Programmed Cell Death and Cancer Therapy
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Programmed Death: A Cat and Mouse Game
Blog | April 08, 2016 Cancer Network
http://www.cancernetwork.com/blog/programmed-death-cat-and-mouse-game
Prologue: The world of cancer care has been shaken up by the news that patients with hard-to-treat tumors benefit from a new type of immunotherapy, called checkpoint inhibition. A key receptor, called programmed death 1 (PD-1), is charged with suppressing the ability of activated T cells and other immune cells to destroy cancer cells, all in the name of preventing damage to normal tissue via autoimmunity. When PD-1 receptors on T cells bind with PD-L1 and PD-L2, complimentary receptors expressed on tumor cells, the immune response (call it the assassination of the cell) is checked and the tumor lives on. The anti PD-1 monoclonal antibodies nivolumab and pembrolizumab keep PD-L1 from turning off T cells, which has produced durable responses in several tumor types including melanoma, lung cancer, and renal cell carcinoma and represents a new hope for many.
Oncologists are excited to relay this news to patients, but is there a way to explain this without putting everyone in the room to sleep? Well, I like to use analogies to make seemingly complicated mechanisms easier to understand and the PD-1/PD-L1 relationship has inspired several colorful examples, to wit:
“Think of T cells as killers that use photographs to identify individual bad guys. Their weakness is that they will not act if the intended victim shakes their hand first. The bad guys used to be born without arms, but over time they evolved to grow arms and hands, thus avoiding elimination. The antibodies are boxing gloves that cover the hands of the T cells. Goodbye, bad guys.”
“Think of T cells as cats specially trained to eliminate mice wherever they hide. Their only weakness is if they smell catnip they will roll over and purr like idiots instead of doing their job. The mice then develop special glands that secrete catnip, thus pacifying the kitties. Solution: plug up the cats’ noses with nivolumab or pembrolizumab. Sayonara, Mr. Mouse.”
“Think of T cells as a fire sprinkler system designed to activate when a metal plug is heated to its melting point, releasing water from a pipe. The fire then emits a toxin that coats the fusible metal, keeping it below its melting point. By fitting a protective shield around the plug we block the toxic molecules and allow the plug to melt in a fire. The shield is the monoclonal antibody against PD-1 and thus the fire is successfully extinguished.”
This is getting exhausting, so I think I will stop, but don’t you agree that the concept of checkpoint inhibition lends itself to a plethora of metaphors? Now for the next lesson: how to explain chimeric antigen receptor T-cell therapy to patients. Hold on—I think I need to explain it to myself first.
As an clinical immunologist, i agree with the concept, looks pretty straightforward but definitely much more complex as our immune system work like a network. I would appreciate clinical trial data with statistical significance.
Much more confusing. Most people understand simplified concepts.
“Some cancer cells turn off your immune systems ability to recognise them. These drugs ramp up the immune system and prevent the cancer cells from hiding. This allows your cells to attack and kill cancer cells”
If i think patient seem to have better ability to understand I say “the drugs block the “off switch” that cancer cells use to escape their detection. This turns your immune systems ability to attack and kill cancer cells back on”
I haven’t had one patient that has looked confused since.
HDAC Inhibitors Enhance Immunotherapy Efficacy in Lung Cancer
http://www.oncotherapynetwork.com/lung-cancer-targets/hdac-inhibitors-enhance-immunotherapy-efficacy-lung-cancer
Histone deacetylase (HDAC) inhibitors like romidepsin might improve the efficacy of programmed cell death-1 (PD-1) blockade in lung cancer, suggest preclinical findings reported in the journal Clinical Cancer Research.
Most lung cancer patients’ tumors do not respond to immune checkpoint blockade agents like those that target PD-1. One possible mechanism underlying tumor resistance to PD-1 blockade is the failure of sufficient numbers of T cells to infiltrate tumor tissue.
Hypothesizing that upregulating T-cell chemokine expression and thereby T-cell infiltration of tumors would improve PD-1 blockade’s efficacy against lung tumors, the research team went hunting for FDA-approved oncology agents that induce chemokine expression. Screening 97 approved agents, they found one class that did: HDAC inhibitors.
