Advertisements
Feeds:
Posts
Comments

Posts Tagged ‘Lung cancer’


What about PDL-1 in oncotherapy diagnostics for NSCLC?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Questions on PD-L1 Diagnostics for Immunotherapy in NSCLC
Alexander M. Castellino, PhD
http://www.medscape.com/viewarticle/862275

Two immunotherapies that target the cell programmed death (PD) pathway are now available, and both nivolumab (Opdivo, Bristol-Myers Squibb Company) and pembrolizumab (Keytruda, Merck Sharp & Dohme Corp) are approved for treating advanced, refractory, non–small cell lung cancer (NSCLC). Across several studies in patients with NSCLC, response to these agents has been correlated with PD-L1 staining, which determines PD-L1 levels in the tumor tissue. How do the available assays for PD-L1 compare?

The linear correlation between three commercially available assays is good across a range of cutoff points, concluded a presentation at the 2016 American Association for Clinical Research Annual Meeting.

Cutoffs are defined as the percentage of cells expressing PD-L1 when analyzed histochemically. “The dataset builds confidence that the assays may be used according to the cutoff clinically validated for the drug in question,” Marianne J. Radcliffe, MD, diagnostic associate director at AstraZeneca, toldMedscape Medical News.

“The correlation is good between the assays across the range examined,” she added.

However, a recently published study showed a high rate of discordance between another set of PD-L1 assays that were tested.

Dr Marianne Radcliffe

“Different diagnostic tests yield different results, depending on the cutoff for each assay. We need to harmonize the assays so clinicians are talking about the same thing,” Brendon Stiles, MD, associate professor of cardiothoracic surgery at Weill Cornell Medicine and New York-Presbyterian Hospital, New York City, told Medscape Medical News.

For Dr Stiles, these studies raise the issue that it is difficult to compare results of diagnostic testing across the different drugs and even with the same drug that are derived from different assays. “More importantly, it raises confusion in clinical practice when a patient’s sample stains positive for PD-L1 with one assay and negative with another,” he said.

“The commercial strategy for developing companion diagnostics for each drug is not in the best interests of the patients. It generates confusion among both clinicians and patients,” Dr Stiles commented. “We need to know if these assays can be used interchangeably,” he said.

As new agents come into the clinic, Dr Stiles believes there should be a universal yes-or-no answer, so that clinicians can use the assay to help decide on the use of immunotherapy.

Three Assays Tested

The study presented by Dr Radcliffe and colleagues investigated three commercially available assays, Ventana SP263, Dako 22C3, and Dako 28-8, with regard to how they compare at different cutoffs. Different studies use different cutoffs to express positivity.

Ventana SP263 was developed as a companion diagnostic for durvalumab (under development by AstraZeneca) using a rabbit monoclonal antibody. Positivity is defined as ≥25% staining of tumor cells.

Dako 22C3 was developed, and is approved, as a companion diagnostic for pembrolizumab. It uses a mouse monoclonal antibody. Positivity is defined as ≥1% and ≥50% staining of tumor cells.

Dako 28-8 was developed as a companion diagnostic for nivolumab and uses a rabbit monoclonal antibody (different from the one used in the Ventana SP263). In clinical practice, this assay is used as a complementary diagnostic for nivolumab, but the drug is approved for use regardless of PD-L1 expression. Positivity is defined as ≥1%, ≥5%, or ≥10% staining of tumor cells.

Ventana SP142 was not included in the study because it is not commercially available, Dr Ratcliffe indicated.
The three assays were used on consecutive sections of 500 archival NSCLC tumor samples obtained from commercial vendors. A single pathologist trained by the manufacturer read all samples in batches on an assay-by-assay basis. Samples were assessed per package inserts provided by Ventana and Dako in a Clinical Laboratory Improvement Amendments program-certified laboratory.

Dr Ratcliffe indicated that between reads of samples from the same patient, there was a washout period for the pathologist to remove bias.

The NSCLC samples included patients with stage I (38%), II (39%), III (20%), and IV (<1%) disease. Histologies included nonsquamous (54%) and squamous (43%) cancers.

All three PD-L1 assays showed similar patterns of staining in the range of 0% to 100%, Dr Ratcliffe indicated.

