/HER2– advanced or metastatic breast cancer with disease progression following endocrine therapy. In addition, abemaciclib was approved as monotherapy for women and men with HR+/HER2– advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Approved by FDA in 2018 in combination with an aromatase inhibitor as initial endocrine-based therapy for postmenopausal women with HR+/HER2– advanced or metastatic breast cancer. Approved by FDA in 2021 for adjuvant treatment of early-stage HR+/HER2– breast cancer in combination with endocrine therapy.
Abemaciclib is administered at a dose of 200 mg (given orally) every 12 hours. The dose-limiting toxicity is fatigue. Neutropenia is also observed but is not rate-limiting. Other severe side effects include diarrhea and nausea.
Currently, palbociclib is being used in 164 active or recruiting clinical trials, ribociclib in 69 trials, and abemaciclib in 98 trials for more than 50 tumor types (139). These trials evaluate combinations of CDK4/6 inhibitors with a wide range of compounds (Table 4). Trials with trilaciclib test the benefit of this compound in preserving bone marrow and the immune system.
Additional target
Inhibitor
Immune checkpoint inhibitor
Tumor type
Trial identifier
Palbociclib
Aromatase
Letrozole, anastrozole,
exemestane
HR+ breast cancer, HR+ ovarian
cancer, metastatic breast cancer,
metastatic endometrial cancer
tumors with ERK1/2
mutations, glioblastoma,
metastatic cancer
NCT04534283,
NCT04391595,
NCT02857270
Trilaciclib
Proliferating cells
Chemotherapy
SCLC: This trial evaluates the
potential clinical benefit of
trilaciclib in preventing
chemotherapy-induced
myelosuppression in patients
receiving chemotherapy
NCT04504513
Proliferating cells +
PD-L1
Carboplatin + etoposide
Atezolizumab
SCLC: This trial investigates the
potential clinical benefit of trilaciclib
in preserving the bone marrow and
the immune system, and enhancing
antitumor efficacy when
administered with chemotherapy
NCT03041311
Proliferating cells
Topotecan
SCLC: This trial investigates the
potential clinical benefit of
trilaciclib in preserving the
bone marrow and the immune
system, and enhancing the
antitumor efficacy of chemotherapy
when administered prior
to chemotherapy
NCT02514447
Proliferating cells
Carboplatin + gemcitabine
Metastatic TNBC: This study
investigates the potential
clinical benefit of trilaciclib in
preserving the bone marrow
and the immune system, and
enhancing the antitumor efficacy
of chemotherapy when administered
prior to chemotherapy
NCT02978716
Lerociclib
ER
ER antagonist: fulvestrant
HR+/HER2– metastatic
breast cancer
NCT02983071
EGFR
Osimertinib
EGFR mutant NSCLC
NCT03455829
SHR6390
ER
ER antagonist: fulvestrant
HR+/HER2– recurrent/
metastatic breast cancer
NCT03481998
Aromatase
Letrozole, anastrozole
HR+/HER2– recurrent/
metastatic breast cancer
NCT03966898,
NCT03772353
EGFR/HER2
Pyrotinib
HER2+ gastric cancer, HER2+
metastatic breast cancer
NCT04095390,
NCT03993964
AR
AR antagonists: SHR3680
metastatic TNBC
NCT03805399
PF-06873600
Endocrine therapy
Single agent and then
in combination with
endocrine therapy
HR+/HER2– metastatic breast
cancer, ovarian and fallopian tube
cancer, TNBC and other tumors
Although CDK4/6 inhibitors represent very effective agents in cancer treatment, nearly all patients eventually develop resistance and succumb to the disease. Moreover, a substantial fraction of tumors show intrinsic resistance to treatment with CDK4/6 inhibitors (Fig. 3).
Fig. 3. Mechanisms of cancer cell resistance to CDK4/6 inhibition.
Known mechanisms include loss of RB1, activation of pathways impinging on CycD-CDK4/6, amplification of the CDK4/6 genes and overexpression of CDK6 protein, activation of CycE-CDK2, and lysosomal sequestration of CDK4/6 inhibitors. Blank pieces of the puzzle denote additional mechanisms that remain to be discovered.
The best-documented mechanism of preexisting and acquired resistance is the loss of RB1 (71, 81, 140). Acquired RB1 loss has been detected in PDXs (141), in circulating tumor DNA (ctDNA) (142, 143), and in tumors from patients treated with CDK4/6 inhibitors (144, 145). However, RB1 mutations are likely subclonal and are seen in only 5 to 10% of patients (143, 145).
Increased expression of CDK6 was shown to underlie acquired resistance to CDK4/6 inhibitors. Amplification of the CDK6 gene and the resulting overexpression of CDK6 protein were found in abemaciclib-resistant ER+ breast cancer cells (146) and in ctDNA of patients with ER+ breast cancers that progressed during treatment with palbociclib plus endocrine therapy (147). Also, CDK4 gene amplification conferred insensitivity to CDK4/6 inhibition in GBM and sarcomas (148–150), whereas overexpression of CDK4 protein was associated with resistance to endocrine therapy in HR+ breast cancers (79).
Resistant breast cancer cells can also up-regulate the expression of CDK6 through suppression of the TGF-β/SMAD4 pathway by the microRNA miR-432-5p. In this mechanism, exosomal expression of miR-432-5p mediates the transfer of the resistance phenotype between neighboring cell populations (151). Another mechanism of CDK6 up-regulation in ER+ breast cancers is the loss of FAT1, which represses CDK6 expression via the Hippo pathway. Loss of FAT1 triggers up-regulation of CDK6 expression by the Hippo pathway effectors TAZ and YAP. Moreover, genomic alterations in other components of the Hippo pathway, although rare, are also associated with reduced sensitivity to CDK4/6 inhibitors (81).
Genetic lesions that activate pathways converging on D-type cyclins can cause resistance to CDK4/6 inhibitors. These include (i) FGFR1/2 gene amplification or mutational activation, detected in ctDNA from patients with ER+ breast cancers that progressed upon treatment with palbociclib plus endocrine therapy (147); (ii) hyperactivation of the MAPK pathway in resistant prostate adenocarcinoma cells, possibly due to increased production of EGF by cancer cells (152); and (iii) increased secretion of FGF in palbociclib-resistant KRAS-mutant NSCLC cells, which stimulates FGFR1 signaling in an autocrine or paracrine fashion, resulting in activation of ERK1/2 and mTOR as well as up-regulation of D-cyclin, CDK6, and cyclin E expression (153). Analyses of longitudinal tumor biopsies from a melanoma patient revealed an activating mutation in the PIK3CA gene that conferred resistance to ribociclib plus MEK inhibitor treatment (154). It is possible that these lesions elevate the cellular levels of active cyclin D–CDK4/6 complexes, thereby increasing the threshold for CDK4/6 inhibition.
Formation of a noncanonical cyclin D1–CDK2 complex was shown to represent another mechanism of acquired CDK4/6 inhibitor resistance. Such a complex was observed in palbociclib-treated ER+ breast cancer cells and was implicated in overcoming palbociclib-induced cell cycle arrest (141). Also, depletion of AMBRA1 promoted the interaction of D-cyclins with CDK2, resulting in resistance to CDK4/6 inhibitors (20, 22); it remains to be seen whether this represents an intrinsic or acquired resistance mechanism in human tumors.
Genetic analyses revealed that activation of cyclin E can bypass the requirement for cyclin D–CDK4/6 in development and tumorigenesis (155, 156). Hence, it comes as no surprise that increased activity of cyclin E–CDK2 is responsible for a large proportion of intrinsic and acquired resistance to CDK4/6 inhibitors. Several different mechanisms can activate cyclin E–CDK2 kinase in resistant tumor cells: (i) Down-regulation of KIP/CIP inhibitors results in increased activity of cyclin E–CDK (54, 157). (ii) Loss of PTEN expression, which activates AKT signaling, leads to nuclear exclusion of p27KIP1. This in turn prevents access of p27KIP1 to CDK2, resulting in increased CDK2 kinase activity (144). (iii) Activation of the PI3K/AKT pathway causes decreased levels of p21CIP1. Co-treatment of melanoma PDXs with MDM2 inhibitors (which up-regulate p21CIP1 via p53) sensitized intrinsically resistant tumor cells to CDK4/6 inhibitors (158). (iv) Up-regulation of cyclin D1 levels triggers sequestration of KIP/CIP inhibitors from cyclin E–CDK2 to cyclin D–CDK4/6, thereby activating the former (158). (v) Amplification of the CCNE1 gene and increased levels of cyclin E1 protein result in elevated activity of E-CDK2 kinase (141). (vi) mTOR signaling has been shown to up-regulate cyclin E1 (and D1) in KRAS-mutated pancreatic cancer cells; CDK2 activity was essential for CDK4/6 inhibitor resistance in this setting (159). (vii) Up-regulation of PDK1 results in activation of the AKT pathway, which increases the expression of cyclins E and A and activates CDK2 (160). (viii) In CDK4/6 inhibitor–resistant melanoma cells, high levels of RNA-binding protein FXR1 increase translation of the amino acid transporter SLC36A1. Up-regulation of SLC36A1 expression activates mTORC1, which in turn increases CDK2 expression (161). All these lesions are expected to allow cell proliferation, despite CDK4/6 inhibition, as a consequence of the activation of the downstream cell cycle kinase CDK2.
The role for cyclin E–CDK2 in CDK4/6 inhibitor resistance has been confirmed in clinical trials. In patients with advanced ER+ breast cancer treated with palbociclib and letrozole or fulvestrant, the presence of proteolytically cleaved cytoplasmic cyclin E in tumor tissue conferred strongly shortened progression-free survival (71). Moreover, analyses of PALOMA-3 trial for patients with ER+ breast cancers revealed lower efficacy of palbociclib plus fulvestrant in patients displaying high cyclin E mRNA levels in metastatic biopsies (80). Amplification of the CCNE1 gene was detected in ctDNA of patients with ER+ breast cancers that progressed on palbociclib plus endocrine therapy (147). Also, amplification of the CCNE2 gene (encoding cyclin E2) was seen in a fraction of CDK4/6 inhibitor–resistant HR+ mammary carcinomas (145, 162).
Collectively, these analyses indicate that resistant cells may become dependent on CDK2 for cell cycle progression. Indeed, depletion of CDK2 or inhibition of CDK2 kinase activity in combination with CDK4/6 inhibitors blocked proliferation of CDK4/6 inhibitor–resistant cancer cells (111, 141, 158–161). Recently, two CDK2-specific inhibitors, PF-07104091 (163) and BLU0298 (164), have been reported. PF-07104091 is now being tested in a phase 2 clinical trial in combination with palbociclib plus antiestrogens. Another recent study identified a novel compound, PF-3600, that inhibits CDK4/6 and CDK2 (165). PF3600 had potent antitumor effects against xenograft models of intrinsic and acquired resistance to CDK4/6 inhibition (165). A phase 2 clinical trial is currently evaluating this compound as a single agent and in combination with endocrine therapy in patients with HR+/HER2– breast cancer and other cancer types.
Whole-exome sequencing of 59 HR+/HER2– metastatic breast tumors from patients treated with CDK4/6 inhibitors and anti-estrogens revealed eight alterations that likely conferred resistance: RB1 loss; amplification of CCNE2 or AURKA; activating mutations or amplification of AKT1, FGFR2, or ERBB2; activating mutations in RAS genes; and loss of ER expression. The frequent activation of AURKA (in 27% of resistant tumors) raises the possibility of combining CDK4/6 inhibitors with inhibitors of Aurora A kinase to overcome resistance (145).
In contrast to ER+ mammary carcinomas, TNBCs are overall resistant to CDK4/6 inhibition (45). A subset of TNBCs display high numbers of lysosomes, which causes sequestration of CDK4/6 inhibitors into the expanded lysosomal compartment, thereby preventing their action on nuclear CDK4/6. Preclinical studies revealed that lysosomotropic agents that reverse the lysosomal sequestration (such as chloroquine, azithromycin, or siramesine) render TNBC cells fully sensitive to CDK4/6 inhibition (71, 111). These observations now need to be tested in clinical trials for TNBC patients.
Outlook
Although D-cyclins and CDK4/6 were discovered 30 years ago, several aspects of cyclin D–CDK4/6 biology, such as their role in antitumor immunity, are only now starting to be appreciated. The full range of cyclin D–CDK4/6 functions in tumor cells remains unknown. It is likely that these kinases play a much broader role in cancer cells than is currently appreciated. Hence, the impact of CDK4/6 inhibition on various aspects of tumorigenesis requires further study. Also, treatment of patients with CDK4/6 inhibitors likely affects several aspects of host physiology, which may be relevant to cancer progression.
In the next years, we will undoubtedly witness the development and testing of new CDK4/6 inhibitors. Because activation of CDK2 represents a frequent CDK4/6 inhibitor resistance mechanism, compounds that inhibit CDK4/6 and CDK2 may prevent or delay the development of resistance. Conversely, selective compounds that inhibit CDK4 but not CDK6 may allow more aggressive dosing, as they are expected not to result in bone marrow toxicity caused by CDK6 inhibition. New, less basic CDK4/6 inhibitor compounds (111) may escape lysosomal sequestration and may be efficacious against resistant cancer types such as TNBC. Degrader compounds, which induce proteolysis of cyclin D rather than inhibit cyclin D–CDK4/6 kinase, may have superior properties, as they would extinguish both CDK4/6-dependent and -independent functions of D-cyclins in tumorigenesis. Moreover, dissolution of cyclin D–CDK4/6 complexes is expected to liberate KIP/CIP inhibitors, which would then inhibit CDK2. D-cyclins likely play CDK-independent functions in tumorigenesis—for example, by regulating gene expression (166). However, their role in tumor biology and the utility of targeting these functions for cancer treatment remain largely unexplored.
An important challenge will be to test and identify combinatorial treatments involving CDK4/6 inhibitors for the treatment of different tumor types. CDK4/6 inhibitors trigger cell cycle arrest of tumor cells and, in some cases, senescence. It will be essential to identify combination treatments that convert CDK4/6 inhibitors from cytostatic compounds to cytotoxic ones, which would unleash the killing of tumor cells. Genome-wide high-throughput screens along with analyses of mouse cancer models and PDXs will help to address this point. Another largely unexplored area of cyclin D–CDK4/6 biology is the possible involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may extend the use of CDK4/6 inhibitors to treatment of other diseases. All these unresolved questions ensure that CDK4/6 biology will remain an active area of basic, translational, and clinical research for several years to come.
CDK inhibitors and Breast Cancer
The U.S. Food and Drug Administration today granted accelerated approval to Ibrance (palbociclib) to treat advanced (metastatic) breast cancer inr postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have not yet received an endocrine-based therapy. It is to be used in combination with letrozole, another FDA-approved product used to treat certain kinds of breast cancer in postmenopausal women.
See Dr. Melvin Crasto’s blog posts on the announcement of approval of Ibrance (palbociclib) at
Palbociclib and LY2835219 are both cyclin-dependent kinase (CDK) 4/6 inhibitors. CDK4 and CDK6 are kinases that, together with cyclin D1, facilitate the transition of dividing cells from the G1 to the S (synthesis) phase of the cell cycle. Preclinical studies have shown that breast cancer cells rely on CDK4 and CDK6 for division and growth, and that selective CDK4/6 inhibitors can arrest the cells at this G1/S phase checkpoint.
The results of the phase II trial of palbociclib and phase I trial of LY2835219 both indicated that hormone receptor (HR)-positive disease appears to be the best marker to predict patient response.
LY2835219 Phase I Trial Demonstrates Early Activity
The CDK4/6 inhibitor LY2835219 has demonstrated early activity in heavily pretreated women with metastatic breast cancer. Nineteen percent of these women (9 out of 47) had a partial response and 51% (24 out of 47) had stable disease following monotherapy with the oral CDK4/6 inhibitor. Patients had received a median of seven prior therapies, and 75% had metastatic disease in the lung, liver, or brain. The median age of patients was 55 years.
All of the partial responses were in patients with HR-positive disease. The overall response rate for this patient subset was 25% (9 of 36 patients). Twenty of the patients with stable disease had HR-positive disease, with 13 patients having stable disease lasting 24 weeks or more.
Despite treatment, disease progression occurred in 23% of the patients.
These results were presented at a press briefing by Amita Patnaik, MD, associate director of clinical research at South Texas Accelerated Research Therapeutics in San Antonio, Texas, at the 2014 American Association for Cancer Research (AACR) Annual Meeting, held April 5–9, in San Diego.
The phase I trial of LY2835219 enrolled 132 patients with five different tumor types, including metastatic breast cancer. Patients received 150-mg to 200-mg doses of the oral drug every 12 hours.
The overall disease control rate was 70% for all patients and 81% among the 36 HR-positive patients.
The median progression-free survival (PFS) was 5.8 months for all patients and 9.1 months for HR-positive patients. Patnaik noted that the median PFS is still a moving target, as 18 patients, all with HR-positive disease, remain on therapy.
“The data are rather encouraging for a very heavily pretreated patient population,” said Patnaik during the press briefing.
Even though the trial was not designed to compare efficacy based on breast cancer subpopulations, the results in HR-positive tumors are particularly encouraging, according to Patnaik.
Common adverse events thought to be treatment-related were diarrhea, nausea, fatigue, vomiting, and neutropenia. These adverse events occurred in 5% or less of patients at grade 3 or 4 toxicity, except neutropenia, which occurred as a grade 3 or 4 toxicity in 11% of patients. Patnaik noted during the press briefing that the neutropenia was uncomplicated and did not result in discontinuation of therapy by any of the patients.
Palbociclib Phase II Data “Impressive”
The addition of the oral CDK4/6 inhibitor palbociclib resulted in an almost doubling of PFS in first-line treatment of postmenopausal metastatic breast cancer patients with HR-positive disease compared with a control population. The patients in this trial were not previously treated for their metastatic breast cancer, unlike the patient population in the phase I LY2835219 trial.