The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor infiltration and impacted lung tumor growth in mouse models, the team reported—and when romidepsin was subsequently combined with PD-1 blockade in several lung tumor models, the combination showed greater antitumor activity than either agent on its own.
“These results suggest that combination of HDAC inhibitors with PD-1 blockade represent a promising strategy for lung cancer treatment,” said senior study author Amer A. Beg, PhD, of the Moffitt Cancer Center’s Immunology Program, in a news release.
Romidepsin and other HDAC inhibitors have already been approved by the FDA for use against lymphoma and other hematologic cancers, Dr. Beg noted.
The combination will next be tested in several clinical trials, including a study of patients diagnosed with non-small cell lung cancer (NSCLC) at Moffitt Cancer Center.
HDAC inhibitors enhance T cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma
Hong Zheng1, weipeng zhao2, Cihui Yan3, Crystina C Watson4,…., Brian Ruffell13, and Amer A Beg4,*
Clin Cancer Res March 10, 2016; http://dx.doi.org:/10.1158/1078-0432.CCR-15-2584
Purpose: A significant limitation of checkpoint blockade immunotherapy is the relatively low response rate (e.g. ~20% with PD-1 blockade in lung cancer). In this study, we tested whether strategies which increase T cell infiltration to tumors can be efficacious in enhancing immunotherapy response. Experimental Design: We performed an unbiased screen to identify FDA-approved oncology agents with ability to enhance T cell chemokine expression with the goal of identifying agents capable of augmenting immunotherapy response. Identified agents were tested in multiple lung tumor models as single agents and in combination with PD-1 blockade. Additional molecular and cellular analysis of tumors was used to define underlying mechanisms. Results: We found that histone deacetylase (HDAC) inhibitors (HDACi) increased expression of multiple T cell chemokines in cancer cells, macrophages and T cells. Using the HDACi romidepsin in vivo, we observed increased chemokine expression, enhanced T cell infiltration, and T cell-dependent tumor regression. Importantly, romidepsin significantly enhanced the response to PD-1 blockade immunotherapy in multiple lung tumor models, including nearly complete rejection in two models. Combined romidepsin and PD-1 blockade also significantly enhanced activation of tumor-infiltrating T cells. Conclusions: These results provide evidence for a novel role of HDACs in modulating T cell chemokine expression in multiple cell types. In addition, our findings indicate that pharmacological induction of T cell chemokine expression represents a conceptually novel approach for enhancing immunotherapy response. Finally, these results suggest that combination of HDAC inhibitors with PD-1 blockade represents a promising strategy for lung cancer treatment.
Cancer Cell Survival Driven by Novel Metabolic Pathway
http://www.genengnews.com/gen-news-highlights/cancer-cell-survival-driven-by-novel-metabolic-pathway/81252584/
Researchers have identified a novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells. [National Cancer Institute, NIH] http://www.genengnews.com/Media/images/GENHighlight/thumb_28216_large1422477191.jpg
Being attached to the extracellular matrix (ECM) provides cells with numerous advantages for survival, for instance, receiving much needed growth stimuli. However, for malignant cells to function, they must overcome their anchorage-dependent growth—a scenario that is associated with increased production of reactive oxygen species (ROS) and altered glucose metabolism.
Now, researchers at the Children’s Medical Center Research Institute at UT Southwestern (CRI) believe they have uncovered a novel metabolic pathway that helps cancer cells thrive in conditions that would otherwise be lethal to healthy cells.
“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” explained senior study author Ralph DeBerardinis, M.D., Ph.D., associate professor, and director of CRI’s Genetic and Metabolic Disease Program. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”
This new study describes an alternate version of two well-known metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle, to defend against ROS that destroy cells via oxidative stress.
The findings from this study were published recently in Nature in an article entitled “Reductive Carboxylation Supports Redox Homeostasis During Anchorage-Independent Growth.”
Previous work from Dr. DeBerardinis’ laboratory found that the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.