 

The correlation between any two of the assays was determined from tumor cell membrane staining. The correlation was linear with Spearman correlation of 0.911 for Ventana SP263 vs Dako 22C3; 0.935 for Ventana SP263 vs Dako 28-8; and 0.954 for Dako 28-8 vs Dako 22C3.

“With an overall predictive value of >90%, the assays have closely aligned dynamic ranges, but more work is needed,” Dr Ratcliffe said. “In general, scoring of immunohistochemical assays can be more variable between 1% and 10%, and we plan to look at this in more detail,” she said. These samples need to be reviewed by an independent pathologist, she added.

Dr Radcliffe said that currently, “Direct clinical efficacy data supporting a specific diagnostic test should still be considered as the highest standard of proof for diagnostic clinical utility.”

Why Correlations Are Needed

Pembrolizumab is approved for use only in patients with PD-L1-positive, previously treated NSCLC. A similar patient profile is being considered for nivolumab, for which testing for PD-L1 expression is not required.

For new PD-immunotherapy agents in clinical development, it is not clear whether PD-L1 testing will be mandated.

However, in clinical practice, it is clear that some patients respond to therapy, even if they are PD-L1 negative, as defined from the study. “Is it a failure of the assay, tumor heterogeneity, or is there another time point when PD-L1 expression is turned on?” Dr Stiles asked.

Dr Stiles also pointed out that a recent publication from Yale researchers showed a high a rate of discordance. In this study, PD-L1 expression was determined using two rabbit monoclonal antibodies. Both of these were different from the ones used in the Ventana SP263 and Dako 28-8 assays.

In this study, whole-tissue sections from 49 NSCLC samples were used, and a corresponding tissue microarray was also used with the same 49 samples. Researchers showed that in 49 NSCLC tissue samples, there was intra-assay variability, with results showing fair to poor concordance with the two antibodies. “Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%.

“Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way,” the study authors conclude.

The Blueprint Proposal

Coincidentally, a blueprint proposal was announced here at the AACR meeting at a workshop entitled FDA-AACR-ASCO Complexities in Personalized Medicine: Harmonizing Companion Diagnostics across a Class of Targeted Therapies.

The blueprint proposal was developed by four pharmaceutical giants (Bristol-Myers Squibb Company, Merck & Co, Inc, AstraZeneca PLC, and Genentech, Inc) and two diagnostic companies (Agilent Technologies, Inc/Dako Corp and Roche/Ventana Medical Systems, Inc).

In this proposal, the development of an evidence base for PD-1/PD-L1 companion diagnostic characterization for NSCLC would be built into studies conducted in the preapproval stage. Once the tests are approved, the information will lay the foundation for postapproval studies to inform stakeholders (eg, patients, physicians, pathologists) on how the test results can best be used to make treatment decisions.

The blueprint proposal is available online.

Dr Ratcliffe is an employee and shareholder of AstraZeneca. Dr Stiles has disclosed no relevant financial relationships.

 American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract LB-094, presented April 18, 2016.
Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non–Small-Cell Lung Cancer
Joseph McLaughlin, 1,2; Gang Han, 3; Kurt A. Schalper, 2; ….,  Roy Herbst, 1; Patricia LoRusso, 1; David L. Rimm, 2

JAMA Oncol. 2016;2(1):46-54.       http://dx.doi.org:/10.1001/jamaoncol.2015.3638.

Importance  Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non–small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.

Objective  To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.

Design, Setting, and Participants  PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non–small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.

Main Outcomes and Measures  PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.

Results  Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).