Patients receiving the combination of palbociclib at 125 mg once daily plus letrozole at 2.5 mg once daily had a median PFS of 20.2 months compared with a median of 10.2 months for patients treated with letrozole alone (hazard ratio = 0.488; P = .0004).
Richard S. Finn, MD, assistant professor of medicine at the University of California, Los Angeles, presented the data from the phase II PALOMA-1 trial at a press briefing at the AACR Annual Meeting.
A total of 165 patients were randomized 1:1 to either the experimental arm or control arm.
Forty-three percent of patients in the combination arm had an objective response compared with 33% of patients in the control arm.
Overall survival (OS), a secondary endpoint in this trial, was encouraging but the results are still preliminary, said Finn during the press briefing. The median OS was 37.5 months in the palbociclib arm compared with 33.3 months in the letrozole alone arm (P = .21). Finn noted that long-term follow-up is necessary to establish the median OS. “This first look of the survival data is encouraging. This is a front-line study, and it is encouraging that there is early [separation] of the curves,” he said.
No new toxicities were reported since the interim trial results. Common adverse events included leukopenia, neutropenia, and fatigue. The neutropenia could be quickly resolved and was uncomplicated and not accompanied by fever, said Finn.
Palbociclib is currently being tested in two phase III clinical trials: The PALOMA-3 trial is testing the combination of palbociclib with letrozole and fulvestrant in late-stage metastatic breast cancer patients who have failed endocrine therapy. The PENELOPE-B trial is testing palbociclib in combination with standard endocrine therapy in HR-positive breast cancer patients with residual disease after neoadjuvant chemotherapy and surgery.
References
Patnaik A, Rosen LS, Tolaney SM, et al. Clinical activity of LY2835219, a novel cell cycle inhibitor selective for CDK4 and CDK6, in patients with metastatic breast cancer. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT232.
Finn RS, Crown JP, Lang I, et al. Final results of a randomized phase II study of PD 0332991, a cyclin-dependent kinase (CDK)-4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2-advanced breast cancer (PALOMA-1; TRIO-18). American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT101.
This article has been cited by other articles in PMC.
Cyclin-dependent kinases (CDKs) drive cell cycle progression and control transcriptional processes. The dysregulation of multiple CDK family members occurs commonly in human cancer; in particular, the cyclin D-CDK4/6-retinoblastoma protein (RB)-INK4 axis is universally disrupted, facilitating cancer cell proliferation and prompting long-standing interest in targeting CDK4/6 as an anticancer strategy. Most agents that have been tested inhibit multiple cell cycle and transcriptional CDKs and have carried toxicity. However, several selective and potent inhibitors of CDK4/6 have recently entered clinical trial. PD0332991, the first to be developed, resulted from the introduction of a 2-aminopyridyl substituent at the C2-position of a pyrido(2,3-d)pyrimidin-7-one backbone, affording exquisite selectivity toward CDK4/6.1 PD0332991 arrests cells in G1 phase by blocking RB phosphorylation at CDK4/6-specfic sites and does not inhibit the growth of RB-deficient cells.2 Phase I studies conducted in patients with advanced RB-expressing cancers demonstrated mild side effects and dose-limiting toxicities of neutropenia and thrombocytopenia, with prolonged stable disease in 25% of patients.3,4 In cyclin D1-translocated mantle cell lymphoma, PD0332991 extinguished CDK4/6 activity in patients’ tumors, resulting in markedly reduced proliferation, and translating to more than 1 year of stability or response in 5 of 17 cases.5
Two recent papers from the Knudsen laboratory make several important observations that will help guide the continued clinical development of CDK4/6 inhibitors. In the study by Dean et al., surgically resected patient breast tumors were grown on a tissue culture matrix in the presence or absence of PD0332991. Crucially, these cultures retained associated stromal components known to play important roles in cancer pathogenesis and therapeutic sensitivities, as well as key histological and molecular features of the primary tumor, including expression of ER, HER2 and Ki-67. Similar to results in breast cancer cell lines,6 the authors demonstrate that only RB-positive tumors have growth inhibition in response to PD0332991, irrespective of ER or HER2 status, while tumors lacking RB were completely resistant. This result underscores RB as the predominant target of CDK4/6 in breast cancer cells and the primary marker of drug response in primary patient-derived tumors. As expected, RB-negative tumors routinely demonstrated robust expression of p16INK4A; however, p16INK4A expression was not always a surrogate marker for RB loss, supporting the importance of direct screening of tumors for RB expression to select patients appropriate for CDK4/6 inhibitor clinical trials.
In the second study, McClendon et al. investigated the efficacy of PD0332991 in combination with doxorubicin in triple-negative breast cancer cell lines. Again, RB functionality was paramount in determining response to either PD0332991 monotherapy or combination treatment. In RB-deficient cancer cells, CDK4/6 inhibition had no effect in either instance. However, in RB-expressing cancer cells, CDK4/6 inhibition and doxorubicin provided a cooperative cytostatic effect, although doxorubicin-induced cytotoxicity was substantially reduced, assessed by markers for mitotic catastrophe and apoptosis. Additionally, despite cytostatic cooperativity, CDK4/6 inhibition maintained the viability of RB-proficient cells in the presence of doxorubicin, which repopulated the culture after removal of drug. These results reflect previous data demonstrating that ectopic expression of p16INK4A can protect cells from the lethal effects of DNA damaging and anti-mitotic chemotherapies.7 Similar results have been reported in MMTV-c-neu mice bearing RB-proficient HER2-driven tumors, where PD0332991 compromised carboplatin-induced regressions,8 suggesting that DNA-damaging treatments should not be combined concomitantly with CDK4/6 inhibition in RB-proficient tumors.
To combine CDK4/6 inhibition with cytotoxics, sequential treatment may be considered, in which CDK4/6 inhibition is followed by DNA damaging chemotherapy; cells relieved of G1 arrest may synchronously enter S phase, where they may be most susceptible to agents disrupting DNA synthesis. Release of myeloma cells from a prolonged PD0332991-mediated G1 block leads to S phase synchronization; interestingly, all scheduled gene expression is not completely restored (including factors critical to myeloma survival such as IRF4), further favoring apoptotic responses to cytotoxic agents.9 Furthermore, in RB-deficient tumors, CDK4/6 inhibitors may be used to maximize the therapeutic window between transformed and non-transformed cells treated with chemotherapy. In contrast to RB-deficient cancer cells, RB-proficient non-transformed cells arrested in G1 in response to PD0332991 are afforded protection from DNA damaging agents, thereby reducing associated toxicities, including bone marrow suppression.8
In summary, the current work provides evidence for RB expression as a determinant of response to CDK4/6 inhibition in primary tumors and highlights the complexity of combining agents targeting the cell cycle machinery with DNA damaging treatments.
To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.
Keywords: ER, PD0332991, breast cancer, cell cycle, ex vivo
Breast cancer is a highly heterogeneous disease.1–4 Such heterogeneity is known to influence patient response to both standard of care and experimental therapeutics. In regards to biomarker-driven treatment of breast cancers, it was initially recognized that the presence of the estrogen receptor α (ER) in a fraction of breast cancer cells was associated with the response to tamoxifen and similar anti-estrogenic therapies.5,6 Since this discovery, subsequent marker analyses and gene expression profiling studies have further divided breast cancer into a series of distinct subtypes that harbor differing and often divergent therapeutic sensitivities.1–3 While clearly important in considering the use of several current standard of care therapies, these markers, or molecular sub-types, do not necessarily predict the response to new therapeutic approaches that are currently undergoing clinical development. Thus, there is the continued need for functional analyses of drug response and the definition of new markers that can be used to direct treatment strategies.
Currently, all preclinical cancer models are associated with specific limitations. It is well known that cell culture models lack the tumor microenvironment known to have a significant impact on tumor biology and therapeutic response.7–9 Xenograft models are dependent on the host response for the engraftment of tumor cells in non-native tissues, which do not necessarily recapitulate the nuances of complex tumor milieu.10 In addition, genetically engineered mouse models, while enabling the tumor to develop in the context of the host, can develop tumors that do not mirror aspects of human disease.10 Furthermore, it remains unclear whether any preclinical model truly represents the panoply of breast cancer subtypes that are observed in the clinic. Herein, we utilized a primary human tumor explant culture approach to interrogate drug response, as well as specific determinants of therapeutic response, in an unselected series of breast cancer cases.
Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.
Click on Video Link for Dr. Tolaney slidepresentation of recent data with CDK4/6 inhibitor trial results https://youtu.be/NzJ_fvSxwGk
Sara Tolaney, MD, MPH, a breast oncologist with the Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute, gives an overview of phase I clinical trials and some of the new drugs being tested to treat breast cancer. This talk was originally given at the Metastatic Breast Cancer Forum at Dana-Farber on Oct. 5, 2013.
A great article on current clinical trials and explanation of cdk inhibitors by Sneha Phadke, DO; Alexandra Thomas, MD at the site OncoLive
cdk4/6 inhibitor Ibrance Has Favorable Toxicity and Adverse Event Profile
As discussed in earlier posts and the Introduction to this chapter on Cytotoxic Chemotherapeutics, most anti-cancer drugs developed either to target DNA, DNA replication, or the cell cycle usually have similar toxicity profile which can limit their therapeutic use. These toxicities and adverse events usually involve cell types which normally exhibit turnover in the body, such as myeloid and lymphoid and granulocytic series of blood cells, epithelial cells lining the mucosa of the GI tract, as well as follicular cells found at hair follicles. This understandably manifests itself as common toxicities seen with these types of agents such as the various cytopenias in the blood, nausea vomiting diarrhea (although there are effects on the chemoreceptor trigger zone), and alopecia.
It was felt that the cdk4/6 inhibitors would show serious side effects similar to other cytotoxic agents and this definitely may be the case as outlined below:
For full details, please see Pharmacology/Toxicology review by Dr. Wei Chen The nonclinical studies adequately support the safety of oral administration of palbociclib for the proposed indication and the recommendation from the team is for approval. Non-clinical studies of palbociclib included safety pharmacology studies, genotoxicity
studies, reproductive toxicity studies, pharmacokinetic studies, toxicokinetic studies and repeat-dose general toxicity studies which were conducted in rats and dogs. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation.
Pharmacology:
As described above, palbociclib is an inhibitor of CDK4 and CDK6. Palbociclib modulates downstream targets of CDK4 and CDK6 in vitro and induces G1 phase cell cycle arrest and therefore acts to inhibit DNA synthesis and cell proliferation. Combination of palbociclib with anti-estrogen agents demonstrated synergistic inhibition
of cell proliferation in ER+ breast cancer cells. Palbociclib showed anti-tumor efficacy in animal tumor model studies. Safety pharmacology studies with palbociclib demonstrated adverse effects on both the respiratory and cardiovascular function of dogs at a dose of 125mg/day (four times and 50-times the human clinical exposure
respectively) based on mean unbound Cmax.
General toxicology:
Palbociclib was studied in single dose toxicity studies and repeated dose studies in rats and dogs. Adverse effects in the bone marrow, lymphoid tissues, and male reproductive organs were observed at clinically relevant exposures. Partial to complete reversibility of toxicities to the hematolymphopoietic and male reproductive systems was demonstrated following a recovery period (4-12 weeks), with the exception of the male reproductive organ findings in dogs. Gastrointestinal, liver, kidney, endocrine/metabolic (altered glucose metabolism), respiratory, ocular, and adrenal effects were also seen.
Genetic toxicology:
Palbociclib was evaluated for potential genetic toxicity in in vitro and in vivo studies. The Ames bacterial mutagenicity assay in the presence or absence of metabolic activation demonstrated non-mutagenicity. In addition, palbociclib did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes in the presence or absence of metabolic activation. Palbociclib was identified as aneugenic based on kinetochore analysis of micronuclei formation in an In vitro assay in CHO-WBL cells. In addition, palbociclib was shown to induce micronucleus formation in male rats at doses 100
mg/kg/day (10x human exposure at the therapeutic dose) in an in vivo rat micronucleus assay.
Reproductive toxicology: No effects on estrous cycle and no reproductive toxicities were noticed in standard assays.
Pharmacovigilance (note please see PDF for more information)
Deaths Associated With Trials: Although a few deaths occurred during some trials no deaths were attributed to the drug.
Non-Serious Adverse Events:
(note a reviewers comment below concerning incidence of pulmonary embolism is a combination trial with letrazole)
Other article in this Open Access Journal on Cell Cycle and Cancer Include:
Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?
Curator/Reporter: Stephen J. Williams, Ph.D.
Therapeutic resistance remains one of the most challenging problems for the oncologist, despite the increase of new therapeutics in the oncologist’s toolkit. As new targeted therapies are developed, and new novel targets are investigated as potential therapies, especially cytostatic therapies which it has become evident our understanding of chemoresistance is expanding beyond mechanisms to circumvent a drug’s pharmacologic mechanism of action (i.e. increased DNA repair and cisplatin) or pharmacokinetic changes (i.e. increased efflux by acquisition of a MDR phenotype).
To recount a bit of background I list the overall points of the one of previous posts on tumor heterogeneity (and an interview with Dr. Charles Swanton) are as follows:
Multiple biopsies of primary tumor and metastases are required to determine the full mutational landscape of a patient’s tumor
The intratumor heterogeneity will have an impact on the personalized therapy strategy for the clinician
Metastases arising from primary tumor clones will have a greater genomic instability and mutational spectrum than the tumor from which it originates
Tumors and their metastases do NOT evolve in a linear path but have a branched evolution and would complicate biomarker development and the prognostic and resistance outlook for the patient
The following is a curation of various talks and abstracts from the 2015 AACR National Meeting in Philadelphia on effects of clonal evolution and intratumoral heterogeneity of a tumor with respect to development of chemoresistance. As this theory of heterogeneity and clonal evolution is particularly new I attempted to present all works (although apologize for the length upfront) to forgo bias and so the reader may extract any information pertinent to their clinical efforts and research. However I will give a brief highlight summary below:
From the 2015 AACR National Meeting in Philadelphia
PresentationNumber:NGO2
Presentation Title:
Polyclonal and heterogeneous resistance to targeted therapy in leukemia
Presentation Time:
Monday, Apr 20, 2015, 10:40 AM -10:55 AM
Location:
Room 201, Pennsylvania Convention Center
Author Block:
Catherine C. Smith, Amy Paguirigan, Chen-Shan Chin, Michael Brown, Wendy Parker, Mark J. Levis, Alexander E. Perl, Kevin Travers, Corynn Kasap, Jerald P. Radich, Susan Branford, Neil P. Shah. University of California, San Francisco, CA, Fred Hutchinson Cancer Research Center, Seattle, WA, Pacific Biosciences, Menlo Park, CA, Royal Adelaide Hospital, Adelaide, Australia, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA, University of California, San Francisco, CA
Abstract Body:
Genomic studies in solid tumors have revealed significant branching intratumoral clonal genetic heterogeneity. Such complexity is not surprising in solid tumors, where sequencing studies have revealed thousands of mutations per tumor genome. However, in leukemia, the genetic landscape is considerably less complex. Chronic myeloid leukemia (CML) is the human malignancy most definitively linked to a single genetic lesion, the BCR-ABL gene fusion. Genome wide sequencing of acute myeloid leukemia (AML) has revealed that AML is the most genetically straightforward of all extensively sequenced adult cancers to date, with an average of 13 coding mutations and 3 or less clones identified per tumor.
In CML, tyrosine kinase inhibitors (TKIs) of BCR-ABL have resulted in high rates of remission. However, despite excellent initial response rates with TKI monotherapy, patients still relapse, including virtually all patients with Philadelphia-positive acute lymphoblastic leukemia and blast crisis CML. Studies of clinical resistance highlight BCR-ABL as the sole genetic driver in CML as secondary kinase domain (KD) mutations that prevent drug binding are the predominant mechanism of relapse on BCR-ABL TKIs.
In AML, a more diverse panel of disease-defining genetic mutations has been uncovered. However, in individual patients, a single oncogene can still drive disease. This is the case in FLT3 mutant AML, in which the investigational FLT3 TKI quizartinib achieved an initial response rate of ~50% in relapsed/refractory AML patients with activating FLT3 internal tandem duplication (ITD) mutations, though most patients eventually relapsed. Confirming the importance of FLT3 in disease maintenance, we showed that 8 of 8 patients who relapsed on quizartinib did so due to acquired drug-resistant FLT3 KD mutations.
Studies in CML have revealed that sequential TKI therapy is associated with additional complexity where multiple mutations can coexist separately in an individual patient (“polyclonality”) or in tandem on a single allele (“compound mutations”). In AML, we observed polyclonal FLT3-ITD KD mutations in 2 of 8 patients examined in our initial study of quizartinib resistance.
In light of the polyclonal KD mutations observed in CML and AML at the time of TKI relapse, we undertook next generation sequencing studies to determine the true genetic complexity in CML and AML patients at the time of relapse on targeted therapy. We used Pacific Biosciences RS Single Molecule Real Time (SMRT) third generation sequencing technology to sequence the entire ABL KD or the entire FLT3 juxtamembrane and KD on a single strand of DNA. Using this method, we assessed a total of 103 samples from 79 CML patients on ABL TKI therapy and 36 paired pre-treatment and relapse samples from 18 FLT3-ITD+ AML patients who responded to investigational FLT3 TKI therapy.
In CML, using SMRT sequencing, we detected all mutations previously detected by direct sequencing. Of samples in which multiple mutations were detectable by direct sequencing, 85% had compound mutant alleles detectable in a variety of combinations. Compound mutant alleles were comprised of both dominant and minor mutations, some which were not detectable by direct sequencing. In the most complex case, 12 individual mutant alleles comprised of 7 different mutations were identified in a single sample.
For 12 CML patients, we interrogated longitudinal samples (2-4 time points per patient) and observed complex clonal relationships with highly dynamic shifts in mutant allele populations over time. We detected compound mutations arising from ancestral single mutant clones as well as parallel evolution of de novo polyclonal and compound mutations largely in keeping with what would be expected to cause resistance to the second generation TKI therapy received by that patient.