Dr. DeBerardinis also noted that cells “are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival.” Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells. Yet, cancer cells seem to have evolved workaround.
A landmark study from 2009 elucidated that healthy cells were destroyed when detached from the ECM. Moreover, in the same study, investigators found that inserting an oncogene into a normal cell caused it to behave like a cancer cell and survive detachment.
“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.
Dr. DeBerardinis and his team worked from the premise that the two findings were pieces of the same puzzle and that a crucial part of the picture seemed to be missing.
It had been well known that the PPP was an important source of nicotine adenine dinucleotide phosphate (NADPH), which provides a source of reducing electrons to scavenge ROS; however, the PPP produces NADPH in the cytosol, whereas the ROS are generated primarily in another subcellular structure called the mitochondria.
“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis noted. But how could NADPH from the PPP help deal with the stress of ROS produced in an entirely different part of the cell? “What we did was to discover how this happens.”
The CRI team was able to demonstrate that cancer cells use a “piggybacking” system to carry the reducing electron from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle.The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing electrons to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.
“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells. But we had no idea that they were linked in this unusual fashion,” Dr. DeBerardinis stated. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”
The researchers stressed that their findings were based on cultured cell models and more research will be necessary to test the role of the pathway in living organisms.
“We are particularly excited to test whether this pathway is required for metastasis because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” Dr. DeBerardinis concluded.
Reductive carboxylation supports redox homeostasis during anchorage-independent growth
Lei Jiang, Alexander A. Shestov, Pamela Swain, Chendong Yang, …., Brian P. Dranka, Benjamin Schwartz & Ralph J. DeBerardinis
Nature(2016) http://dx.doi.org:/10.1038/nature17393
Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)1. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells2. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism2. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.
Liquid Biopsy Accurately Detects Mutations in Advanced NSCLC
http://www.oncotherapynetwork.com/lung-cancer/liquid-biopsy-accurately-detects-mutations-advanced-nsclc#sthash.bEPxRkAq.dpuf
Droplet digital polymerase chain reaction (ddPCR)-based plasma genotyping—referred to as liquid biopsy—exhibited perfect specificity in identifying EGFR and KRAS mutations in patients with advanced non–small-cell lung cancer (NSCLC), according to the results of a study published in JAMA Oncology.
“We see plasma genotyping as having enormous potential as a clinical test, or assay—a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said senior author, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital, in a press release. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients.”
According to the press release, the test proved so reliable in the study that the Dana-Farber/Brigham and Women’s Cancer Center this week became the first medical facility in the country to offer it to all patients with NSCLC, either at the time of first diagnosis or of relapse following previous treatment.
Oxnard and colleagues enrolled 180 patients with advanced NSCLC. Patients were either newly diagnosed with the disease (n = 120) or had acquired resistance to prior EGFR kinase inhibitors (n = 60) and were planned for rebiopsy. Patients underwent initial blood sampling and immediate plasma ddPCR screening for EGFR exon 19 deletion, L858R, the EGFR T790M acquired resistance mutation, or KRAS G12X. In addition, patients underwent biopsy for tissue genotyping used to compare the accuracy of ddPCR.
Among the enrolled patients, 80 had EGFR exon19/L858R mutations, 35 had T790M mutations and 25 had KRAS G12X mutations. The median test turnaround time for liquid biopsy was 3 days. In comparison, the median turnaround time for tissue genotyping was 12 days for newly diagnosed patients and 27 days for patients with acquired EGFR inhibitor resistance.
“This long turnaround time is due largely to the practical reality that many patients with newly diagnosed NSCLC require a repeat biopsy to obtain tissue for genotyping, as do all patients with acquired resistance,” the researchers noted.
The liquid biopsy showed 100% positive predictive value for detecting EGFR 19 deletion, L858R, and KRAS mutations. However, it had only a positive predictive value of 79% for T790M mutations. The sensitivity of the test was lower. ddPCR had a sensitivity of 82% for EGFR 19 deletion, 74% for L858R, 77% for T790M, and 64% for KRAS.