Conclusions and Relevance  Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

 

 
Introduction We are in an era of rapid incorporation of basic scientific discoveries into the drug development pipeline. Currently, numerous sponsors are developing therapeutic products that may use similar or identical biomarkers for therapeutic selection, measured or detected by an in vitro companion diagnostic device. The current practice is to independently develop a companion diagnostic for each therapeutic. Thus, the matrix of therapeutics and companion diagnostics, if each therapeutic were approved in conjunction with a companion diagnostic, may present a complex challenge for testing and decision making in the clinic, potentially putting patients at risk if inappropriate diagnostic tests were used to make treatment decisions. To address this challenge, there is a desire to understand assay comparability and/or standardize analytical and clinical performance characteristics supporting claims that are shared across companion diagnostic devices. Pathologists and oncologists also need clarity on how to interpret test results to inform downstream treatment options for their patients.
Clearly using each of the companion diagnostics to select one of the several available targeted therapies in the same class is not practical and may be impossible. Likewise, having a single test or assay as a sole companion test for all of the multiple therapeutic options within a class is also impractical since the individual therapies have differing modes of action, intended use populations, specificities, safety and efficacy outcomes. Thus, a single assay or test may not adequately capture the appropriate patient population that may benefit (or not) from each individual therapeutic option within a class of therapies. Furthermore, aligning multiple sponsors’ study designs and timelines in order that they all adopt a single companion test may inadvertently slow down development of critical therapeutic products and delay patient access to these life-saving products.
Any solution to this challenge will be multifaceted and will, by necessity, involve multiple stakeholders. Thus, the US Food and Drug Administration (FDA), the American Association for Cancer Research (AACR) and American Society of Clinical Oncology (ASCO) convened a workshop titled “Complexities in Personalized Medicine: Harmonizing Companion Diagnostics Across a Class of Targeted Therapies” to draw out and assess possible solutions. Recognizing that the complex scientific, regulatory and market forces at play here require a collaborative effort, an industry workgroup volunteered to develop a blueprint proposal of potential solutions using nonsmall cell lung cancer (NSCLC) as the use case indication.
Goal and Scope of Blueprint The imminent arrival to the market of multiple PD1 / PD-L1 compounds and the possibility of one or more associated companion diagnostics is unprecedented in the field of oncology. Some may assume that since these products target the same biological pathway, they are interchangeable; however, each PD1/PD-L1 compound is unique with respect to its clinical pharmacology and each compound is being developed in the context of a unique biological scientific hypothesis and registration strategy. Similarly, each companion diagnostic has been optimized within the individual therapeutic development programs to meet specific development goals, e.g., 1) validation for patient selection, 2) subgroup analysis as a prognostic variable, or 3) enrichment.
Further, each companion diagnostic test is optimized for its specific therapy and with its own unique performance characteristics and scoring/interpretation guidelines.
The blueprint development group recognizes that to assume that any one of the available tests could be used for guiding the treatment decision with any one or all of the drugs available in this class presents a potential risk to patients that must be addressed.
The goal of this proposal is to agree and deliver, via cross industry collaboration, a package of information /data upon which analytic comparison of the various diagnostic assays may be conducted, potentially paving the way for post-market standardization and/or practice guideline development as appropriate.
A comparative study of PD-L1 diagnostic assays and the classification of patients as PD-L1 positive and PD-L1 negative
Presentation Time: Monday, Apr 18, 2016, 8:00 AM -12:00 PM
Location: Section 10
Poster Board Number: 18
Author Block: Marianne J. Ratcliffe1, Alan Sharpe2, Anita Midha1, Craig Barker2, Paul Scorer2, Jill Walker2. 1AstraZeneca, Alderley Park, United Kingdom; 2AstraZeneca, Cambridge, United Kingdom
Abstract Body: Background: PD-1/PD-L1 directed antibodies are emerging as effective therapeutics in multiple oncology settings. Keynote 001 and Checkmate 057 have shown more frequent response to PD-1 targeted therapies in NSCLC patients with high tumour PD-L1 expression than patients with low or no PD-L1 expression. Multiple diagnostic PD-L1 tests are available using different antibody clones, different staining protocols and diverse scoring algorithms. It is vital to compare these assays to allow appropriate interpretation of clinical outcomes. Such understanding will promote harmonization of PD-L1 testing in clinical practice.
Methods: Approximately 500 tumour biopsy samples from NSCLC patients, including squamous and non-squamous histologies, will be assessed using three leading PD-L1 diagnostics assays. PD-L1 assessment by the Ventana SP263 assay that is currently being used in Durvalumab clinical trials (positivity cut off: ≥25% tumour cells with membrane staining) will be compared with the Dako 28-8 assay (used in the Nivolumab Checkmate 057 trial at the 1%, 5% and 10% tumour membrane positivity cut offs), and the Dako 22C3 assay (used in the Pembrolizumab Keynote 001 trial) at the 1% and 50% cut offs).
Results: Preliminary data from 81 non-squamous patients indicated good concordance between the Ventana SP263 and Dako 28-8 assays. Optimal overall percent agreement (OPA) was observed between Dako 28-8 at the 10% cut off and the Ventana SP263 assay (OPA; 96%, Positive percent agreement (PPA); 91%, Negative percent agreement (NPA); 98%), where the Ventana SP263 assay was set as the reference. Data on the full cohort will be presented for all three assays, and a lower 95% confidence interval calculated using the Clopper-Pearson method.
Conclusions: This study indicates that the patient population defined by Ventana SP263 at the 25% cut off is similar to that identified by the Dako-28-8 assay at the 10% tumour membrane cut off. This, together with data on the 22C3 assay, will enable cross comparison of studies using different PD-L1 tests, and widen options for harmonization of PD-L1 diagnostic testing.