We used a phospho-flow cytometric technique to assesses the phosphorylation status of the BCR-ABL substrate CRKL in as a method to test the ex vivo biochemical responsiveness of individual mutant cell populations to TKI therapy and assess functional cellular heterogeneity in a given patient at a given timepoint. Using this technique, we observed co-existing cell populations with differential ex vivo response to TKI in 2 cases with detectable polyclonal mutations. In a third case, we identified co-existence of an MLL-AF9 containing cell population that retained the ability to modulate p-CRKL in response to BCR-ABL TKIs along with a BCR-ABL containing only population that showed biochemical resistance to all TKIs, suggesting the co-existence of BCR-ABL independent and dependent resistance in a single patient.
In AML, using SMRT sequencing, we identified acquired quizartinib resistant KD mutations on the FLT3-ITD (ITD+) allele of 9 of 9 patients who relapsed after response to quizartinib and 4 of 9 patients who relapsed after response to the investigational FLT3 inhibitor, PLX3397. In 4 cases of quizartinib resistance and 3 cases of PLX3397 resistance, polyclonal mutations were observed, including 7 different KD mutations in one patient with PLX3397 resistance. In 7 quizartinib-resistant cases and 3 PLX3397-resistant cases, mutations occurred at the activation loop residue D835. When we examined non-ITD containing (ITD-) alleles, we surprisingly uncovered concurrent drug-resistant FLT3 KD mutations on ITD- alleles in 7 patients who developed quizartinib resistance and 4 patients with PLX3397 resistance. One additional PLX3397-resistant patient developed a D835Y mutation only in ITD- alleles at the time of resistance, suggesting selection for a non-ITD containing clone. All of the individual substitutions found on ITD- alleles were the same substitutions identified on ITD+ alleles for each individual patient.
Given that the same individual mutations found on ITD- alleles were also found on ITD+ alleles, we sought to determine whether these mutations were found in the same cell or were indicative of polyclonal blast populations in each patient. To answer this question, we performed single cell sorting of viably frozen blasts from 3 quizartinib-resistant patients with D835 mutations identified at the time of relapse and genotyped single cells for the presence or absence of ITD and D835 mutations. This analysis revealed striking genetic heterogeneity. In 2/3 cases, polyclonal D835 mutations were found in both ITD+ and ITD- cells. In all cases, FLT3-ITD and D835 mutations were found in both heterozygous and homozygous combinations. Most surprisingly, in all 3 patients, approximately 30-40% of FLT3-ITD+ cells had no identified quizartinib resistance-causing FLT3 KD mutation to account for resistance, suggesting the presence of non-FLT3 dependent resistance in all patients.
To determine that ITD+ cells lacking FLT3 KD mutations observed in patients relapsed on quizartinib are indeed consistent with leukemic blasts functionally resistant to quizartinib and do not instead represent a population of differentiated or non-proliferating cells, we utilized relapse blasts from another patient who initially achieved clearance of bone marrow blasts on quizartinib and developed a D835Y mutation at relapse. We performed a colony assay in the presence of 20nM quizartinib. As expected, this dose of quizartinib was unable to suppress the colony-forming ability of blasts from this relapsed patient when compared to DMSO treatment. Genotyping of individual colonies grown from this relapse sample in the presence of 20nM quizartinib again showed remarkable genetic heterogeneity, including ITD+ and ITD- colonies with D835Y mutations in homozygous and heterozygous combinations as well as ITD+ colonies without D835Y mutations, again suggesting the presence of blasts with non-FLT3 dependent resistance. Additionally, 4 colonies with no FLT3 mutations at all were identified in this sample, suggesting the presence of a quizartinib-resistant non-FLT3 mutant blast population. To see if we could identify specific mechanisms of off-target resistance, we performed targeted exome sequencing 33-AML relevant genes from relapse and pre-treatment DNA from all four patients and detected no new mutations in any genes other than FLT3 acquired at the time of disease relapse. Clonal genetic heterogeneity is not surprising in solid tumors, where multiple driver mutations frequently occur, but in CML and FLT3-ITD+ AML, where disease has been shown to be exquisitely dependent on oncogenic driver mutations, our studies suggest a surprising amount of clonal diversity. Our findings show that clinical TKI resistance in these diseases is amazingly intricate on the single allele level and frequently consists of both polyclonal and compound mutations that give rise to an complicated pool of TKI-resistant alleles that can change dynamically over time. In addition, we demonstrate that cell populations with off-target resistance can co-exist with other TKI-resistant populations, underscoring the emerging complexity of clinical TKI resistance. Such complexity argues strongly that monotherapy strategies in advanced CML and AML may be ultimately doomed to fail due to heterogeneous cell intrinsic resistance mechanisms. Ultimately, combination strategies that can address both on and off target resistance will be required to effect durable therapeutic responses.
Session Title:
Tumor Heterogeneity and Evolution
Session Type:
Educational Session
Session Start/End Time:
Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM
Location:
Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
CME:
CME-Designated
CME/CE Hours:
2
Session Description:
One of the major challenges for both the measurement and management of cancer is its heterogeneity. Recent studies have revealed both extensive inter- and intra-tumor heterogeneity at the genotypic and phenotypic levels. Leaders in the field will discuss this challenge, its origins, dynamics and clinical importance. They will also review how we can best measure and deal with tumor heterogeneity, particularly intra-tumor heterogeneity.
Bozic I, Reiter JG, Allen B, Antal T, Chatterjee K, Shah P, Moon YS, Yaqubie A, Kelly N, Le DT, Lipson EJ, Chapman PB, Diaz LA Jr, Vogelstein B, Nowak MA.
Elife. 2013 Jun 25;2:e00747. doi: 10.7554/eLife.00747.
Despite dramatic advances in the treatment of cancer, therapy resistance remains the most significant hurdle in improving the outcome of cancer patients. In this session, we will discuss many different aspects of therapy resistance, including a summary of our current understanding of therapy resistant tumor cell populations as well as analyses of the challenges associated with intratumoral heterogeneity and adaptive responses to targeted therapies.
Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance
Presentation Time:
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Location:
Section 30
Poster Board Number:
29
Author Block:
Xiaowei Xu1, Agostina Nardone1, Huizhong Hu1, Lanfang Qin1, Sarmistha Nanda1, Laura Heiser2, Nicholas Wang2, Kyle Covington1, Edward Chen1, Alexander Renwick1, Tamika Mitchell1, Marty Shea1, Tao Wang1, Carmine De Angelis1, Alejandro Contreras1, Carolina Gutierrez1, Suzanne Fuqua1, Gary Chamness1, Chad Shaw1, Marilyn Li1, David Wheeler1, Susan Hilsenbeck1, Mothaffar Fahed Rimawi1, Joe Gray2, C.Kent Osborne1, Rachel Schiff1. 1Baylor College of Medicine, Houston, TX; 2Oregon Health & Science University, Portland, OR
Abstract Body:
Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant
trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line
models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a
subset of pre-treatment specimens from TBCRC006.
Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed.
Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ~30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ~30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02µM vs. L 3 µM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation.
Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S
levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group
with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC.
Presentation Number:
SY07-04
Presentation Title:
The evolutionary landscape of CLL: Therapeutic implications
Presentation Time:
Sunday, Apr 19, 2015, 2:25 PM – 2:45 PM
Location:
Grand Ballroom (300 Level), Pennsylvania Convention Center
Author Block:
Catherine J. Wu. Dana-Farber Cancer Institute, Boston, MA
Abstract Body:
Clonal evolution is a key feature of cancer progression and relapse. Recent studies across cancers have demonstrated the extensive degree of intratumoral heterogeneity present within individual cancers. We hypothesized that evolutionary dynamics contribute to the variations in disease tempo and response to therapy that are highly characteristic of chronic lymphocytic leukemia (CLL). We have recently investigated this phenomenon by developing a pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequence (WES) and local copy number data (Landau et al., Cell 2013). By applying this analysis approach to 149 CLL cases, we discovered earlier and later cancer drivers, uncovered patterns of clonal evolution in CLL and linked the presence of subclones harboring driver mutations with adverse clinical outcome. Thus, our study, generated from a heterogeneous sample cohort, strongly supports the concept that CLL clonal evolution arises from mass extinction and therapeutic bottlenecks which lead to the emergence of highly fit (and treatment resistant) subclones. We further hypothesized that epigenetic heterogeneity also shapes CLL clonal evolution through interrelation with genetic heterogeneity. Indeed, in recent work, we have uncovered stochastic methylation disorder as the primary cause of methylation changes in CLL and cancer in general, and that this phenomena impacts gene transcription, genetic evolution and clinical outcome. Thus, integrated studies of genetic and epigenetic heterogeneity in CLL have revealed the complex and diverse evolutionary trajectories of these cancer cells.
Immunotherapy is exquisitely suited for specifically and simultaneously targeting multiple lesions. We have developed an approach that leverages whole-exome sequencing to systematically identify personal tumor mutations with immunogenic potential, which can be incorporated as antigen targets in multi-epitope personalized therapeutic vaccines. We are pioneering this approach in an ongoing trial in melanoma and will now expand this concept to address diverse malignancies. Our expectation is that the choice of tumor neoantigens for a vaccine bypasses thymic tolerance and thus generates highly specific and potent high-affinity T cell responses to eliminate tumors in any cancer, including both ‘trunk’ and ‘branch’ lesions.
Abstract Number:
LB-056
Presentation Title:
TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer
Presentation Time:
Sunday, Apr 19, 2015, 4:50 PM – 5:05 PM
Location:
Room 122, Pennsylvania Convention Center
Author Block:
Ping Mu, Zhen Cao, Elizabeth Hoover, John Wongvipat, Chun-Hao Huang, Wouter Karthaus, Wassim Abida, Elisa De Stanchina, Charles Sawyers. Memorial Sloan Kettering Cancer Center, New York, NY
Abstract Body:
Castration-resistant prostate cancer (CRPC) is one of the most difficult cancers to treat with conventional methods and is responsible for nearly all prostate cancer deaths in the US. The Sawyers laboratory first showed that the primary mechanism of resistance to antiandrogen therapy is elevated androgen receptor (AR) expression. Research based on this finding has led to the development of next-generation antiandrogen: enzalutamide. Despite the exciting clinical success of enzalutamide, about 60% of patients exhibit various degrees of resistance to this agent. Highly variable responses to enzalutamide limit the clinical benefit of this novel antiandrogen, underscoring the importance of understanding the mechanisms of enzalutamide resistance. Most recently, an unbiased SU2C-Prostate Cancer Dream Team metastatic CRPC sequencing project led by Dr. Sawyers and Dr. Chinnaiyan revealed that mutations in the TP53 locus are the most significantly enriched alteration in CRPC tumors when compared to primary prostate cancers. Moreover, deletions and decreased expressions of the TP53 and RB1 loci (co-occurrence and individual occurrence) are more commonly associated with CRPC than with primary tumors. These results established that alteration of the TP53 and RB1 pathways are associated with the development of antiandrogen resistance.
By knockdowning TP53 or/and RB1 in the castration resistant LNCaP/AR model, we demonstrate that the disruption of either TP53 or RB1 alone confers significant resistance to enzalutamide both in vitro and in vivo. Strikingly, the co-inactivation of these pathways confers the most dramatic resistance. Since up-regulation of either AR or AR target genes is not observed in the resistant tumors, loss of TP53 and RB1 function confers enzalutamide resistance likely through an AR independent mechanism. In the clinic, resistance to enzalutamide is increasingly being associated with a transition to a poorly differentiated or neuroendocrine-like histology. Interestingly, we observed significant up-regulations of the basal cell marker Ck5 and the neuroendocrine-like cell marker Synaptophysin in the TP53 and RB1 inactivated cells, as well as down-regulation of the luminal cell marker Ck8. The differences between these markers became even greater after enzalutamide treatment. By using the p53-stabilizing drug Nutlin, level of p53 is rescued and consequently the the decrease of AR protein caused by RB1 and TP53 knockdown is reversed. These results strongly suggest that interference of TP53 and RB1 pathways confers antiandrogen resistance by “priming” prostate cancer cells to reprogramming or transdifferentiation, likely neuroendocrine-like differentiation, in response to treatment. Futher experiments will be performed to assess the molecular mechanism of TP53/RB1 alterations in mediating cell programming and conferring antiandrogen resistance.
Abstract Number:
LB-146
Presentation Title:
TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells
Presentation Time:
Monday, Apr 20, 2015, 1:00 PM – 5:00 PM
Location:
Section 41
Author Block:
Naoki Oshimori1, Daniel Oristian1, Elaine Fuchs2. 1Rockefeller University, New York, NY; 2HHMI/Rockefeller University, New York, NY
Abstract Body:
Among the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression.
Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatin-induced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs.
Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity.
Abstract Number:
LB-129
Presentation Title:
Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts
Presentation Time:
Monday, Apr 20, 2015, 1:00 PM – 5:00 PM
Location:
Section 41
Author Block:
Paul Savage1, Sadiq M. Saleh1, Ernesto Iacucci1, Timothe Revil1, Yu-Chang Wang1, Nicholas Bertos1, Anie Monast1, Hong Zhao1, Margarita Souleimanova1, Keith Szulwach2, Chandana Batchu2, Atilla Omeroglu1, Morag Park1, Ioannis Ragoussis1. 1McGill University, Montreal, QC, Canada; 2Fluidigm Corporation, South San Francisco, CA
Abstract Body:
Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. Functional studies distinguishing these subpopulations are ongoing. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells showed high and homogeneous target enrichment with >75% of reads mapping uniquely on target. Variant calling using GATK and Samtools revealed founder mutations in key genes as BRCA1 and TP53, as well as subclonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 cancer driver genes. These findings may be important in understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance.
Abstract Number:
2847
Presentation Title:
High complexity barcoding to study clonal dynamics in response to cancer therapy
Presentation Time:
Monday, Apr 20, 2015, 4:35 PM – 4:50 PM
Location:
Room 118, Pennsylvania Convention Center
Author Block:
Hyo-eun C. Bhang1, David A. Ruddy1, Viveksagar Krishnamurthy Radhakrishna1, Rui Zhao2, Iris Kao1, Daniel Rakiec1, Pamela Shaw1, Marissa Balak1, Justina X. Caushi1, Elizabeth Ackley1, Nicholas Keen1, Michael R. Schlabach1, Michael Palmer1, William R. Sellers1, Franziska Michor2, Vesselina G. Cooke1, Joshua M. Korn1, Frank Stegmeier1. 1Novartis Institutes for BioMedical Research, Cambridge, MA; 2Dana-Farber Cancer Institute, Boston, MA
Abstract Body:
Targeted therapies, such as erlotinib and imatinib, lead to dramatic clinical responses, but the emergence of resistance presents a significant challenge. Recent studies have revealed intratumoral heterogeneity as a potential source for the emergence of therapeutic resistance. However, it is still unclear if relapse/resistance is driven predominantly by pre-existing or de novo acquired alterations. To address this question, we developed a high-complexity barcode library, ClonTracer, which contains over 27 million unique DNA barcodes and thus enables the high resolution tracking of cancer cells under drug treatment. Using this library in two clinically relevant resistance models, we demonstrate that the majority of resistant clones pre-exist as rare subpopulations that become selected in response to therapeutic challenge. Furthermore, our data provide direct evidence that both genetic and non-genetic resistance mechanisms pre-exist in cancer cell populations. The ClonTracer barcoding strategy, together with mathematical modeling, enabled us to quantitatively dissect the frequency of drug-resistant subpopulations and evaluate the impact of combination treatments on the clonal complexity of these cancer models. Hence, monitoring of clonal diversity in drug-resistant cell populations by the ClonTracer barcoding strategy described here may provide a valuable tool to optimize therapeutic regimens towards the goal of curative cancer therapies.
Abstract Number:
3590
Presentation Title:
Resistance mechanisms to ALK inhibitors
Presentation Time:
Tuesday, Apr 21, 2015, 8:00 AM -12:00 PM
Location:
Section 31
Poster Board Number:
13
Author Block:
Ryohei Katayama1, Noriko Yanagitani1, Sumie Koike1, Takuya Sakashita1, Satoru Kitazono1, Makoto Nishio1, Yasushi Okuno2, Jeffrey A. Engelman3, Alice T. Shaw3, Naoya Fujita1. 1Japanese Foundation for Cancer Research, Tokyo, Japan; 2Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Massachusetts General Hospital Cancer Center, Boston, MA
Abstract Body:
Purpose: ALK-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Approximately 3-5% of NSCLCs harbor an ALK gene rearrangement. The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with advanced ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising antitumor activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to one next-generation ALK-TKI – alectinib – and potential strategies to overcome this resistance.
Experimental Procedure: We established a cell line model of alectinib resistance, and analyzed resistant tumor specimens from patients who had relapsed on alectinib. Cell lines were also established under an IRB-approved protocol when there was sufficient fresh tumor tissue. We established Ba/F3 cells expressing EML4-ALK and performed ENU mutagenesis to compare potential crizotinib or alectinib-resistance mutations. In addition, we developed Ba/F3 models harboring ALK resistance mutations and evaluated the potency of multiple next-generation ALK-TKIs including 3rd generation ALK inhibitor in these models and in vivo. To elucidate structure-activity-relationships of ALK resistance mutations, we performed computational thermodynamic simulation with MP-CAFEE.
Results: We identified multiple resistance mutations, including ALK I1171N, I1171S, and V1180L, from the ENU mutagenesis screen and the cell line model. In addition we found secondary mutations at the I1171 residue from the Japanese patients who developed resistance to alectinib or crizotinib. Both ALK mutations (V1180L and I1171 mutations) conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Based on thermodynamics simulation, each resistance mutation is predicted to lead to distinct structural alterations that decrease the binding affinity of ALK-TKIs for ALK.
Conclusions: We have identified multiple alectinib-resistance mutations from the cell line model, patient derived cell lines, and tumor tissues, and ENU mutagenesis. ALK secondary mutations arising after alectinib exposure are sensitive to other next generation ALK-TKIs. These findings suggest a potential role for sequential therapy with multiple next-generation ALK-TKIs in patients with advanced, ALK-rearranged cancers.