The researchers pointed out that “a key limitation of plasma ddPCR is that although this method is adept at rapidly detecting specific targetable mutations, it cannot easily detect copy number alterations and rearrangements. The ddPCR panel assessed in this study thus cannot currently detect targetable alterations in either ALK or ROS1,” two other common mutations in NSCLC.
In an editorial that accompanied the article, P. Mickey Williams, PhD, of Frederick National Laboratory for Cancer Research, and Barbara A. Conley, MD, from the National Cancer Institute, questioned whether or not these results, and the rapid turnaround time for liquid biopsy, could be replicated widely by other institutions.
“However, if this performance were generally applicable, this would indeed be an advance in clinical care, reducing the proportion of patients requiring biopsy, at least in the resistance setting,” Williams and Conley wrote.
“This study is a step in the right direction of preparing needed clinical validation for the use of ctDNA for detection and serial monitoring of clinically relevant tumor mutations. Owing to low sensitivity and high positive predictive value and specificity, this approach is probably best suited for detection of resistance mutations and for serial plasma testing to assess treatment response, and should not replace tumor biopsy assessment for initial treatment decision-making,” they concluded.
Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer
Adrian G. Sacher, MD1,2; Cloud Paweletz, PhD3; Suzanne E. Dahlberg, PhD4,5; Ryan S. Alden, BSc1; Allison O’Connell, BSc3; Nora Feeney, BSc3; Stacy L. Mach, BA1; Pasi A. Jänne, MD, PhD1,2,3; Geoffrey R. Oxnard, MD1,2
JAMA Oncol. Published online April 07, 2016. http://dx.doi.org:/10.1001/jamaoncol.2016.0173
Importance Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.
Objective To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.
Design, Setting, and Participants Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.
Main Outcomes and Measures Plasma ddPCR assay sensitivity, specificity, and TAT.
Results Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.
Conclusions and Relevance Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.
Plasma genotyping uses tumor-derived cell-free DNA (cfDNA) to allow for rapid noninvasive genotyping of tumors. This technology is currently poised to transition into a treatment decision-making tool in multiple cancer types. It is particularly relevant to the treatment of advanced non–small-cell lung cancer (NSCLC), in which therapy hinges on rapid and accurate detection of targetable epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and ROS1 alterations.1– 6Plasma genotyping is capable of circumventing many limitations of standard tissue genotyping including slow turnaround time (TAT), limited tissue for testing, and the potential for failed biopsies. It may be particularly useful in directing the rapid use of new targeted therapies for acquired resistance in advanced EGFR-mutant NSCLC, where the need for a repeat biopsy to test for resistance mechanisms has amplified the inherent limitations of traditional genotyping.7,8
The need to carefully validate the test characteristics of each of the myriad individual plasma genotyping assays before use in clinical decision making is paramount. We have previously reported the development of a quantitative droplet digital polymerase chain reaction (ddPCR)-based assay for the detection of EGFR kinase mutations andKRAS codon 12 mutations in plasma.9 The detection of these mutations has the potential to guide treatment by either facilitating targeted therapy with an EGFR tyrosine kinase inhibitor (TKI) or ruling out the presence of other potentially targetable alterations in the case of KRAS.5 Alternative platforms including Cobas, peptide nucleic acid–mediated PCR, multiplexed next-generation sequencing (NGS), high-performance liquid chromatography, and Scorpion–amplified refractory mutation system have also been examined in retrospective analyses of patient samples.10– 22 The test characteristics of these assays have been variable and may be attributable to differences in testing platforms, as well as the retrospective nature of these studies, their smaller size, and the timing of blood collection with respect to disease progression and therapy initiation. The absence of reliable prospective data on the use of specific plasma genotyping assays in advanced NSCLC has left key aspects of its utility largely undefined and slowed its uptake as a tool for clinical care in patients with both newly diagnosed NSCLC and EGFR acquired resistance.