http://www.abstractsonline.com/Plan/ViewAbstract.aspx

Table 1
Reference: Ventana SP-263 (≥25% tumour membrane staining)
Dako 28-8 assay cut off PPA
(%)
NPA
(%)
OPA
(%)
>1% 58 100 81
>5% 72 100 90
>10% 91 98 96
Advertisements

Read Full Post »


PLD1 tests for Lung Cancer

Larry H. Bernstein, MD, FCAP, Curator

PLBI

 

AACR: Three PD-L1 Biomarker Tests Give Similar Results in Lung Cancer

http://www.oncotherapynetwork.com/lung-cancer-targets/aacr-three-pd-l1-biomarker-tests-give-similar-results-lung-cancer?GUID=08B7ACA4-07B7-4253-8ACC-0C9AAFF0371A&rememberme=1&ts=22042016#sthash.1sWQZ14d.dpuf

 

Three commercial tests that measure levels of PD-L1 on tumors showed similar results on non-small cell lung cancer (NSCLC) tumor samples, according to a study presented at the American Association of Cancer Research (AACR) Annual Meeting, being held April 16-20, 2016, in New Orleans.
Two anti-PD-1 immunotherapy antibodies are now approved for certain patients with metastatic NSCLC (see FDA Approves Keytruda for Metastatic Non-Small Cell Lung Cancer and FDA Approves Nivolumab (Opdivo) for NSCLC).

Several pharmaceutical companies are developing diagnostic tests to measure the levels of PD-L1 protein expression on patients’ tumors. Two clinical trials, with both nivolumab (Opdivo) and pembrolizumab (Keytruda), support the notion that those patients whose tumors have higher levels of PD-L1 on their surface are more likely to respond to anti-PD-1 treatment compared to those patients whose tumors have low or no PD-L1 expression.

“Before our study, we did not know whether the different assays identified the same patients,” said study author Marianne Ratcliffe, PhD, MA, a diagnostic associate director at AstraZeneca, which is developing durvalumab/MEDI4736, an anti-PD-L1 immunotherapy antibody.

The current study compared the Ventana SP263 assay (developed by Ventana) in collaboration with AstraZeneca for use in conjunction with patients treated with durvalumab, the Dako 22C3 assay (by Dako), approved for the US Food and Drug Administration (FDA) as a companion diagnostic to identify patients who are most likely to benefit from pembrolizumab, and the Dako 28-8 assay, approved by the FDA as a complementary diagnostic for nivolumab. All three tests measure the percentage of tumor cells within a sample that stain positive for the surface protein PD-L1. Each test sets a unique cut-off point of test positivity which corresponds to a greater likelihood of response to the immunotherapy among lung cancer patients.

The researchers evaluated the three tests on 500 patient biopsy samples, including both squamous and nonsquamous histologies. The comparison showed that a 25% cutoff using the Ventana SP263 test was similar to the results using the Dako 28-8 test at a 10% cutoff mark. There were similar results between the SP263 and the 22C3 tests at a 50% cut off mark.