Session Title:
Mechanisms of Resistance: From Signaling Pathways to Stem Cells
Session Type:
Major Symposium
Session Start/End Time:
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Location:
Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
CME:
CME-Designated
CME/CE Hours:
2
Session Description:
Even the most effective cancer therapies are limited due to the development of one or more resistance mechanisms. Acquired resistance to targeted therapies can, in some cases, be attributed to the selective propagation of a small population of intrinsically resistant cells. However, there is also evidence that cancer drugs themselves can drive resistance by triggering the biochemical- or genetic-reprogramming of cells within the tumor or its microenvironment. Therefore, understanding drug resistance at the molecular and biological levels may enable the selection of specific drug combinations to counteract these adaptive responses. This symposium will explore some of the recent advances addressing the molecular basis of cancer cell drug resistance. We will address how tumor cell signaling pathways become rewired to facilitate tumor cell survival in the face of some of our most promising cancer drugs. Another topic to be discussed involves how drugs select for or induce the reprogramming of tumor cells toward a stem-like, drug resistant fate. By targeting the molecular driver(s) of rewired signaling pathways and/or cancer stemness it may be possible to select drug combinations that prevent the reprogramming of tumors and thereby delay or eliminate the onset of drug resistance.
Tumour heterogeneity and therapy resistance in melanoma
Presentation Time:
Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM
Location:
Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
Author Block:
Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom
Abstract Body:
Solid tumors are structurally very complex; they consist of heterogeneous cancer cell populations, other non-cancerous cell types and a distinct extracellular matrix. Interactions of cancer cells with non-cancerous cells is well investigated, and our recent work in melanoma has demonstrated that the cellular environment that surrounds cancer cells has a major impact on the way a patient responds to MAP-kinase pathway targeting therapy.
We have shown that intra-tumor signaling within a heterogeneous tumor can have a major impact on the efficacy of BRAF and MEK inhibitors. With the increasing evidence of genetic and phenotypic heterogeneity within tumors, intra-tumor signaling between individual cancer-cell subpopulations is therefore a crucial factor that needs to be considered in future therapy approaches. Our work has identified the ‘melanocyte-lineage survival oncogene’ MITF as an important player in phenotypic heterogeneity (MITFhigh and MITFlow cells) in melanoma, and MITF expression levels are crucial for the response to MAP-kinase pathway targeted therapy. We found that ‘MITF heterogeneity’ can be caused by cell-autonomous mechanisms or by the microenvironment, including the immune-microenvironment.
We have identified various mechanisms underlying MITF action in resistance to BRAF and MEK inhibitors in melanoma. In MITFhigh expressing cells, MITF confers cell-autonomous resistance to MAP-kinase pathway targeted therapy. Moreover, it appears that in melanomas heterogeneous for MITF expression (MITFhigh and MITFlow cells), individual subpopulations of resistant and sensitive cells communicate and MITF can contribute to overall tumor-resistance through intra-tumor signaling. Finally, we have identified a novel approach of interfering with MITF action, which profoundly sensitizes melanoma to MAP-kinase pathway targeted therapy.
Cellular Reprogramming in Carcinogenesis: Implications for Tumor Heterogeneity, Prognosis, and Therapy
Session Type:
Major Symposium
Session Start/End Time:
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Location:
Room 103, Pennsylvania Convention Center
CME:
CME-Designated
CME/CE Hours:
2
Session Description:
Cancers, both solid and liquid, consist of phenotypically heterogeneous cell types that make up the full cellular complement of disease. Deep sequencing of bulk cancers also frequently reveals a genetic intratumoral heterogeneity that reflects clonal evolution in space and in time and under the influence of treatment. How the distinct phenotypic and genotypic cells contribute to individual cancer growth and progression is incompletely understood. In this symposium, we will discuss issues of cancer heterogeneity and effects on growth and treatment resistance, with emphasis on cancer cell functional properties and influences of the microenvironment, interclonal genomic heterogeneity, and lineage relationships between cancer cells with stem cell and differentiated properties. Understanding these complex cellular relationships within cancers will have critical implications for devising more effective treatments.
Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.
Myelodysplastic syndrome and acute myeloid leukemia following adjuvant chemotherapy
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Myelodysplastic syndrome and acute myeloid leukemia following adjuvant chemotherapy with and without granulocyte colony stimulating factors for breast cancer
Gregory S. Calip1,2,3 • Judith A. Malmgren3,4 • Wan-Ju Lee1 • Stephen M. Schwartz2,3 • Henry G. Kaplan5
Abstract Risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) post-breast cancer treatment with adjuvant chemotherapy and granulocyte colonystimulating factors (G-CSF) is not fully characterized. Our objective was to estimate MDS/AML risk associated with specific breast cancer treatments. We conducted a retrospective cohort study of women aged C66 years with stage I–III breast cancer between 2001 and 2009 using the Surveillance, Epidemiology, and End Results-Medicare database. Women were classified as receiving treatment with radiation, chemotherapy, and/or G-CSF. We used multivariable Cox proportional hazards models to estimate adjusted hazard ratios (HR) and 95 % confidence intervals (CI) for MDS/AML risk. Among 56,251 breast cancer cases, 1.2 % developed MDS/AML during median followup of 3.2 years. 47.1 % of women received radiation and 14.3 % received chemotherapy. Compared to breast cancer cases treated with surgery alone, those treated with chemotherapy (HR = 1.38, 95 %-CI 0.98–1.93) and chemotherapy/radiation (HR = 1.77, 95 %-CI 1.25–2.51) had increased risk of MDS/AML, but not radiation alone (HR = 1.08, 95 % CI 0.86–1.36). Among chemotherapy regimens and G-CSF, MDS/AML risk was differentially associated with anthracycline/cyclophosphamide-containing regimens (HR = 1.86, 95 %-CI 1.33–2.61) and filgrastim (HR = 1.47, 95 %-CI 1.05–2.06), but not pegfilgrastim (HR = 1.10, 95 %-CI 0.73–1.66). We observed increased MDS/AML risk among older breast cancer survivors treated with anthracycline/cyclophosphamide chemotherapy that was enhanced by G-CSF. Although small, this risk warrants consideration when determining adjuvant chemotherapy and neutropenia prophylaxis for breast cancer patients.
Electronic supplementary material The online version of this article (http://dx.doi.org:/10.1007/s10549-015-3590-1) contains supplementary material, which is available to authorized users.
Introduction Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) following cancer treatment with cytotoxic therapy with radiation and chemotherapy account for approximately 10–20 % of all cases of MDS/AML [1]. The clinical course of these cases is typically aggressive with worse prognostic features and survival compared to de novo MDS/AML [2]. Given the growing population of breast cancer survivors successfully treated with radiation and chemotherapy, uncommon but lethal iatrogenic MDS and AML (MDS/AML) are important concerns for oncologists and their patients [3].
Several studies report increased risk of MDS/AML in breast cancer patients after treatment with radiation and adjuvant chemotherapy [4–8]. For some commonly used classes of breast cancer chemotherapy agents such as alkylating agents (i.e., cyclophosphamide) and anthracyclines (i.e., doxorubicin and epirubicin), an increased incidence of treatment-related leukemia with multiple signature chromosomal abnormalities has been observed [9, 10].
Another possible association with increased MDS/AML risk in breast cancer patients has been reported for granulocyte colony-stimulating factors (G-CSF) as prophylactic treatment following adjuvant chemotherapy [11–13]. G-CSF stimulates proliferation and differentiation of white blood cells and is used to prevent chemotherapy-induced neutropenia [14–17]. While G-CSF reduces the need for treatment delays or dose reductions, the anti-apoptotic effects of G-CSF could potentially spare some lineagespecific mutant stem cells resulting from cytotoxic therapy and permit survival in subsets of mature myeloid cells with chromosomal alterations [18]. Prior population-based studies of G-CSF use with adjuvant chemotherapy and MDS/AML risk among older women in the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database show mixed results with increased risk of MDS/ AML reported in one study [12] and no association with risk of AML in another [4]. In the Cancer and Leukemia Group B 9741 phase III trial [19], patients randomized to dose-dense chemotherapy with filgrastim support had no increased risk of developing MDS/AML, but in a review of randomized clinical trials by Lyman et al. [13], chemotherapy patients treated with G-CSF had a doubling in risk of MDS/AML post-treatment.
Secondary treatment-related myeloid cancers are rare, and the overall number of cases following breast cancer is low. However, consideration of MDS/AML risk postbreast cancer treatment may be important for subgroups of patients susceptible due to age [20] and/or use of potentially leukemogenic therapies [21]. Our purpose is to evaluate current MDS/AML risk among women treated for invasive breast cancer in the SEER-Medicare linked database between 2001 and 2009, a period in which MDS became a reportable disease, new treatment regimens became standard and use of G-CSF increased [14, 15, 22].
Results Characteristics of the 56,251 incident stage I–III breast cancer patients included in our study are shown in Table 1. Median age was 75 years at breast cancer diagnosis and most were Non-Hispanic White (87 %) with Charlson comorbidity scores C1 (84 %).
The majority of incident breast cancer was stage I (57 %) or stage II (34 %), lymph nodenegative (74 %), estrogen/progesterone receptor positive (79 %), and treated with breast-conserving surgery (59 %). The majority of breast cancer patients were treated with surgery only (46 %), with 40 % receiving surgery/radiation, 7 % surgery/chemotherapy and 7 % surgery/radiation/chemotherapy (Table 1).
Table 1 (not shown) Descriptive characteristics of female Medicare beneficiaries diagnosed with incident stage I–III breast cancer between 2001 and 2009 by subsequent primary MDS/AML status
No MDS/AML (n = 55,596) MDS/AML (n = 655) P value All women (N = 56,251)
Age (years) at breast cancer diagnosis Median (interquartile range)
Race
Hispanic ethnicity
AJCC Stage
Lymph node status
Hormone receptor status
Charlson comorbidity index
Surgical procedure
Combined radiation and chemotherapy
Chemotherapy type
Granulocyte colony-stimulating factors
Number of G-CSF doses
Person-years of follow-up
Median (interquartile range)
————————————–
MDS myelodysplastic syndrome, AML acute myeloid leukemia
a To test for differences of characteristics between the 2 groups, we used v2 test for categorical variables and Wilcoxon rank-sum test for medians
b Other ethnicity includes Asian/Pacific Islander, American Indian, and Alaskan Native
c Chemotherapy agents: A anthracyclines (doxorubicin or epirubicin); C cyclophosphamide; T taxanes (docetaxel or paclitaxel); F fluorouracil; M methotrexate; chemotherapy regimens listed are mutually exclusive groups with or without trastuzumab; AC-containing regimens = AC, ACT, FAC
d Other/non-standard = single-agent therapy or one of the regimens listed above and vinca alkaloids (vincristine, vinorelbine, or vinblastine), etoposide, bevacizumab, or azacitidine together or in combination
e Use of G-CSF products not mutually exclusive
Table 2 MDS/AML cases and use of G-CSF products among female Medicare beneficiaries diagnosed with incident stage I–III breast cancer between 2001 and 2009 by chemotherapy regimena
Any chemotherapy regimen, n = 8050
All AC-containing regimensb, n = 3686 (45.8 %)
TC, n = 1491 (18.5 %)
CF or CMF, n = 1086 (13.5 %)
Otherc, n = 1787 (22.2 %)
MDS/AMS cases
176 (2.2)
94 (2.6)
18 (1.2)
27 (2.5),
37 (2.1)
Any G-CSFd
4835 (60.1)
2552 (69.2)
1137 (76.3)
422 (38.9)
756 (42.3)
Filgrastim
1865 (23.2)
1025 (27.8)
276 (18.5)
263 (24.2)
301 (16.8)
Pegfilgrastim
3554 (44.1)
1865 (50.6)
960 (64.4)
192 (17.7)
537 (30.1)
a Chemotherapy agents: A anthracyclines (doxorubicin or epirubicin); C cyclophosphamide; T taxanes (docetaxel or paclitaxel); F fluorouracil; M methotrexate; chemotherapy regimens listed are mutually exclusive groups with or without trastuzumab
b AC-containing regimens = AC, ACT, FAC
c Other/non-standard = single-agent therapy or one of the regimens listed above and vinca alkaloids (vincristine, vinorelbine, or vinblastine), etoposide, bevacizumab, or azacitidine together or in combination
d Use of G-CSF products not mutually exclusive
Among women receiving chemotherapy treatment, the most common regimen was anthracycline/cyclophosphamide/taxane (ACT) (28 %). Overall, 46 % of breast cancer patients receiving chemotherapy had AC-containing regimens. The remaining 56 % received taxane/cyclophosphamide (TC) (19 %), cyclophoshamide/fluorouracil ?/- methotrexate (CF or CMF) (13.5 %) and other/non-standard regimens (22 %). Other non-standard regimens were single-agent therapies or one of the other listed regimens with vinca alkaloids, etoposide, bevacizumab, or azacitidine together or in combination. Sixty percent of those treated with chemotherapy received G-CSF treatment with 23 % receiving filgrastim and 44 % receiving pegfilgrastim (Table 2). Treatment with G-CSF varied by adjuvant chemotherapy regimen with TC regimen most often accompanied by G-CSF treatment (79 %) followed by the AC regimens (69 %).
Median follow-up was 3 years (interquartile range 2–7) and varied by diagnosis date with women diagnosed during the earlier study years having the longest follow-up: median 8 years for women diagnosed in 2001–2003, 6 years for 2004–2006, and 3 years for 2007–2009. Among the women in our study, 655 (1.2 %) developed MDS/ AML (n = 515 MDS and n = 140 AML), including 38 MDS cases that were later diagnosed with AML. Age-adjusted cumulative hazards of MDS/AML are shown in Fig. 1. MDS/AML incidence varied by index breast cancer treatment; 2.4 % of women treated with surgery/radiation/chemotherapy and 1.0 % of women treated with surgery only were diagnosed with MDS/AML during follow-up. Breast cancer patients with subsequent MDS/AML diagnosis were more likely to have breast cancer that was stage II or III, lymph node-positive, ER-negative/PR-negative, higher Charlson score and treated with chemotherapy and G-CSF (Table 1).
Fig. 1 (not shown) Cumulative hazard of MDS/AML among female Medicare beneficiaries diagnosed with incident stage I–III breast cancer between 2001 and 2009 by initial breast cancer treatment
In analyses from multivariable Cox models, we observed a significant association between treatment with chemotherapy and risk of MDS/AML (Fig. 2).
Fig. 2 (not shown) Risk of MDS/AML among female Medicare beneficiaries diagnosed with incident stage I–III breast cancer between 2001 and 2009 in relation to primary breast cancer initial treatment and to chemotherapy regimen.
HR (95% CI)
Combined Radiation
and Chemotherapy
None
1.00 (reference)
Radiation only
1.08 (0.86-1.36)
Chemotherapy only
1.38 (0.98-1.93)
Radiation and Chemotherapy
1.77 (1.25-2.51)
Chemotherapy
Regimen *
No Chemotherapy
1.00 (reference)
All AC-containing
regimens **
1.86 (1.33-2.61)
AC
1.48 (0.99-2.23)
ACT
1.91 (1.29-2.84)
FAC
1.79 (0.82-3.94)
TC
1.50 (0.84-2.67)
CF or CMF
1.26 (0.83-1.95)
1HR (log scale)
Abbreviations: MDS myelodysplastic syndrome; AML acute myeloid leukemia; HR hazard ratio; CI confidence interval. Chemotherapy agents: A anthracyclines (doxorubicin or epirubicin); C cyclophosphamide; T taxanes (docetaxel or paclitaxel); F fluorouracil; M methotrexate. Note all hazard ratios are adjusted for age at diagnosis (66–70, 71–75, 76–80, 81–85, 86–95 years); diagnosis year; race (White, Black, other, unknown); Hispanic ethnicity (yes, no, unknown); AJCC stage (I, II, III); hormone receptor status (ER-positive or PR-positive, ER-negative/PR-negative, unknown); surgical procedure (mastectomy, breast-conserving surgery, surgery NOS); Charlson comorbidity index score (0, 1, 2?); any granulocyte colony-stimulating factors received (yes/no). *Hazard ratios for chemotherapy regimens (AC, ACT, FAC, TC, CF, or CMF) are from a separate model adjusted for radiation therapy (yes/no). **AC-containing regimens = AC, ACT, FAC
Compared to women who received surgery only, women who received chemotherapy (HR = 1.38, 95 % CI 0.98–1.93) and chemotherapy/radiation (HR = 1.77, 95 % CI 1.25–2.51) had increased MDS/AML risk. Risk was not significantly elevated among surgery/radiation only patients. Compared to women not treated with chemotherapy, women who received anthracycline/cyclophosphamide-containing regimens had higher risk of MDS/AML (HR = 1.86, 95 % CI 1.33–2.61). Patients in the other adjuvant chemotherapy regimen categories, TC and CF/CMF, did not have significantly increased risk of MDS/AML post-treatment. G-CSF use increased over time during the study period, with use of pegfilgrastim surpassing filgrastim (Supplemental Figure S2). Adjusted for chemotherapy type, there was a non-significant increased risk of MDS/AML in women who received G-CSF with chemotherapy (HR = 1.33, 95 % CI 0.94–1.89) (Fig. 3).