To our knowledge, we have conducted the first prospective study of the use of ddPCR-based plasma genotyping for the detection of EGFR and KRAS mutations. This study was performed in the 2 settings where we anticipate clinical adoption of this assay: (1) patients with newly diagnosed advanced NSCLC and (2) those with acquired resistance to EGFR kinase inhibitors. The primary aim of this study was to prospectively evaluate the feasibility and accuracy of this assay for the detection ofEGFR/KRAS mutations in patients with newly diagnosed NSCLC and EGFR T790M in patients with acquired resistance in a clinical setting. Additional end points included test TAT and the effect of sample treatment conditions on test accuracy.
Key Points
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Question What is the sensitivity, specificity, turnaround time, and robustness of droplet digital polymerase chain reaction (ddPCR)-based plasma genotyping for the rapid detection of targetable genomic alterations in patients with advanced non–small-cell lung cancer (NSCLC)?
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Findings In this study of 180 patients with advanced NSCLC (120 newly diagnosed, 60 with acquired resistance to epidermal growth factor receptor [EGFR] kinase inhibitors), plasma genotyping exhibited perfect specificity (100%) and acceptable sensitivity (69%-80%) for the detection of EGFR-sensitizing mutations with rapid turnaround time (3 business days). Specificity was lower for EGFR T790M (63%), presumably secondary to tumor heterogeneity and false-negative tissue genotyping.
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Meaning The use of ddPCR-based plasma genotyping can detect EGFR mutations with the rigor necessary to direct clinical care. This assay may obviate repeated biopsies in patients with positive plasma genotyping results.
CYP3A7*1C Allele Associated With Poor Outcomes in CLL, Breast, and Lung Cancer
http://www.oncotherapynetwork.com/breast-cancer-targets/cyp3a71c-allele-associated-poor-outcomes-cll-breast-and-lung-cancer#sthash.7uiD8XFD.dpuf
Patients with the CYP3A7*1C allele suffer higher rates of cancer progression and mortality, possibly because of worse outcomes among patients treated with chemotherapy drugs that are broken down by the enzyme encoded by CYP3A7, according to authors of a retrospective study published in the journal Cancer Research.
“We found that individuals with breast cancer, lung cancer, or CLL [chronic lymphocytic leukemia] who carry one or more copy of the CYP3A7*1C allele tend to have worse outcomes,” said Olivia Fletcher, PhD, a senior investigator at the Breast Cancer Now Toby Robins Research Centre at the Institute of Cancer Research in London, England, in an American Association for Cancer Research (AACR) news release.
Approximately 8% of cancer patients harbor the CYP3A7*1C allele, the coauthors noted. For these patients, it is possible that standard chemotherapy with CYP3A substrates “may not be optimal,” they cautioned.
The team analyzed DNA samples from 1,008 patients with breast cancer, 1,128 patients with lung cancer, and 347 patients with CLL. They found that the CYP3A7*1C-associated single nucleotide polymorphism (SNP) rs45446698 is associated with increased breast cancer mortality (hazard ratio [HR] 1.74; P = .03), all-cause mortality among patients with lung cancer (HR 1.43; P = .009), and progression of CLL (HR 1.62; P = .03). The rs45446698 SNP is one of seven SNPs that form the CYP3A7*1C allele.
The CYP3A7*1C allele is expressed in adults, whereas other variants of CYP3A7 are expressed during fetal development. CYP3A7 encodes an enzyme that degrades estrogen and testosterone, and some anticancer drugs.
“We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (P = .06),” they noted.
“Even though we did not see a statistically-significant difference when stratifying patients by treatment with a CYP3A7 substrate, the fact that we see the same effect in three very different cancer types suggests to me that it is more likely to be something to do with treatment than the disease itself,” commented Dr. Fletcher. “However, we are looking at ways of replicating these results in additional cohorts of patients and types of cancer, as well as overcoming the limitations of this study.”
Limitations included the retrospective nature of the study and the absence of data on how quickly individual patients metabolized chemotherapeutic agents, she said.