No filgrastim
1.00 (reference)
Filgrastim
1.47 (1.05-2.06)
1-5 doses
1.30 (0.79-2.14)
6+ doses
1.64 (1.10-2.46)
P-trend: 0.021
No pegfilgrastim
1.00 (reference)
Pegfilgrastim
1.10 (0.73-1.66)
1-5 doses
1.04 (0.66-1.65)
6+ doses
1.30 (0.76-2.23)
P-trend: 0.845
1HR (log scale)
Fig. 3 Risk of MDS/AML among female Medicare beneficiaries diagnosed with incident stage I-III breast cancer between 2001 and 2009 that received chemotherapy in relation to G-CSF treatment. Abbreviations: MDS myelodysplastic syndrome; AML acute myeloid leukemia; HR hazard ratio; CI confidence interval; G-CSF granulocyte colony-stimulating factors. Note all hazard ratios are adjusted for age at diagnosis (66–70, 71–75, 76–80, 81–85, 86–95 years); diagnosis year; race (White, Black, other, unknown); Hispanic ethnicity (yes, no, unknown); AJCC stage (I, II, III); hormone receptor status (ER-positive or PR-positive, ER-negative/PR-negative, unknown); surgical procedure (mastectomy, breast-conserving surgery, unknown); radiation (yes/no); Charlson comorbidity index score (0, 1, 2?); chemotherapy regimen received (AC, ACT, FAC, TC, CF or CMF, other)
Stratified by G-CSF type, increased risk was observed with filgrastim use (HR = 1.47, 95 % CI 1.05–2.06) which when stratified by dosage was exclusive to the 6? dose-category (HR = 1.64, 95 % CI 1.10–2.46) (log-linear dose–response trend, P = 0.021). Pegfilgrastim use was not associated with increased MDS/AML risk with any use or in dose-stratified analyses.
In analyses of G-CSF use stratified by chemotherapy regimen, we observed a significantly increased risk of MDS/AML post-treatment among AC-containing regimens with any G-CSF treatment (n = 2552, HR = 1.78, 95 % CI 1.07–3.02) and AC-containing regimens with filgrastim treatment (n = 1025, HR = 2.01, 95 % CI 1.23–3.27) but not with pegfilgrastim treatment (n = 1865, HR = 1.20, 95 % CI 0.67–2.15) (Table 2). In additional analyses of the AC-containing regimens with filgrastim treatment by dose (1–5 doses/6+ doses), risk was increased for both categories but only significant for the 6+ filgrastim dose-category (1–5 doses: HR = 1.82, 95 % CI 0.94–3.39; 6+ doses: HR = 2.70, 95 % CI 1.33–5.28, P-trend = 0.036). No increased risk was observed among any of the other chemotherapy regimens with G-CSF treatment or with filgrastim or pegfilgrastim individually (Table 3).
Table 3 (not shown) Risk of MDS/AML in relation to G-CSF treatment among female Medicare beneficiaries diagnosed with incident stage I-III breast cancer between 2001 and 2009 by grouped chemotherapy regimens
1 G-CSF
2 Filgrastim
3 Pegfilgrastim
P-trend
AC-containing regimens
N = 3686
n = 94 MDS/AML cases
TC
N = 1491
n = 18 MDS/AML cases
CF or CMF
N = 1086
n = 27 MDS/AML cases
Chemotherapy regimen-specific hazard ratios are adjusted for age at diagnosis (66–70, 71–75, 76–80, 81–85, 86–95 years); diagnosis year;
race (White, Black, Other, unknown);
Hispanic ethnicity (yes, no, unknown);
AJCC stage (I, II, III);
hormone receptor status (ER-positive or PRpositive, ER-negative/PR-negative, unknown);
surgical procedure (mastectomy, breast-conserving surgery, unknown);
radiation (yes/no);
Charlson comorbidity score at diagnosis (0, 1, 2+)
MDS myelodysplastic syndrome,
AML acute myeloid leukemia,
HR hazard ratio,
CI confidence interval
a Chemotherapy agents: A anthracyclines (doxorubicin or epirubicin); C cyclophosphamide; T taxanes (docetaxel or paclitaxel); F fluorouracil; M methotrexate
b AC-containing regimens = AC, ACT, FAC
In sensitivity analyses, we evaluated changes in our results when criteria were varied for chemotherapy agent exposure by 1+, 2+, or 3+ HCPCS/ CPT codes, and MDS/ AML diagnoses identified from Medicare claims (1+ or 2+ ICD-9 codes). Results were not substantively different in these analyses.
Discussion In a large population-based cohort of older breast cancer patients, we found an association between chemotherapy treatment and increased risk of MDS/AML with evidence that the association may be exclusive to anthracycline/cyclophosphamide-containing regimens. In our analysis of G-CSF use by chemotherapy type and number of doses, we found increased risk of MDS/AML specific to filgrastim use and a possible dose–response effect. The MDS/AML incidence of 2.4 % among women treated with combined radiation and chemotherapy was more than double the incidence observed in women treated with surgery only and may represent a significant issue for elderly breast cancer patients. No significantly increased risk was observed among elderly patients treated with surgery and radiation only.
The absence of significant risk of MDS/AML among surgical patients treated with radiation without chemotherapy runs counter to our previous studies of MDS/AML post-treatment for breast cancer [7, 8]. However, the patients treated with chemotherapy and radiation had a higher risk than surgical patients treated with chemotherapy and no radiation. This may be due to the restriction of our observations to elderly women only in whom the risk associated with radiation may be less than that of younger women [32].
Our findings of MDS/AML risk associated with chemotherapy align with findings from previous population-based studies [4, 33] and clinical trials [5, 34]. In a prior analysis of older women using SEER-Medicare linked data (1992–2002) [4], adjuvant chemotherapy with alkylating agents and anthracyclines was associated with increased risk of AML, but MDS was not included in the analysis. The absence of association between antimetabolites (i.e., fluorouracil, methotrexate) or taxanes with risk of MDS/AML has been reported previously [4, 35–38].
Concern for leukemogenesis with the use of cytokines has existed for some time [39–43]. In a SEER-Medicare database study of breast cancer cases between 1991 and 1999 [12], increased risk of MDS/AML was observed with use of G-CSF. Another SEER-Medicare study [4] of breast cancer cases from 1992 to 2002 found no increased risk of AML with use of G-CSF in breast cancer treatment, but MDS was not included in that study. Studies in healthy stem cell donors that receive G-CSF have shown short-term induced DNA instability without increased risk of MDS/ AML [44–47]. Patients with congenital chronic neutropenia (CCN) have a high risk of leukemic transformation which may increase with long-term exposure to G-CSF [40, 41, 43]. Touw et al. hypothesized that genomic instability, including G-CSF receptor mutations, is responsible for the high rate of leukemic transformation in CCN patients treated regularly with G-CSF [42]. Slovak et al. [48] conducted a small study of clonal hematopoiesis following neoadjuvant chemotherapy for breast cancer but did not observe changes suggesting MDS/AML development. Our observation of a possible leukemogenic effect of G-CSF may represent leukemogenesis potentiation in hematopoietic cells genetically damaged by chemotherapy.
In the current study, we observed an increased risk of MDS/AML with the use of filgrastim but not pegfilgrastim. During our study period, the pegylated form of recombinant human G-CSF analog filgrastim (i.e., pegfilgrastim) was introduced. G-CSF is administered differentially with pegfilgrastim given as a fixed dose once per chemotherapy cycle and filgrastim administered daily until absolute neutrophil counts increase [14–16]. Pegfilgrastim has different pharmacokinetics than filgrastim but a similar mechanism of action [49, 50].
Strengths of this study include use of a population-based cohort from the SEER-Medicare linked database. As elderly women are rarely included in clinical trials, information on treatment risks and surveillance for rare adverse events like MDS/AML are only answered by large population-based cohort studies like ours.
A limitation of our study is the identification of subsequent diagnoses of MDS and AML based on Medicare claims and SEER data. Concerns with misclassification of MDS/AML outcomes from claims data (ICD-9 codes) are based on the tendency for false-positive MDS/AML when based on a single claim. To minimize possible misclassification, we used a recommended case definition with high specificity [31]. Using SEER as the criterion measure, ascertainment of cases using classification of two or more ICD-9 codes had sensitivity and specificity of 90.1 % and 99.2 %, respectively, for MDS; 89.3 % and 99.7 %, respectively, for AML in our study. We conducted stratified analysis to adjust for the potential confounding effect of simultaneous indication for adjuvant chemotherapy and G-CSF treatment to prevent neutropenia, both with possible leukemogenic effects. The presence of elevated risk in both the adjusted multivariable model and the stratum specific categories supports our findings.
American Society of Clinical Oncology guidelines recommend G-CSF treatment before adjuvant chemotherapy if a 20 % or greater chance of neutropenia exists [15]. Growth factor products (G-CSF) continue to be among the top 15 drug expenditures in the United States [22]. The benefits of adjuvant chemotherapy made possible with G-CSF outweigh the risk of MDS/AML in patients with high risk of breast cancer relapse [8, 17, 51–54]. It is unclear why a differential risk of MDS/AML between filgrastim and pegfilgrastim was observed. Con- firming and understanding possible differences in longterm safety of G-CSF by product are extremely important. Further studies of MDS/AML risk post-breast cancer treatment with G-CSF and adjuvant chemotherapy need to include all age groups to characterize risk attributable to specific therapies and identify patient groups that may be at greater leukemic risk.
2. Larson RA (2007) Etiology and management of therapy-related myeloid leukemia. Hematol Am Soc Hematol Educ Program. doi:10.1182/asheducation-2007.1.453
3. Wolff AC, Blackford AL, Visvanathan K, Rugo HS, Moy B, Goldstein LJ, Stockerl-Goldstein K, Neumayer L, Langbaum TS, Theriault RL, Hughes ME, Weeks JC, Karp JE (2015) Risk of marrow neoplasms after adjuvant breast cancer therapy: the national comprehensive cancer network experience. J Clin Oncol 33(4):340–348. doi:10.1200/JCO.2013.54.6119
4. Patt DA, Duan Z, Fang S, Hortobagyi GN, Giordano SH (2007) Acute myeloid leukemia after adjuvant breast cancer therapy in older women: understanding risk. J Clin Oncol 25(25):3871–3876. doi:10.1200/JCO.2007.12.0832
A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy
Xuejun Fan1 , Randall J Brezski2 , Ming Fa1 , Hui Deng1 , Allison Oberholtzer2, et al. Breast Cancer Research 2012, 14:R116 http://dx.doi.org:/10.1186/bcr3240
Introduction
Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.
Methods
Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.
Results
scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.
Conclusion
Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.
Introduction
Trastuzumab is a humanized IgG1 monoclonal antibody for the treatment of primary and metastatic breast cancers that overexpress HER2 [1]. Both antigen engagement by the Fab region, which results in HER2 signaling inhibition, as well as induction of immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC) mediated by the Fc region play important roles in the mechanisms of action of trastuzumab [2–4]. Despite the clinical success of trastuzumab in treating high HER2 breast cancers, primary and acquired resistance to the therapy is widespread in the clinic [5]. Previous studies on resistance to trastuzumab have focused in large part on cell signaling escape mechanisms. These studies have included loss of phosphatase and tensin homolog function, gain of function mutations in signaling molecules such as phosphatidylinositol 3-kinase and protein kinase B (AKT) [6,7], activation of HER family member receptors epidermal growth factor receptor and HER3 [8], and upregulation of other receptor tyrosine kinases such as insulin-like growth factor 1 receptor [9], hepatocyte growth factor receptor (cMET) [10], and ephrin-A family tyrosine kinase receptor 2 [11].
IgG antibody is known to be susceptible to specific cleavage within the hinge region by proteinasesin vitro [12,13]. Extracellular proteinases secreted by certain human bacterial pathogens can cleave human IgGs within the lower hinge region, and these proteinases are suggested to function as virulence factors by evading the host immune response to bacterial infections [14–17]. Recent reports have also demonstrated that certain human matrix metalloproteinases (MMP-3, MMP-7, MMP-9, MMP-12 and MMP-13) can catalyze a single-strand cleavage of human IgG1 antibodies in the lower hinge region in vitro [15,18], although the rate of cleavage varies among the different MMPs. Purified single-cleaved IgG1 antibodies were shown to have substantially depressed immune effector functions such as ADCC and complement-dependent cytotoxicity [18–20]. The loss of antibody Fc effector function was correlated with a decreased binding to Fcγ receptors that are expressed on immune effector cells such as natural killer (NK) cells and monocytes [18,21]. Since ADCC is considered one of the key mechanisms of action for trastuzumab [3,22–26], factors that compromise Fc-mediated immune functions of trastuzumab are expected to decrease its efficacy.
The study described in this report investigated the impact of trastuzumab hinge cleavage on its anti-HER2 signaling function and anti-tumor efficacy in vitro and in vivo. The results demonstrated that single cleavage of trastuzumab within the lower hinge severely impaired Fc-mediated immune effector cell function in vitro and resulted in significantly reduced anti-cancer efficacy in vivo. These findings underscore the potential effects of proteolytic hinge cleavage of trastuzumab and other therapeutic antibodies in the tumor microenvironment by compromising their clinical efficacy.
Materials and methods
Enzymes, antibodies and cell lines
Trastuzumab was purchased from a specialty pharmacy. Single hinge cleaved trastuzumab (scIgG-T) was prepared by enzymatic digestion with a bacterial proteinase, IgG-degrading enzyme S (IdeS), as previously described [15,18]. Recombinant IdeS was expressed in Escherichia coli and purchased from Genovis AB (Lund, Sweden). The isotype control monoclonal antibody (human IgG1) was expressed at Janssen R&D, LLC (Radnor, PA, USA). The cancer cell lines SKOV-3 and BT474 were obtained from American Type Culture Collection (Manassas, VA, USA), and were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin in an incubator with 5% CO2 at 37°C. Antibodies for total HER2, pHER2 (Y1248), pHER3 (Y1289), pEGFR (Y1068), pAKT (S473), and pErk1/2 (T202/Y204) were purchased from Epitomics (Burlingame, CA, USA).
Capillary gel electrophoresis
The method used for antibody hinge cleavage and gel analysis were reported previously [15]. Briefly, trastuzumab was subjected to proteolysis by recombinant human MMPs or the bacterial protease IdeS in Tris-buffered saline plus calcium chloride. Electrophoresis separation was performed using an Agilent 2100 microfluidics-based Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Proteins were applied at 1 mg/ml in Tris-buffered saline and run on Agilent 230 chips following the manufacturer’s protocol [27]. Trastuzumab digests were run under nonreducing conditions to best visualize the single-cleaved products.
Flow cytometry
Binding of trastuzumab and scIgG-T on HER2-expressing breast cancer cells was measured using a Guava easyCyte HT instrument based on the manufacturer’s instructions (Millipore, Danvers, MA, USA). Briefly, 5×105 cells were dispensed in 100 µl aliquots and trastuzumab as primary antibody was added for 1 hour at 25°C, followed by the addition of phycoerythrin-conjugated anti-human-Fc (Jackson ImmuneResearch Laboratories, Inc., West Grove, PA, USA). After washing with PBS buffer, the cells were analyzed for fluorescence intensity and human isotype IgG1 antibody was used as the reference sample control.
Fcγ receptor binding assays
Binding of Fcγ receptors with antibodies was determined by ELISA as reported previously [28]. Fcγ receptors were from R&D Systems (Minneapolis, MN, USA). The fluorescence signal was detected at excitation 340 nm and emission 460 nm using a plate reader (Molecular Devices, Sunnyvale, CA, USA).
Cell lysate preparation from tumor tissues
Tumor tissues from mouse xenograft studies or breast cancer patients were homogenized using gentle MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s procedure in a cell lysis buffer in the presence of proteinase inhibitor cocktails (Calbiochem, San Diego, CA, USA). Trastuzumab was affinity enriched from the tissue lysate using HER2 extracellular domain protein (Sino Biologicals, Beijing, China) and used for SDS-PAGE and western blotting analysis.
Western blotting
Cell lysates prepared from cell culture or tumor tissues were subjected to SDS-PAGE separation. Gels were either stained with Coomassie blue (Bio-Rad, CA, USA) or transferred to a polyvinylidene fluoride membrane by standard procedures. Membranes were blotted using a goat-anti-human Fc-horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) and images were detected with FluorChem M imager (Cell BioSciences, Santa Clara, CA, USA) using enhanced chemiluminescence substrate (GE Healthcare, Piscataway, NJ, USA).
Antibody-dependent cellular cytotoxicity assay
ADCC activities were assayed as described previously [29] using the xCELLigence instrument (Roche, Mannheim, Germany). Briefly, the high HER2 expression cancer cell line SKOV-3 was seeded in E-plate 96 (ACEA Biosciences, Inc., San Diego, CA, USA), followed by the addition of human peripheral blood mononuclear cells (PBMC) cells as immune effector cells. Human PBMC were isolated from fresh blood samples collected from consented healthy volunteers using procedures reported previously [18]. Antibodies were added in three-fold titration starting from 5 µg/ml, and the cell index (cell growth) was monitored continuously for 2 days. Cancer cells with PBMC only were used as the control group and the ratio of effector cells to target cells was 50:1. The cell index recorded after 24 hours of treatment with trastuzumab and scIgG-T was used to calculate the percentage of cell lysis using the formula:
Each treatment contains three replicates and the standard deviation is shown in the plots.
Cell proliferation assay
Inhibition of breast cancer cell proliferation by trastuzumab and scIgG-T was measured based on a reported procedure [29]. Briefly, BT474 cells (5,000 cells/well) were seeded in a 96-well plate and cultured overnight in RPMI medium with 10% fetal bovine serum at 37°C and 5% CO2. Treatment antibodies were added into the cell culture in a series of concentrations (0 to 10 µg/ml) and continued incubation for 72 hours. Alamar Blue (Invitrogen, Carlsbad, CA, USA) was added according to the manufacturer’s instructions and fluorescence signal was read using a SpectraMax M4 plate reader (Molecular Devices) using excitation at 535 nm and emission at 590 nm.
Mouse xenograft tumor model
Mouse xenograft studies were carried out in accordance with the animal care and use guidelines and the protocol was approved by the Animal Welfare Committee of the University of Texas Medical School at Houston. HER2-overexpressing BT474 breast cancer cells were used in the xenograft model as previously described [29]. Immunodeficient nu/nu mice from a homogeneous BALB/c background were from Charles River Laboratories (Wilmington, MA, USA). When tumors reached 50 to 100 mm3, mice were randomized into treatment groups with five mice per group. Antibodies were administered weekly intraperitoneally at 5 mg/kg dosing level for 6 weeks. Tumor size was measured and recorded every 3 or 4 days using a Vernier scale caliper. Tumor tissue and sera were collected and stored at -80°C until analysis.
Immunohistochemistry
Xenograft tumor tissues were excised freshly and fixed in 4% paraformaldehyde and embedded in paraffin. Serial 4-μm thick sections were made for staining. Anti-mouse integrin αM/CD11b and F4/80 antibodies (R&D Systems) were applied as primary antibody, and positive staining was visualized using a three-step staining procedure with an Elite ABC kit (Vector Laboratories, Peterborough, UK) and counterstained with hematoxylin (Vector Laboratories). Six tumor tissue sections (n = 6) were made from each treatment group and the tissue slides were viewed using Nikon Eclipse E200 microscopy and the entire tumor sections were scanned at 10× (magnification = 10×10) to identify all positive stained cells. Representative images from each treatment group were taken under ×40 magnification. The average number of immune cell infiltration per tumor slide under ×40 magnification was plotted for each treatment groups and standard error was calculated among the six tissue slides.
Mass spectrometry analysis
Mass spectral analyses were performed in the Translational Proteomics Core at University of Texas Health Science Center at Houston. Protein bands were excised and in-gel digestion was performed as previously described [30]. Tryptic digested proteins were taken to dryness in a Thermo SpeedVac and dissolved in 20 µl of 2% acetonitrile, 0.1% formic acid (solvent A). Aliquots of the digest were analyzed by liquid chromatography/mass spectrometry (MS)/MS on an Agilent 6538 UHD Accurate-Mass Quadrupole Time-of-Flight mass spectrometer equipped with an Agilent 1260 nanoLC system. The reverse-phase chromatography was performed on an Agilent High Capacity Chip (143 mm) using solvent A as the initial mobile phase and varying percentages of solvent B (90% acetonitrile, 0.1% formic acid) to constitute a gradient elution. Electrospray ionization was operated at the spray voltage of 1.75 kV. Mass spectral data were extracted with the MassHunter Quantitative Analysis package (Agilent Technologies, Inc., Santa Clara, CA USA) and peptides were identified from MS/MS spectra with MASCOT (Matrix Science Inc., Boston, MA, USA). The MASCOT search was performed with a peptide tolerance of 5 ppm and an MS/MS tolerance of 0.05 Da, fixed modification with carbamidomethyl and variable methionine oxidation. Identification of nontryptic fragments was performed manually with an initial search on the basis of predicted peptide masses of all possible fragments resulting from novel cleavage sites in the hinge region. The MS/MS spectra of suspect peaks were verified by manual de novo peptide sequencing to confirm their identities.
Patient samples
Individuals enrolled in the study protocol gave informed consent and all procedures and the protocol were reviewed and approved by the institutional review board of the University of Texas Medical School at Houston. Breast tumor tissue and adjacent normal tissues 2 cm from the tumor sites were obtained from breast cancer patients immediately following surgery by a certified pathologist and were snap-frozen in liquid nitrogen until analysis.
Detection of MMP expression in human breast cancer tissues
MMP expression was measured using the Quantibody reverse-phase human MMP array kit according to the manufacturer’s instructions (RayBiotech, Norcross, GA, USA). Fluorescence images were detected using a GenePix 4100A Scanner, and data were analyzed using the QAH-MMP-1 GAL software based on the instruction provided by the array manufacturer.
Statistical analysis
Where appropriate, statistical analysis was performed using paired Student’s t test. P < 0.05 between treatment groups is considered significantly different.
Results
Trastuzumab can be cleaved within the lower hinge in vitro by human MMPs
An increased expression of human MMPs has been reported in the tumor microenvironment of various cancer types, including breast cancer [31]. To investigate whether MMPs catalyze specific single hinge cleavage of trastuzumab, a panel of four human MMPs were individually incubated with trastuzumab in vitro, and hinge cleavage was detected using a capillary electrophoresis separation system. As shown in Figure 1A, trastuzumab was readily cleaved at the hinge by all four MMPs tested, as indicated by the production of scIgG-T. Based on previous studies of other IgG1 antibodies [18,32], IdeS produced by Streptococcus pyogenes can also cleave human IgGs at the hinge region. We treated trastuzumab with IdeS and, as expected, IdeS efficiently cleaved trastuzumab within the hinge (Figure 1B). Since the solution-phase kinetics of IgG single hinge cleavage by IdeS can be better controlled in vitro compared with the human MMPs tested in this study, the scIgG-T used for the in vitro characterization and in vivo efficacy studies was prepared by incubating trastuzumab with IdeS. The resultant scIgG-T was purified as previously reported[18].
Figure 1.Detection of trastuzumab proteolytic cleavage in vitro. (A)Capillary electropherograms (CEs) of trastuzumab and matrix metalloproteinase (MMP)-digested trastuzumab under nonreducing and denatured running conditions. (B) CE of trastuzumab and IgG-degrading enzyme S (IdeS)-digested single hinge cleaved trastuzumab (scIgG-T).(C) Purified scIgG-T under nonreducing and denaturing running conditions and stained with Coomassie blue. (D) Trastuzumab and scIgG-T under reducing and denaturing running conditions and stained with Coomassie blue. Fab, fragment antigen binding; Fc(m), Fc monomer; HC, heavy chain; LC, light chain.
The purity of scIgG-T was analyzed by SDS-PAGE separation under both nonreducing conditions (Figure 1C) and reducing conditions (Figure 1D). Under nonreducing/denaturing conditions, the intact trastuzumab was detected as a single band of ~150 kDa. The IdeS scIgG-T was detected as two major bands, with the upper band representing the single hinge cleaved intermediate (135 kDa band) and the lower band (~26 kDa) corresponding to the detached Fc monomer (Fc(m)) in the denaturing conditions (Figure 1C). A faint band below 135 kDa was visible and is most probably the (Fab)2 fragment that migrates as a 110 kDa band by SDS-PAGE under nonreducing conditions (Figure 1C). The proteolytic activity of IdeS is quite active to cleave at the hinge of the first heavy chain, but the hinge of the second heavy chain is relatively resistant to the IdeS proteolytic cleavage. As a result, a small fraction of the double-cleaved (Fab)2 fragment exists in the scIgG-T preparation.
Under reducing/denaturing conditions, the intact trastuzumab is separated into a heavy chain band (~50 kDa) and a light chain band (~25 kDa) (Figure 1D, right lane), whereas the IdeS single hinge cleavage of trastuzumab resulted in four fragments: the ~50 kDa intact heavy chain, the ~25 kDa light chain, and the two heavy chain proteolytic cleavage fragments (Fc(m) and scHC) migrated slightly above the light chain band (Figure 1D).
Sequence analysis of the tryptic digestion products of scIgG-T using MS confirmed the IdeS cleavage site at G236-/-G237 (Figure 2A). In comparison, the tryptic digestion of intact trastuzumab showed only a hinge peptide from T223 to K248 as expected (Figure 2B).
Figure 2.Detection of single hinge cleaved trastuzumab and trastuzumab proteolytic cleavage using mass spectrometry. (A)Sequence of the hinge region of trastuzumab showing the major trypsin cleavage sites (black arrows) and the IgG-degrading enzyme S (IdeS) cleavage site (red arrow). Bottom graph: liquid chromatography (LC)/mass spectrometry (MS)/MS analysis of trypsin-treated single hinge cleaved trastuzumab (scIgG-T), identifying the two peptides: THTCPPCPAPELLG and GPSVFLFPPKPK. Spectra are the MS2 spectra showing the b and y ions for the two peptides, confirming their sequences. (B) Sequence of the hinge region of trastuzumab. Black arrows, major trypsin cleavage sites. Bottom graph: LC/MS/MS analysis of trypsin-treated trastuzumab that identifies the peptide THTCPPCPAPELLGGPSVFLFPPKPK. Spectra are the MS2 spectra showing the b and y ions for the peptide, confirming its sequence.
scIgG-T retains HER2 antigen engagement activity
To study the effect of single hinge cleavage on trastuzumab HER2 binding and downstream signaling, we carried out a series of in vitro experiments using purified scIgG-T. The histograms collected from a flow cytometer for the binding of trastuzumab and scIgG-T on BT474 high HER2 expression breast cancer cells showed a concentration-dependent increase of the fluorescence signal (Figure 3A). The concentration-dependent binding to BT474 cells showed overlapping sigmoidal curves when plotted with fluorescence signals against antibody concentrations, and the half-maximal effective concentration values for trastuzumab and scIgG-T were 0.27 nM and 0.31 nM, respectively (Figure 3B). The effects of trastuzumab and scIgG-T on HER2 signaling were detected by western blotting (Figure 3C). Similar to trastuzumab, scIgG-T decreased phosphorylation of pHER2 (Y1248), pAKT (S473) and pErk1/2 in BT474 cells (Figure 3C). There were minimal effects on total HER2 levels by either scIgG-T or the intact trastuzumab (Figure 3C). Consistent with inhibition of pAKT (S473) and pErk1/2, the intact trastuzumab and scIgG-T showed similar inhibition of cancer cell proliferation (Figure 3D). Collectively, these results indicated that a single cleavage of trastuzumab within the lower hinge did not impact antibody binding to the HER2 antigen, inhibition of downstream signaling, and inhibition of cell proliferation.
Figure 3.Biological characterization of single hinge cleaved trastuzumab compared with the intact trastuzumab antibody in vitro. (A) Histograms of trastuzumab and single hinge cleaved trastuzumab (scIgG-T) binding to HER2 expressed on BT474 cells using a flow cytometer. (B) Concentration-dependent binding of trastuzumab and scIgG-T to HER2 expressed on BT474 breast cancer cells as measured by flow cytometer. Mean fluorescence intensity (MFI) is plotted against each antibody concentration (nM) on the xaxis. (C) Effect of trastuzumab and scIgG-T on total HER2 expression, pHER2 (Y1289), pAKT (S473), and pErk1/2 in BT474 cells as determined by western blotting. (D) Inhibition of BT474 breast cancer cell proliferation by trastuzumab and scIgG-T (n = 4). Percentage of cell growth inhibition calculated as: (fluorescence signal of control group – signal of treatment group)/signal of control group×100.
scIgG-T reduced binding to Fc gamma receptors and diminished Fc-mediated ADCC activities
Fc gamma receptors (FcγRs) are expressed on diverse immune cell types and are important for antibody engagement of immune effector cell functions such as ADCC. Binding of the intact trastuzumab and scIgG-T to ADCC-activating receptors (FcγRI, FcγRIIA, and FcγRIIIA) was assessed using ELISA. Intact trastuzumab demonstrated binding to all three FcγRs in a concentration-dependent manner (Figure 4A to 4C). In contrast, scIgG-T had minimal binding to the FcγRIIA and FcγRIIIA receptors (Figure 4B,C). Binding to the high-affinity FcγRI was also reduced by scIgG-T, although the reduction was not as marked when compared with FcγRIIA and FcγRIIIA (Figure 4A). ADCC activities mediated by the intact trastuzumab and scIgG-T were determined using human PBMC as effector cells and the high HER2 expression SKOV-3 ovarian cancer cells as target cells. Cancer cell lysis mediated by the intact trastuzumab reached over 80% at a concentration as low as 1 nM; however, cell lysis induced by scIgG-T reached a 20% maximum at the same concentration (Figure 4D). The decrease in ADCC activity mediated by scIgG-T was consistent with its reduced binding affinity to FcγRs expressed on immune effector cells, most notably the loss of binding to FcγRIIIa that is known for its contribution to ADCC and is the main FcγR expressed on NK cells.
Figure 4.Trastuzumab and single hinge cleaved trastuzumab binding to Fcγ receptors and antibody-dependent cellular cytotoxicity activity. (A) Trastuzumab and single hinge cleaved trastuzumab (scIgG-T) binding to Fc gamma receptor (FcγR) I. (B)Trastuzumab and scIgG-T binding to FcγRIIA. (C) Trastuzumab and scIgG-T binding to FcγRIIIA. (D) Antibody-dependent cellular cytotoxicity (ADCC) activity induced by trastuzumab and scIgG-T using the high-HER2-expressing SKOV-3 ovarian cancer cells as target cells and human peripheral blood mononuclear cells (PBMC) as effector cells (n= 3). Cell index was compared after treatment of trastuzumab and scIgG-T for 24 hours. Percentage of cell lysis calculated as: (cell index of control group – cell index of treatment group)/cell index of control group×100. RFU, relative fluorescence units.
scIgG-T had reduced anti-tumor efficacy in a mouse xenograft tumor model
The impact of single hinge cleavage on trastuzumab efficacy in vivo was determined by comparing tumor growth inhibition by the intact trastuzumab and scIgG-T in a mouse xenograft tumor model using high-HER2-expressing BT474 breast cancer cells. The antibody was dosed at 5 mg/kg and administered once weekly for 4 weeks (Figure 5A). Compared with the isotype IgG control groups, trastuzumab completely inhibited tumor growth (Figure 5A). However, the scIgG-T-treated mice showed significantly lower inhibition of tumor growth than that of trastuzumab with a maximum of ~30% inhibition of tumor growth when compared with the isotype control IgG (Figure 5A). To understand the mechanism of tumor growth inhibition by scIgG-T and trastuzumab, we conductedex vivo studies to investigate the effect of trastuzumab and scIgG-T on HER2-mediated downstream signaling in xenograft tumors. Both intact trastuzumab and scIgG-T-treated tumor extracts showed downregulation of total HER2 levels, and inhibition of pHER2 (Y1248), pAKT(S473), and pErk1/2 when compared with tumors treated with the isotype control (Figure 5B). These results suggest that scIgG-T retained its HER2 antigen engagement and inhibitory effects on cancer cell signaling.
Figure 5.Trastuzumab and single hinge cleaved trastuzumab tumor inhibition efficacy in breast cancer xenograft mouse model. (A)BALB/c nu/nu mice (n = 5) were subcutaneously inoculated with 5×106BT474 human breast cancer cells. Mice were treated with the antibodies at 5 mg/kg weekly for a total of five doses when tumors reached an average size of 100 mm3. Tumor sizes were measured and compared among the treatment groups. *P < 0.05 and **P < 0.005. (B) Tumor lysates were prepared from the frozen xenograft tumor tissues. Total HER2 expression, pHER2 (Y1248), pAKT (S473), and pErk1/2 were determined by western blotting. scIgG-T, single hinge cleaved trastuzumab.
scIgG-T reduced infiltration of immune effector cells in the mouse xenograft tumor
To study whether scIgG-T lost Fc-mediated immune cell effector function in vivo, we studied the effect of trastuzumab and scIgG-T on the infiltration of mouse immune effector cells in the xenograft tumors. Mouse immune cell infiltration was determined by immunohistochemistry using both anti-CD11b and F4/80 primary antibodies that can recognize infiltrated mouse macrophages and monocyte-derived immune cells. Trastuzumab-treated tumor tissues showed infiltrated immune cells when detected with both macrophage markers (Figure 6A,B). In contrast, tumor tissue treated with scIgG-T showed much fewer numbers of mouse macrophage infiltration and presented no difference from the tumor tissues treated with the isotype IgG control (Figure 6). Statistical analysis of the number of immune cells detected in tumor tissue slides in each group of three mice showed a significant increase of infiltrated immune cells in the intact trastuzumab-treated group (Figure 6, right bar graph). The reduced immune cell infiltration in scIgG-T treated tumors is consistent with the in vitro low ADCC activity mediated by scIgG-T (Figure 4D). These results imply that the decreased anti-tumor activity of scIgG-T in vivo is the result of reduced ADCC-mediated by immune effector cells.
Figure 6.Immune cell infiltration in xenograft tumors measured by immunohistochemistry. Mouse xenograft tumors treated with trastuzumab, single hinge cleaved trastuzumab (scIgG-T), and the isotype control were fixed in 4% paraformaldehyde. Six immunohistochemistry slides were prepared from the paraffin-embedded tissues of two mice from each treatment group. Monocytes/macrophages were stained using the anti-mouse integrin αM/CD11b and anti-mouse F4/80 antibodies. (A) Representative images (×40) of CD11b-stained tumor tissue and positive-stained immune cells indicated by black arrows. Right bar graph: average number of CD11b-positive cells per tumor tissue slide (n = 6). Pairwise t test used to compare different treatment groups. **P < 0.005. (B) Representative images (×40) of F4/80-stained tumor tissues from different treatment groups. Right bar graph: Average number of positive stained cells detected per tissue slides (n = 6). Pairwise ttest used to compare different treatment groups. **P < 0.005.
Discussion
The correlation between accumulation of proteinases, such as MMPs, in the tumor microenvironment and cancer progression and metastasis is well documented [31,33–37]. However, the role of MMPs in antibody hinge cleavage and the consequence of such antibody cleavage events in tumor microenvironment are unknown. Primary and acquired resistance to cancer therapeutic monoclonal antibodies such as trastuzumab in the clinic is one of the major challenges in cancer therapy [10,38]. Mechanisms resulting in refractory and acquired resistance to trastuzumab are poorly understood, and different models have been proposed including genetic mutations among key genes in the HER signaling pathway such as phosphatase and tensin homolog as well as upregulation of oncogenes such as cMET, epidermal growth factor receptor and HER3 [8,10,39]. The ability of MMPs to cleave IgG1 antibodies specifically in the lower hinge regionin vitro suggests a possible link between MMP accumulation and loss of antibody Fc-mediated immune effector function in the tumor microenvironment. Using trastuzumab and high-HER2-expressing breast cancer cells, this study provided for the first time in vitro and in vivoexperimental evidence that proteinase cleavage of trastuzumab at the lower hinge compromised its Fc-mediated immune effector functions such as ADCC in vitro, which resulted in reduced anti-tumor efficacy in vivo. We used the bacterial proteinase IdeS to prepare scIgG-T in our studies for two reasons. First, IdeS cleaves the IgG1 antibody with high specificity and generates a scIgG intermediate similar to IgG1 cleavage products by human MMPs. Second, IdeS cleaves the first strand rapidly and the second strand slowly, which presents a practical window for the isolation of homogeneous scIgG-T for in vitro and in vivo studies [14,18,40].