Cytochrome P450 AlleleCYP3A7*1C Associates with Adverse Outcomes in Chronic Lymphocytic Leukemia, Breast, and Lung Cancer
Nichola Johnson1,2, Paolo De Ieso3, Gabriele Migliorini4,….., Gillian Ross12, Richard S. Houlston, and Olivia Fletcher1,2,*
Cancer Res March 10, 2016; http://dx.doi.org:/10.1158/0008-5472.CAN-15-1410
CYP3A enzymes metabolize endogenous hormones and chemotherapeutic agents used to treat cancer, thereby potentially affecting drug effectiveness. Here, we refined the genetic basis underlying the functional effects of a CYP3A haplotype on urinary estrone glucuronide (E1G) levels and tested for an association betweenCYP3A genotype and outcome in patients with chronic lymphocytic leukemia (CLL), breast, or lung cancers. The most significantly associated SNP was rs45446698, an SNP that tags the CYP3A7*1Callele; this SNP was associated with a 54% decrease in urinary E1G levels. Genotyping this SNP in 1,008 breast cancer, 1,128 lung cancer, and 347 CLL patients, we found that rs45446698 was associated with breast cancer mortality (HR, 1.74; P = 0.03), all-cause mortality in lung cancer patients (HR, 1.43; P = 0.009), and CLL progression (HR, 1.62; P= 0.03). We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (Pinteraction = 0.06). The CYP3A7*1C allele, which results in adult expression of the fetal CYP3A7 gene, is likely to be the functional allele influencing levels of circulating endogenous sex hormones and outcome in these various malignancies. Further studies confirming these associations and determining the mechanism by which CYP3A7*1C influences outcome are required. One possibility is that standard chemotherapy regimens that include CYP3A substrates may not be optimal for the approximately 8% of cancer patients who are CYP3A7*1C carriers. Cancer Res; 76(6); 1–9. ©2016 AACR.
Specific Form of CYP3A7 Gene Associated With Poor Outcomes for Patients With Several Cancer Types
3/10/2016
PHILADELPHIA — Among patients with breast cancer, lung cancer, or chronic lymphocytic leukemia (CLL), those who had a specific form of the CYP3A7 gene (CYP3A7*1C) had worse outcomes compared with those who did not have CYP3A7*1C, and this may be related to how the patients metabolize, or break down, the therapeutics used to treat them, according to a study published in Cancer Research, a journal of the American Association for Cancer Research.
“The CYP3A7 gene encodes an enzyme that breaks down all sorts of naturally occurring substances—such as sex steroids like estrogen and testosterone—as well as a wide range of drugs that are used in the treatment of cancer,” saidOlivia Fletcher, PhD, a senior investigator at the Breast Cancer Now Toby Robins Research Centre at The Institute of Cancer Research in London. “The CYP3A7 gene is normally turned on in an embryo and then turned off shortly after a baby is born, but individuals who have one or more copy of the CYP3A7*1C form of the gene [the CYP3A7*1C allele] turn on their CYP3A7 gene in adult life.
“We found that individuals with breast cancer, lung cancer, or CLL who carry one or more copy of the CYP3A7*1C allele tend to have worse outcomes,” continued Fletcher. “One possibility is that these patients break down the drugs that they are given to treat their cancer too fast. However, further independent studies that replicate our findings in larger numbers of patients and rule out biases are needed before we could recommend any changes to the treatment that cancer patients with the CYP3A7*1C allele receive.”
To find out whether the CYP3A7*1C allele was associated with outcome for patients with breast cancer, lung cancer, or CLL, Fletcher and colleagues analyzed DNA samples from 1,008 breast cancer patients, 1,142 patients with lung cancer, and 356 patients with CLL for the presence of a single nucleotide polymorphism (SNP), rs45446698. Fletcher explained that a SNP is a type of genetic variant and that because of the way that we inherit our genetic material from our parents, we tend to inherit clusters of genetic variants. She went on to say that rs45446698 is one of seven SNPs that cluster together, forming the CYP3A7*1C allele.
The researchers found that among the breast cancer patients, rs45446698 (and, therefore, the CYP3A7*1C allele) was associated with a 74 percent increased risk of breast cancer mortality. Among the lung cancer patients, it was associated with a 43 percent increased risk of death from any cause, and among the CLL patients, it was associated with a 62 percent increased risk of disease progression.
Patients who were treated with a chemotherapeutic broken down by CYP3A7 tended to have worse outcomes compared with those treated with other chemotherapeutics, but the difference was not statistically significant.
“Even though we did not see a statistically significant difference when stratifying patients by treatment with a CYP3A7 substrate, the fact that we see the same effect in three very different cancer types suggests to me that it is more likely to be something to do with treatment than the disease itself,” said Fletcher. “However, we are looking at ways of replicating these results in additional cohorts of patients and types of cancer, as well as overcoming the limitations of this study.”
Fletcher explained that the main limitation of the study is that the researchers used samples and clinical information collected for other studies. Therefore, they did not have the same clinical information for each patient, and the samples were collected at different time points and for patients treated with various chemotherapeutics. She also noted that the team were not able to determine how quickly the patients broke down the therapeutics they received as treatment.
The study was supported by Breast Cancer Now, Leukaemia and Lymphoma Research (now known as Bloodwise), Cancer Research UK, the Medical Research Council, the Cridlan Trust, the Helen Rollason Cancer Charity, and Sanofi-Aventis. Funding for the authors’ institutions was received from the National Health Service of the United Kingdom. Fletcher declares no conflicts of interest.
Liquid Biopsy for NSCLC
‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.
http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276
A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations.
The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment.
…….
“Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.”
The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies.
The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients.
Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies.
Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFRand KRAS Mutations in Advanced Lung Cancer
Adrian G. Sacher, MD1,2; Cloud Paweletz, PhD3; Suzanne E. Dahlberg, PhD, et al. JAMA Oncol. Published online April 07, 2016. http://dx.doi.org::/10.1001/jamaoncol.2016.0173
Importance Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.
Objective To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.
Design, Setting, and Participants Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.
Main Outcomes and Measures Plasma ddPCR assay sensitivity, specificity, and TAT.
Results Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.
Conclusions and Relevance Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.
In this prospective study, we demonstrate the highly specific and rapid nature of plasma genotyping. No false-positive test results were seen for driver mutations inEGFR or KRAS, and TAT from when the specimen was obtained to result was a matter of days. This assay exhibited 100% positive predictive value for the detection of these mutations. Sensitivity was more modest and was directly correlated with both number of metastatic sites and the presence of liver or bone metastases. This newly demonstrated relationship is likely related to increased cfDNA shed in the setting of more extensive disease where tumor cfDNA shed is the chief driver of assay sensitivity and determines its upper limit. The characteristics of plasma ddPCR prospectively demonstrated in this study were similar or improved compared with previous retrospective reports of other cfDNA genotyping assays.10– 13,15,16,24,25 These retrospective studies are smaller, frequently examined a mix of tumor types and/or stages, and lack the careful prospective design needed to demonstrate the readiness of this technology to transition to a tool for selecting therapy. Studies that use retrospective samples from clinical trials that enrolled only EGFR-mutant patients are further limited by an inability to both blind laboratory investigators to tissue genotype and to generalize their assay test characteristics to a genetically heterogeneous real-world patient population.11 These differences and the multiple platforms examined previously have led to variable test characteristics and uncertainty regarding the clinical application of these technologies. This study is the first to prospectively demonstrate the ability of a ddPCR-based plasma genotyping assay to rapidly and accurately detect EGFR and KRAS mutations in a real-world clinical setting with the rigor necessary to support the assertion that use of this assay is capable of directing clinical care.
Even with a diagnostic sensitivity of less than 100%, such a rapid assay with 100% positive predictive value carries the potential for immense clinical utility. The 2- to 3-day TAT contrasts starkly with the 27-day TAT for tumor genotyping seen in patients needing a new tumor biopsy. This long TAT is due largely to the practical reality that many patients with newly diagnosed NSCLC require a repeat biopsy to obtain tissue for genotyping, as do all patients with acquired resistance. Consider the case of 1 study participant, an octogenarian with metastatic NSCLC who had developed acquired resistance to erlotinib with painful bone metastases (Figure 3). Due to the patient’s age and comorbidities, significant concerns existed about the risks of a biopsy and further systemic therapy. A plasma sample was obtained, and within 24 hours ddPCR demonstrated 806 copies/mL of EGFR T790M. A confirmatory lung biopsy was performed, which confirmed EGFR T790M. Treatment with a third-generation EGFR kinase inhibitor, osimertinib mesylate, was subsequently initiated and the patient had a partial response to therapy that was maintained for more than 1 year. The potential of this technology to obviate repeated biopsy in both patients with newly diagnosed NSCLC with insufficient tissue, as well as patients with acquired resistance, is considerable.