In this study, trastuzumab was readily cleaved in vitro by a panel of human MMPs (MMP-3, MMP-7, MMP-12, and MMP-13), which was consistent with previous reports that human IgG1 antibodies are susceptible to proteinase cleavage [14,15,18,20,21,40]. However, MMPs are complex proteinases and their proteolytic activities are tightly regulated in vivo, and in vitro assays may not reflect theirin vivo activities [34]. It is noted that relatively high levels of MMP9 and MMP3 in the patient tumor sample were detected. Further studies are needed to establish the specificity and activity of the MMPs in the tumor microenvironment with respect to trastuzumab hinge cleavage.
To carry out downstream experiments, we prepared a large batch of scIgG-T by incubating trastuzumab with IdeS, which cleaves the antibody at the G236-/-G237 site. In comparison with trastuzumab, the IdeS-generated scIgG-T showed comparable functions related to HER2-mediated activities but exhibited reduced Fc-mediated ADCC activity in vitro. A previous report demonstrated that a single hinge cleaved IgG intermediate had a complete loss of ADCC function in vitro [18], whereas the scIgG-T tested in this study demonstrated significantly reduced ADCC but not a complete loss of function. One potential explanation for this discrepancy was that different methods were used for ADCC detection. The ADCC assay reported in this study was monitored over the course of 120 hours, whereas the ADCC assay from the previous report was carried out for 2 hours. Numerous studies indicate that cell lysis in a 2-hour ADCC assay is mediated by NK cells alone [41,42], and the only activating FcγR expressed by NK cells is FcγRIIIa [43]. The complete loss of function seen in a 2-hour ADCC assay is therefore consistent with the loss of binding to FcγRIIIa. When ADCC assays are performed for time periods exceeding 12 hours, myeloid linage cells can also mediate in vitro ADCC [44]. The detection of ADCC activity with scIgG-T in the present report, albeit at a much reduced level relative to the intact version of IgG1, perhaps reflects some degree of recruitment of myeloid-mediated ADCC activity in addition to NK function. This low level of ADCC activity is consistent with the ability of scIgG-T to bind to the high-affinity FcγRI expressed in the monocyte population.
The mechanisms of action for trastuzumab can be largely grouped into two categories: one is associated with antagonizing HER2 signaling through Fab region of the IgG1 molecule, and the other is through Fc-mediated immune effector cell functions. The partial anti-tumor efficacy conferred by scIgG-T in the mouse xenograft model can be attributed to the direct HER2 engagement by the Fab region of scIgG-T, as indicated by downregulation of HER2 in tumor tissues and inhibition of pHER2 (Y1248), pAKT (S473), and pErk1/2. These results are consistent with previous findings that trastuzumab mechanisms of action include inhibition of HER2 downstream signaling [8,39,45] and downregulation of HER2 receptor level [46].
Because scIgG-T showed diminished Fc-mediated ADCC in vitro, it is expected that scIgG-T will be less effective in engaging immune cell-mediated cancer cell killing in vivo. As an IgG1 isotype antibody, trastuzumab mediates ADCC by recruiting immune effector cells to HER2-overexpressing tumor cells [25]. Xenograft studies in FcγR knockout mice showed that interaction with the ADCC activation receptor (FcγRIII) is essential for the efficacy of trastuzumab [3]. Patients with the higher binding affinity FcγRIIIa-158V/V genotype showed an improved response to trastuzumab than patients with the lower affinity FcγRIIIa-158 F/F and F/V genotypes, again suggesting that Fc-mediated effector function plays a key role in the clinical efficacy of trastuzumab [4]. Even though the correlation between FcγRIIIa polymorphisms and outcome in trastuzumab-treated cancer patients was not confirmed in a recent clinical study [47], the important role of FcγRIIIa in Fc-mediated immune function is not in dispute. The reduced anti-tumor efficacy by scIgG-T strongly supports that Fc-mediated effector function is a key mechanism of action for in vivo efficacy of trastuzumab. The significantly reduced immune cell infiltration shown in scIgG-T-treated mouse xenograft tumor as compared with that treated with intact trastuzumab again supports the notion that scIgG-T has compromised Fc effector function due to lack of engagement of immune cells at the tumor site. Mouse macrophages are phagocytic and express CD11b and F4/80 [48]. Macrophage recruitment to the tumor site has been documented in a PyMT mouse breast cancer model [48,49]. Even though the nude mouse used in the xenograft tumor model is believed devoid of T cells, the results in the present study clearly indicate that monocyte-derived immune cell infiltration to tumor tissue occurs in the nude mouse tumor model when treated with trastuzumab. The association of reduced anti-tumor efficacy by scIgG-T with the decreased number of infiltrated immune cells further suggests that infiltrated mouse immune cells in xenograft tumor tissue are capable of mediating anti-tumor activities when high HER2 expression cancer cells are treated with trastuzumab. This finding is consistent with previous reports on the correlation of immune effector cell infiltration and trastuzumab efficacy in the clinic [24,25].
This study showed that trastuzumab can be readily cleaved by MMPs in the lower hinge in vitro and that scIgG-T showed much reduced anti-tumor efficacy in comparison with the intact trastuzumabin vivo. Further investigation is necessary to determine whether the hinge cleavage of trastuzumab occurs in the tumor microenvironment in the clinical setting. It is interesting to note that a patient who was diagnosed with HER2-overexpressing breast cancer and was under adjuvant treatment with trastuzumab and chemotherapy for 6 months showed evidence of hinge cleavage of trastuzumab in the tumor tissue (Figure 7A). More importantly, single hinge cleavage of trastuzumab was detected in the tumor tissue but was not visible in the plasma sample where other host IgG antibody is abundant (Figure 7A). Expression of MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, and MMP-13) was also analyzed using a reverse-phase protein array. Among the seven MMPs tested, both MMP-3 and MMP-9 were elevated in samples from the tumor site when compared with that from the adjacent normal tissues (Figure 7B).
Figure 7.Detection of single hinge cleaved trastuzumab and matrix metalloproteinases in breast cancer patient samples. (A) Same amounts of enriched IgGs from each sample were separated by SDS-PAGE under nonreducing/denaturing conditions. Arrow, single hinge cleaved trastuzumab (scIgG-T) as referenced to standard preparation in the right lane. (B) Detection of matrix metalloproteinase (MMP) expression in breast cancer patient tumor tissues and normal adjacent 2 cm tissue using a reverse protein array method (n = 4). *P < 0.05 and **P < 0.005. MW, molecular weight.
While the extent of the hinge cleavage of trastuzumab in cancer patients during the antibody treatment is yet to be established in more patient samples, our data demonstrated that the cleavage of trastuzumab and generation of scIgG-T can occur in the tumor microenvironment. Furthermore, we hypothesize that hinge cleavage from trastuzumab to scIgG-T in the tumor microenvironment may lead to reduced anti-tumor efficacy by weakening the antibody Fc interaction with immune effector cells. However, the extent of the hinge cleavage of trastuzumab in breast cancer patients is yet to be established in a large patient cohort. Further investigation in clinical settings is warranted to determine whether single hinge cleavage represents a novel mechanism for cancer cells to invade immune functions mediated by trastuzumab in vivo.
Conclusion
Studies of resistance mechanisms of trastuzumab have been focused on genetic mutations in the HER2 signaling pathways and little is known on the role of immune evasion in the development of trastuzumab resistance. This study demonstrated that trastuzumab can be cleaved by MMPs within the lower hinge and that the hinge cleaved trastuzumab (scIgG-T) showed a significantly reduced anti-tumor efficacy due to the weakened immune effector function, including ADCC. Taken together, the results in this study suggest that hinge cleavage from intact trastuzumab to scIgG-T in the tumor microenvironment may lead to reduced anti-tumor efficacy by weakening the antibody’s ability to mediate immune effector functions. Further studies are warranted to understand the significance of single hinge cleavage on trastuzumab efficacy in the clinic.
Abbreviations
ADCC: antibody-dependent cellular cytotoxicity; AKT: protein kinase B; ELISA: enzyme-linked immunosorbent assay; Erk: extracellular-signal-regulated kinase; Fab: fragment antigen binding; Fc: fragment crystallizable; Fc(m): Fc monomer; FcγR: Fc gamma receptor; HER: human epidermal growth factor receptor; IdeS: immunoglobulin G-degrading enzyme S; IgG: immunoglobulin G; MMP: matrix metalloproteinase; MS: mass spectrometry; NK: natural killer; PBMC: human peripheral blood mononuclear cells; PBS: phosphate-buffered saline; scIgG-T: single hinge cleaved trastuzumab.
Competing interests
This study was partially funded by a grant from Janssen R&D, LLC. RJB, AO, WRS, and REJ are employees of Janssen R&D, LLC. The remaining authors declare that they have no competing interests.
Authors’ contributions
XF participated in the study design and writing the Materials and methods sections, and conducted mouse tumor xenograft studies and in vitro ADCC and western blotting assays. RJB contributed to scIgG-T preparation, study design, results interpretation and editing the manuscript. MF conducted MS analysis and prepared mass spectra graphs. HD conducted in vitro cancer cell proliferation assays and FcγR assays. AO conducted hinge cleavage of trastuzumab by human MMPs and IdeS proteinase. AG is the medical investigator for the approved institutional review board protocol and consented patients, and provided patient clinical samples and information. WPD supervised and provided expertise in MS. WRS was involved in study design and manuscript review. REJ participated in the study design, supervised scIgG-T preparation and edited the manuscript. NZ designed experiments, supervised in vitro and in vivo assay development and wrote the manuscript. ZA conceived the study, interpreted data, and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Dr Emily K Robinson and Mr Ryan W Schniederjan for providing surgical samples from breast cancer patients and Dr Sheri Skinner for coordinating the schedules between our research laboratory and the clinic. Thanks also go to Dr Michelle Kinder and Dr Katharine Grugan for critical review of the manuscript. This study was partially funded by grants from Janssen R&D, LLC, the Texas Emerging Technology Fund, and the Welch Foundation (AU00024 to ZA).
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The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized Medicine
Writer and Curator: Larry H Bernstein, MD, FCAP
LPBI
3.3 The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized Medicine
Jeffrey S. Ross, Elzbieta A. Slodkowska, W. Fraser Symmans, et al.
The Oncologist 2009; 14:320 –368 http://cme.theoncologist.com
Objectives:
Contrast the current strengths and limitations of the three main slide-based techniques (IHC, FISH, and CISH) currently in clinical use for testing breast cancer tissues for HER-2 status.
Compare the efficacy of trastuzumab- and lapatinib-based regimens in the adjuvant and metastatic settings as reported in published clinical trials and regulatory approval databases.
Contrast the list of biomarkers that have been associated with clinical resistance to trastuzumab and lapatinib and describe their current level of validation.
The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major classifier of invasive breast cancer and target of therapy for the disease. The validation of the general prognostic significance of HER-2 gene amplification and protein overexpression in the absence of anti–HER-2 targeted therapy is discussed in a study of 107 published studies involving 39,730 patients, which produced an overall HER-2– positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74. The issue of HER-2 status in primary versus metastatic breast cancer is considered along with a section on the features of metastatic HER- 2–positive disease. The major marketed slide-based HER-2 testing approaches, immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization, are presented and contrasted in detail against the background of the published American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 testing. Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER-2 testing, are also discussed. Emerging novel HER-2 testing techniques, including mRNA-based testing by real-time polymerase chain reaction and DNA microarray methods, HER-2 receptor dimerization, phosphorylated HER-2 receptors, and HER-2 status in circulating tumor cells, are also considered. A series of biomarkers potentially associated with resistance to trastuzumab is discussed with emphasis on the phosphatase and tensin homologue deleted on chromosome ten/Akt and insulin-like growth factor receptor pathways. The efficacy results for the more recently approved small molecule HER- 1/HER-2 kinase inhibitor lapatinib are also presented along with a more limited review of markers of resistance for this agent. Additional topics in this section include combinations of both anti–HER-2 targeted therapies together as well as with novel agents including bevacizumab, everolimus, and tenespimycin. A series of novel HER-2–targeting agents is also presented, including pertuzumab, ertumaxomab, HER-2 vaccines, and recently discovered tyrosine kinase inhibitors. Biomarkers predictive of HER-2 targeted therapy toxicity are included, and the review concludes with a consideration of HER-2 status in the prediction of response to non–HER-2 targeted treatments including hormonal therapy, anthracyclines, and taxanes.
Biology, Pathology, Diagnosis, And Clinical Significance Of Her-2–Positive Breast Cancer
The human epidermal growth factor receptor 2 (HER-2, HER-2/neu, c-erbB-2) gene, first discovered in 1984 by Weinberg and associates [1], is localized to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein that is a member of the epidermal growth factor receptor (EGFR) or HER family (Fig. 1) [2]. This family of receptors is involved in cell– cell and cell–stroma communication primarily through a process known as signal transduction, in which external growth factors, or ligands, affect the transcription of various genes, by phosphorylating or dephosphorylating a series of transmembrane proteins and intracellular signaling intermediates, many of which possess enzymatic activity. Signal propagation occurs as the enzymatic activity of one protein turns on the enzymatic activity of the next protein in the pathway [3]. Major pathways involved in signal transduction, including the Ras/mitogen-activated protein kinase pathway, the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, the Janus kinase/signal transducer and activator of transcription pathway, and the phospholipase C pathway, ultimately affect cell proliferation, survival, motility, and adhesion. Receptor activation requires three variables, a ligand, a receptor, and a dimerization partner [4]. After a ligand binds to a receptor, that receptor must interact with another receptor of identical or related structure in a process known as dimerization in order to trigger phosphorylation and activate signaling cascades. Therefore, after ligand binding to an EGFR family member, the receptor can dimerize with various members of the family (EGFR, HER-2, HER-3, or HER-4). It may dimerize with a like member of the family (homodimerization) or it may dimerize with a different member of the family (heterodimerization). The specific tyrosine residues on the intracellular portion of the HER-2/neu receptor that are phosphorylated, and hence the signaling pathways that are activated, depend on the ligand and dimerization partner. The wide variety of ligands and intracellular crosstalk with other pathways allow for significant diversity in signaling. Although no known ligand for the HER-2 receptor has been identified, it is the preferred dimerization partner of the other family members. HER-2 heterodimers are more stable [5, 6] and their signaling is more potent [7] than receptor combinations without HER-2. HER-2 gene amplification and/or protein overexpression has been identified in 10%–34% of invasive breast cancers [1]. Unlike a variety of other epithelial malignancies, in breast cancer, HER-2 gene amplification is uniformly associated with HER-2 (p185neu) protein overexpression and the incidence of single copy overexpression is exceedingly rare [8]. HER-2 gene amplification in breast cancer has been associated with increased cell proliferation, cell motility, tumor invasiveness, progressive regional and distant metastases, accelerated angiogenesis, and reduced apoptosis [9].When classified by routine clinicopathologic parameters and compared with HER-2– negative tumors, HER-2–positive breast cancer is more often of intermediate or high histologic grade, more often lacking estrogen receptors (ERs) and progesterone receptors (PgRs) (ER and PgR negative), and featuring positive lymph node metastases at presentation [1]. In the recent molecular classification of breast cancer, positive HER-2 status does not constitute a unique molecular category and is identified in both the “HER-2” and “luminal” tumor classes [10].
Figure 1 (not shown)
Figure 1. The human epidermal growth factor receptor (HER) gene family. This image depicts the complex crosstalk between members of the HER family of receptor tyrosine kinases and intracellular signaling. Activated HER receptors can function to both stimulate and inhibit downstream signaling of members of other biologic pathways. Note that HER-2 has no activating ligands and HER-3 lacks a tyrosine kinase domain. HER-2–mediated signaling is associated with cell proliferation, motility, resistance to apoptosis, invasiveness, and angiogenesis. The figure shows the complexity of signaling pathways initiated by, and influenced by, HER family protein receptors at the cell surface.
HER-2 Status and Prognosis in Breast Cancer Both morphology-based and molecular-based techniques have been used to measure HER-2/neu status in breast cancer clinical samples [11–117]. By a substantial majority, abnormalities in HER-2 expression at the gene, message, or protein level have been associated with adverse prognosis in both lymph node–negative and lymph node–positive breast cancer. Of the 107 studies considering 39,730 patients listed in Table 1, 95 (88%) of the studies determined that either HER-2 gene amplification or HER-2 (p185 neu) protein overexpression predicted breast cancer outcome on either univariate or multivariate analysis. In 68 (73%) of the 93 studies that featured multivariate analysis of outcome data, the adverse prognostic significance of HER-2 gene, message, or protein overexpression was independent of all other prognostic variables. In only 13 (12%) of the studies, no correlation between HER-2 status and clinical outcome was identified. Of these 13 noncorrelating studies, eight (62%) used immunohistochemistry (IHC) on paraffin-embedded tissues as the HER-2/protein detection technique, two (15%) used fluorescence in situ hybridization (FISH), two (15%) used Southern analysis, and one (7%) used a real-time polymerase chain reaction (RT-PCR) technique. Of the 15 studies that used the FISH technique, 13 (87%) showed univariate prognostic significance of gene amplification, and 11 of these (85%) showed prognostic significance on multivariate analysis as well. The two studies that used chromogenic in situ hybridization (CISH) HER-2 gene amplification detection techniques both found that HER-2 amplification was an independent predictor of outcome on multivariate analysis [100, 112]. However, interpretation of these studies is complicated by the fact that most studies included patients who received variable types of systemic adjuvant therapy; therefore, the pure prognostic value of HER-2 overexpression in the absence of any systemic adjuvant therapy is incompletely understood.