A key limitation of plasma ddPCR is that although this method is adept at rapidly detecting specific targetable mutations, it cannot easily detect copy number alterations and rearrangements. The ddPCR panel assessed in this study thus cannot currently detect targetable alterations in either ALK or ROS1. This limitation may potentially be addressed by using targeted NGS of cfDNA for broad, multiplexed detection of complex genomic alterations including ALK and ROS1 rearrangements, although this method is potentially slower than ddPCR-based methods and has been less thoroughly evaluated.23 The potential exists to use these technologies in tandem in advanced NSCLC to facilitate rapid initiation of therapy. Tissue genotyping and repeated biopsy would be specifically used to direct therapy in cases in which plasma genotyping was uninformative due to limitations of assay sensitivity. This approach would be particularly useful in cases of EGFR acquired resistance in which a repeated biopsy for T790M testing could be avoided entirely in many patients. Beyond detecting targetable alterations in order to drive therapy, the identification of nontargetable oncogenic drivers such as KRAS mutations that preclude the presence of other targetable alterations may guide a clinician to rapidly initiate alternative therapies such as chemotherapy or immunotherapy.5 The finding that assay sensitivity is highest in patients with more extensive metastatic disease suggests that those patients most in need of rapid treatment initiation would also be least likely to have false-negative results.
One surprising result of our study was evidence of recurrent false-positive results forEGFR T790M in patients with acquired resistance, despite no false-positive test results for other mutations studied. The sensitivity of the EGFR T790M assay was comparable to that of the EGFR sensitizing mutation assays and similarly related to both disease burden and the presence of liver or bone metastases, which are likely predictive of increased tumor cfDNA shed. We hypothesize that the lower assay specificity is due to the genomic heterogeneity whereby the T790M status of the biopsied site is not representative of all metastatic sites in a patient, a phenomenon supported by mounting evidence in the acquired resistance setting.26,27 This is consistent with the finding that a minority of patients with apparently EGFR T790M tissue-negative disease respond to therapy with third-generation EGFR kinase inhibitors.7,8,28 These observations raise questions regarding the fallibility of tissue-based genotyping as the reference standard for T790M status. The use of plasma genotyping to detect EGFR T790M thus has great potential to identify patients who would benefit from newly approved third-generation EGFR kinase inhibitors but would be unable to access them based on falsely negative tissue genotyping results. Indeed, plasma genotyping may allow more reliable assessment of both T790M status as well as the mechanisms of resistance across all sites of a heterogeneous cancer as opposed to a tissue biopsy and is likely to be an essential tool for future trials targeting drug resistance. The potential to avoid a repeat biopsy entirely in patients in whom plasma ddPCR detects T790M further strengthens the utility of this technology, although a repeat biopsy would still be needed in patients with uninformative plasma ddPCR due to limitations with respect to assay sensitivity.
This study also examined the potential of the quantitative nature of ddPCR-based plasma genotyping to allow for the early prediction of treatment response. Distinct patterns of change in mutant allele copy number were observed as early as 2 weeks after treatment and were similar to those reported in other tumor types.19,20 We hypothesize that these distinct patterns of change in this study will correlate with specific patterns of radiographic response and emergence of acquired resistance and plan to report these data once mature. The observed differences in treatment discontinuation rates observed in this study comparing patients with complete resolution of detectable mutant cfDNA with those with incomplete resolution support this hypothesis. The use of this technology to monitor disease status in real time has potential utility for both routine clinical care, as well as use as an integrated biomarker in early-phase clinical trials.10
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