Table 1 HER-2 status and prognosis in breast cancer (not shown)
HER-2 Positivity Rates The frequency of HER-2 positivity in all of the studies presented in Table 1 was 22.2%, with a range of 9%–74%. The HER-2–positive rate was similar for IHC, at 22% (range, 10%–74%), and FISH, at 23.9% (range, 14.7%– 68%). In current practice, HER-2–positive rates have trended below 20%, with most investigators currently reporting that the true positive rate is in the range of 15%–20%. The HER-2– positive rate may be higher when metastatic lesions are tested, and tertiary hospitals and cancer centers report slightly higher rates than community hospitals and national reference laboratories. Relative Risk and Hazard Ratio In Table 1, a number of studies provided data as to the relative risk (RR) of untreated HER-2–positive breast cancer being associated with an adverse clinical outcome. For OS, the mean RR was 2.74 (range, 1.39 – 6.93) and the median was 2.33; for disease-free survival (DFS), the mean RR was 2.04 (range, 1.30 –3.01) and the median was 1.8. In several studies, the RR was estimated with a hazard ratio (HR) model. The mean HR was 2.12 (range, 1.6 –2.7) and the median was 2.08. HER-2 Expression and Breast Pathology The association of HER-2–positive status with specific pathologic conditions of the breast is summarized in Table 2. HER-2 overexpression has been consistently associated with higher grades and extensive forms of ductal carcinoma in situ (DCIS) and DCIS featuring comedo-type necrosis [118 –121]. The incidence of HER-2 positivity in DCIS has varied in the range of 24%–38% in the published literature, which appears to be slightly higher than that for invasive breast cancer [118 –121]. Routine testing for HER-2 status in DCIS is not widely performed. However, should anti– HER-2 targeted therapies directed at HER-2–positive DCIS result in a reduction in the development of invasive disease, the widespread use of HER-2 testing in DCIS would be adopted. Finally, the invasive carcinoma that develops in association with HER-2–positive DCIS may, on occasion, not feature a HER-2–positive status, a finding that has led investigators to believe that HER-2 gene amplification may not be required for the local progression of breast cancer [122]. Compared with invasive ductal carcinoma (IDC), HER-2 gene amplification occurs at a significantly lower rate in invasive lobular carcinoma (ILC) (10%), but has also been linked to an adverse outcome [85]. HER-2 positivity is linked exclusively to the pleomorphic variant of ILC and is not encountered in classic ILC [123]. HER-2 amplification is strongly correlated with tumor grade in both IDC and ILC. For example, in one study, only one of 73 grade I IDC cases and one of 67 low-grade classic ILC cases showed HER-2 amplification detected by FISH [86]. HER-2 overexpression and HER-2 amplification have been a consistent feature of both mammary and extramammary Paget’s disease [124, 125] (Fig. 2). HER-2 amplification and HER-2 overexpression have been associated with adverse outcome in some studies of male breast carcinoma [126 –129], but not in others [130 –132]. The incidence of HER-2 positivity appears to be lower in male breast cancer than in female breast cancer [126 –132]. Documented responses in male breast cancer to HER-2–targeting agents have been described, and therefore treatment with trastuzumab is an acceptable option for these patients, but the true activity rate remains uncertain [133]. The rate of HER-2 overexpression in mucinous (colloid) breast cancers is extremely low, although, on occasion, it has been associated with aggressive disease [134 –136]. In medullary breast carcinoma, HER-2 testing has consistently found negative results [137]. Similarly, HER-2 positivity is extremely rare in cases of tubular carcinoma [138]. HER-2 status has not been consistently linked to the presence of inflammatory breast cancer [139, 140]. Molecular studies of hereditary breast cancer including cases with either BRCA1 or BRCA2 germline mutations have found a consistently lower incidence of HER-2–positive status for these tumors [141].
Figure 2 not shown
Figure 2. Human epidermal growth factor receptor (HER)-2–positive Paget’s disease of the nipple. In this patient, who presented with HER-2–positive invasive duct carcinoma, classic clinical features of Paget’s disease of the nipple were present. A section of the nipple from the mastectomy specimen shows 3+ continuous cell membrane immunoreactivity for HER-2 protein. Nearly 100% of Paget’s disease of the breast cases are HER-2 positive (see text).
Breast sarcomas and phyllodes tumors have consistently been HER-2 negative [142]. Finally, low-level HER-2/neu overexpression has been identified in benign breast disease biopsies and is associated with a greater risk for subsequent invasive breast cancer [143].
HER-2 Status in Primary Versus Metastatic Breast Cancer The majority of studies that have compared the HER-2 status in paired primary and metastatic tumor tissues have found an overwhelming consistency in the patient’s status regardless of the method of testing (IHC versus FISH) [144 –151]. However, several recent studies indicated 20%–30% discordance rates between the HER-2 status of primary and metastatic lesions. Some of these studies have featured relatively high HER-2–positive rates on both paired specimens (> 35% positive), which has created concern about the conclusions of these reports [152]. Also, considering that 10%–30% discordance rates have been reported even when the same tumor is tested repeatedly, it remains uncertain if the discordance rates seen between primary and metastatic sites is higher than expected by the less than perfect reproducibility of the various HER-2 assays. Increasingly, emerging data suggest that there are changes in HER-2 expression between primary and metastatic disease. This is particularly true after intervening HER-2– directed therapy, but also happens in the absence of such treatment. In cases where the original primary HER-2 test result is questioned because of technical or interpretive issues and in patients where there has been an unusually long (i.e., > 5-year) interval between the primary occurrence and the detection of metastatic disease, retesting of a metastatic lesion may be warranted. Thus, although routine HER-2 testing of metastatic disease is advocated by some investigators, the preponderance of data indicates that the HER-2 status remains stable and that routine retesting of HER-2 may not be needed for most patients with metastatic disease.
Features of Metastatic HER-2–Positive Breast Cancer Metastatic HER-2–positive breast cancer retains the phenotype of the primary tumor not only in HER-2 status, but also is typically ER/PgR negative, moderate to high tumor grade, DNA aneuploid with high S phase fraction, and featuring ductal rather than lobular histology. In the era prior to the initiation of HER-2–targeted therapy, HER-2–positive breast cancer was more likely to spread early to major visceral sites including the axillary lymph nodes, bone marrow, lungs, liver, adrenal glands, and ovaries [153]. In the post–HER-2 targeted therapy era, the incidence of progressive visceral metastatic disease in HER-2–positive tumors has diminished and has frequently been superseded by the development of clinically significant central nervous system (CNS) metastatic disease [154 –157]. It is widely held that the success in the control of visceral disease with trastuzumab has unmasked previously occult CNS disease and, because of the inability of the therapeutic antibody to cross the blood– brain barrier, allowed brain metastases to progress during the extended OS duration of treated patients [154, 155]. The small-molecule drug lapatinib has shown some promise for targeting HER-2–positive CNS metastases that are resistant to trastuzumab-based therapies in initial studies [158].
Interaction of HER-2 Expression with Other Prognosis Variables HER-2 gene amplification and protein overexpression have been associated consistently with high tumor grade, DNA aneuploidy, high cell proliferation rate, negative assays for nuclear protein receptors for estrogen and progesterone, p53 mutation, topoisomerase IIa amplification, and alterations in a variety of other molecular biomarkers of breast cancer invasiveness and metastasis [159 –161].
Figure 3. Human epidermal growth factor receptor (HER)-2 testing.
(not shown) (A): Immunohistochemistry (IHC). This panel depicts the four categories of HER-2 IHC staining including 0 and 1+ (negative), 2+ (equivocal), and 3+ (positive) using the American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 IHC scoring. (B): Fluorescence in situ hybridization (FISH). This panel demonstrates a case of invasive duct carcinoma, on the left, negative for HER-2 gene amplification (gene copy number < 4) and a case of HER-2 gene–amplified breast cancer (gene copy number > 6),
FISH. The FISH technique (Fig. 3B), like IHC, is a morphology-driven slide-based DNA hybridization assay using fluorescent-labeled probes. Both the hybridization steps and the slide scoring can be automated. FISH has the advantages of a more objective scoring system and the presence of a built-in internal control consisting of the two HER-2 gene signals present both in benign cells and in malignant cells that do not feature HER-2 gene amplification.
IHC Versus FISH. Although the FISH method is more expensive and time-consuming than IHC, numerous studies have concluded that this cost is well borne by the greater accuracy and more precise use of anti–HER-2 targeted therapies [179 –180, 182–183]. FISH is considered to be more objective and reproducible in a number of systematic reviews [165, 180, 183–186]. In one study, the concordance rates between IHC and FISH were highest in tumors scored by IHC as 0 and 1+ and lowest for 2+ and 3+ cases [183]. Currently, the majority (approximately 80%) of HER-2 testing in the U.S. commences with a screen by IHC, with results of 0 and 1+ considered negative, 2+ considered equivocal and referred for FISH testing, and 3+ considered positive. In a pharmacoeconomic study of patients being considered for trastuzumab-based treatment for HER-2– positive tumors, FISH was found to be a cost-effective diagnostic approach “from a societal perspective” [187].
CISH and Silver In Situ Hybridization. The CISH method (Fig. 3E) and silver in situ hybridization (SISH) method feature the advantages of both IHC (routine microscope, lower cost, familiarity) and FISH (built-in internal control, subjective scoring, the more robust DNA target) [190, 191]. The CISH technique uses a single HER-2 probe, detects HER-2 gene copy number only, and was recently approved by the FDA to define patient eligibility for trastuzumab treatment. The SISH method employs both HER-2 and chromosome 17 centromere probes hybridized on separate slides and is currently under review by the FDA. Numerous studies have confirmed a very high concordance between CISH and FISH, typically in the 97%–99% range [191–203]. Similar to FISH, CISH has its highest correlation with IHC 0, 1+, and 3+ results and lowest correlation with IHC 2+ staining.
Chromosome 17 Polysomy. The incidence of chromosome 17 polysomy has varied from as low as 4% to as high as 30% in studies of invasive breast cancer [204 –208]. This may reflect differences in the definition of polysomy ranging from a low-level definition of more than two copies per cell to a high of more than four copies per cell of the chromosome. Most studies have linked chromosome 17 polysomy with greater HER-2 protein overexpression [204 –207], but some have found that protein overexpression only occurs in the presence of selective HER-2 gene amplification [204].
The 2007 ASCO-CAP Guidelines. In early 2007, a combined task force from ASCO and the CAP issued a series of recommendations designed to improve the accuracy of tissue-based HER-2 testing in breast cancer [212]. A summary of the ASCO-CAP guidelines is provided in Table 4. Highlights of these recommendations include (a) standardizing fixation in neutral-buffered formalin for no less than 6 hours and no more than 48 hours, (b) unlike their respective FDA-approval specifications, defining equivocal zones for the IHC, FISH, and CISH tests, (c) establishing a standardized quality assurance program for testing laboratories, and (d) requiring the participation of these laboratories in a proficiency testing program [212]. The published guidelines were designed to improve the overall precision and reliability of all types of slide-based HER-2 tests and remained neutral as to the relative superiority of one test over the others.
Figure 4. Real-time polymerase chain reaction (RT-PCR). In this RT-PCR assay using the Taqman RT-PCR System (Applied Biosystems Inc., Foster City, CA), note the detection of increased human epidermal growth factor receptor(HER)-2 mRNA expression in green detected at lower numbers of amplification cycles compared with the two housekeeping genes shown in red and blue.
Figure 5. DNA microarray. In this image, increased expression of human epidermal growth factor receptor (HER)-2 mRNA has been detected using a proprietary DNA microarray system (Millennium Pharmaceuticals, Inc., Cambridge, MA). The microarray demonstrates the coexpression of seven genes (HER-2 is second from the bottom) related to the amplification of HER-2 DNA in this case of HER-2–positive breast cancer.
Her-2–Targeted Therapy and the Treatment of Her-2–Positive Breast Cancer
Trastuzumab: HER-2 Testing and the Prediction of Response to Trastuzumab Therapy Using recombinant technologies, trastuzumab (Herceptin; Genentech, South San Francisco, CA), a monoclonal IgG1 class humanized murine antibody, was developed by the Genentech Corporation to specifically bind the extracellular portion of the HER-2 transmembrane receptor. This antibody therapy was initially targeted specifically for patients with advanced relapsed breast cancer that overexpresses HER-2 protein [262]. Since its launch in 1998, trastuzumab has become an important therapeutic option for patients with HER-2–positive breast cancer and is widely used for its approved indications in both the adjuvant and metastatic settings (Fig. 6) [185, 263–265]. Although trastuzumab is approved as a single-agent regimen, most patients are treated with trastuzumab plus cytotoxic agents. Table 5 summarizes the significant clinical trials that contributed to the regulatory approvals of trastuzumab.
This topic is scheduled for another article.
Trastuzumab Combinations. Since the FDA approval in 1998 of two trastuzumab plus chemotherapy combinations, a number of additional approaches have gained favor in the clinical practice community. The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines [284] currently recommend the following regimens for the first-line treatment of HER-2–positive MBC: trastuzumab plus single agents— either paclitaxel (every 3 weeks or weekly), docetaxel (every 3 weeks or weekly), or vinorelbine (weekly). For combination therapies, the NCCN recommends trastuzumab plus paclitaxel and carboplatin (every 3 weeks) or docetaxel plus carboplatin. Recently, carboplatin-based trastuzumab combinations have gained interest as a result of both the apparent boost in efficacy as measured by a higher overall response rate and longer progression-free survival time and the cardioprotective benefits of avoiding an anthracycline-containing regimen [285].
Neoadjuvant Setting The results of trastuzumab-based neoadjuvant studies (Table 5) have received significant recent interest in the oncology community [289]. Virtually all completed and in progress clinical trials have demonstrated a significant enhancement in the rate of pathologic complete response (pCR), the primary endpoint in these studies, in cases of patients with HER-2–positive breast cancer that received trastuzumab in the neoadjuvant setting [290 –297]. This benefit of the addition of trastuzumab in the neoadjuvant setting appears to be independent of, if not enhanced by, the coexistence of ER positivity [297]. Among the potential explanations for the apparent greater chemosensitivity of HER-2–positive tumors cotreated with trastuzumab in the neoadjuvant setting is the concept that HER-2 gene amplification is in some way related to the growth and survival of breast cancer stem cells [298, 299].
Biomarkers of Trastuzumab Resistance Since trastuzumab was introduced for the treatment of MBC in 1998, there has been growing interest in the discovery and potential clinical utility of biomarkers designed to predict resistance to the drug. Current approaches to HER-2 testing provide a negative predictor of drug response: the test does not predict which patients will respond to trastuzumab, it predicts which patients are unlikely to benefit.
Neoadjuvant Setting The Neo-ALTTO trial is a randomized, open-label, multicenter, phase III study comparing the efficacy of neoadjuvant lapatinib plus paclitaxel with that of trastuzumab plus paclitaxel and with concomitant lapatinib and trastuzumab plus paclitaxel given as neoadjuvant treatment in HER-2– positive primary breast cancer [337].
Biomarkers of Lapatinib Resistance In that lapatinib was approved 9 years after trastuzumab, considerably less information has been published concerning markers of efficacy or resistance to the drug [331, 341– 343].
Trastuzumab Since its introduction in the MBC setting and continuing throughout its advance into use in both the adjuvant and neoadjuvant settings, trastuzumab has been associated with the development of a variety of toxicities [384]. In the original registration trial for MBC, trastuzumab was associated with a variety of adverse events, including pain, gastrointestinal disturbances, minor hematologic deficiencies, pulmonary symptoms, and congestive heart failure (CHF) [265]. Cardiac toxicity has remained the most significant limiting factor for the use of trastuzumab [384 –389]. A major consideration in the development of cardiac toxicity in patients treated with trastuzumab has been their prior or concomitant exposure to anthracycline drugs, also associated with dose-dependent irreversible heart damage [384 – 389].
Lapatinib The most frequent adverse reactions in the lapatinib– capecitabine registration trial for MBC combination were diarrhea (65%), palmar–plantar erythrodysesthesia (53%), nausea (44%), rash (28%), vomiting (26%), and fatigue (23%) [332]. In a comprehensive analysis of the clinical trials featuring lapatinib in combination with various other agents, the overall incidence of LVEF decline was 1.6%, with 0.2% of patients experiencing symptomatic CHF [389].
HER-2 Status and the Prediction of Response to Non–HER-2 Targeted Therapy The use of HER-2 status to predict responsiveness or resistance to hormonal therapies, advocated by a number of oncologists, remains controversial. It has been reported that ER-positive/HER-2–positive patients are either less responsive or completely resistant to single-agent tamoxifen [391–393]. When measured as continuous variables, the expression of HER-2 appears to be inversely related to the expression of ER and PgR even in hormone receptor–positive tumors [394].
Anthracyclines HER-2 overexpression has also been associated with enhanced response rates to anthracycline-containing chemotherapy regimens in most, but not all, studies [42, 410 – 414].
Radiation Therapy Initially, in the era prior to the introduction of anti–HER-2 targeted therapy, HER-2–positive status was associated with a higher rate of local recurrence in some studies of breast cancer treated with surgery and radiation therapy alone, but not in others [427– 429]. However, although large-scale, randomized, prospective studies are lacking, HER-2–positive tumors treated with trastuzumab-based neoadjuvant chemotherapy combined with external-beam radiation have indicated a favorable response in locally advanced breast cancer [430].
Summary The history of the discovery of the HER-2 oncogene in an animal model in 1984, the translation of this finding to the clinical behavior of human breast cancer, and the introduction of the first anti-HER targeted therapy in 1998 is clearly a triumph of “bench to bedside” medicine. In the 10 years that have now passed since the regulatory approval of the first anti–HER-2 targeted therapy, trastuzumab, thousands of preclinical and clinical studies have considered HER-2 as a prognostic factor, its ability to predict response to hormonal and cytotoxic treatments, the best way to test for it in routine specimens, and the clinical efficacy of targeting it in a wide variety of clinical settings. Given the proven efficacy of trastuzumab and lapatinib for the treatment of MBC, and also in the adjuvant and neoadjuvant settings, the critical issue as to which test (IHC versus FISH versus CISH versus mRNA based) is the most accurate and reliable method to determine HER-2 status in breast cancer has continued to increase in importance.
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