The three tests achieved overall percentage agreement of more than 90%, according to Radcliffe. She also noted that this study points to the ability to extrapolate results from one test to another, which in the future, could allow physicians to use the tests interchangeably.

 

Comparison of Three Different PD-L1 Diagnostic Tests Shows a High Degree of Concordance

4/18/2016

NEW ORLEANS — Three commercially available diagnostic tests were similarly effective in measuring  PD-L1 protein expression on non-small cell lung cancer (NSCLC) tumor samples, indicating that health care providers may someday be able to use these tests interchangeably when determining which patients will respond best to anti-PD-L1/PD-1 immunotherapeutic drugs, according to research presented here at the AACR Annual Meeting 2016, April 16-20.Marianne Ratcliffe

“PD-L1-directed antibodies are emerging as effective therapeutics in monotherapy and in combination in multiple oncology settings,” said the study’s lead author, Marianne Ratcliffe, MA, PhD, diagnostic associate director at AstraZeneca. She explained that several different diagnostic tests, or assays, are effective in determining which patients’ tumors express high levels of PD-L1 and might, therefore, respond best to targeted treatment.

“Before our study, we did not know whether the different assays identified the same patients,” Ratcliffe said, adding that the tests were developed on different platforms and use different antibody clones and testing protocols.

“Clearly, for the oncology community, this presents a number of issues, including a lack of confidence in being able to identify appropriate patients for treatment with these targeted therapies,” Ratcliffe said. “Our current study complements the ongoing Blueprint initiative, which is also tackling the issue of PD-L1 assay harmonization.”

This study compared the Ventana SP263 assay, developed by Ventana in collaboration with AstraZeneca for use in evaluating patients for the immunotherapeutic drug durvalumab, an anti-PD-L1; the Dako 22C3 assay, made by Dako and approved for the U.S. Food and Drug Administration (FDA) as a companion diagnostic to identify patients for pembrolizumab (Keytruda), an anti-PD-1; and the Dako 28-8 assay, made by Dako and approved by the FDA as a complementary diagnostic for nivolumab (Opdivo), an anti-PD-1.

All three tests assess the percentage of tumor cells whose membranes stain positive for the PD-L1 protein. A cut-off point is set for each test, and patients whose tumors score above the cut-off point are determined to be more likely to respond to the corresponding therapy, Ratcliffe explained.

In this study, the largest to date, approximately 500 tumor biopsy samples from patients with NSCLC were assessed because all three tests have been previously validated for that disease.
The study determined that the patient population defined by Ventana SP263 at the 25 percent cutoff point is similar to the group identified by the Dako 28-8 at the 10 percent cutoff. The study also showed a high degree of concordance between the SP263 and 22C3 assays if a 50 percent cutoff point was applied in both cases.

Ratcliffe said that the three tests achieved overall percentage agreement of more than 90 percent.  The results also indicate which cutoff points should be used to optimize agreement between a positive or negative PD-L1 result, which will help the medical community to compare results from clinical studies that have used different tests.

Ratcliffe said the study results indicate that it may be possible to extrapolate the results from one test to that of another test, and could someday allow physicians to use the tests interchangeably, though she added that further research is needed to confirm the findings.

 

Read Full Post »


 

ATCC Announces First Isogenic Cell Line Produced by the CRISPR/Cas9 Technology

CellPassages-1-2016_cancer

 

 

 

 

Reporter: Stephen J. Williams, PhD.

 

 

EML4-ALK Isogenic Cells — New!

ATCC is proud to announce its first product developed using CRISPR/Cas9 technology, the EML4-ALK Fusion-A549 Isogenic Cell Line Human (ATCC® CCL-185IG™). This cell line was derived from the parental A549 (ATCC® CCL-185™) non-small cell lung cancer cell line. EML4-ALK Fusion-A549 Isogenic Cell Line has been intensively validated on the genome, transcript, and protein level, and is otherwise identical to the parental line. This isogenic cell line is more sensitive to ALK inhibitor crizotinib when compared to A549, and serves as a vital model to study cell signaling pathways in cancer as well as in drug screening when used side-by-side with A549 cells.

Further your lung cancer research with the EML4-ALK Fusion-A549 Isogenic Cell Line Human ATCC® CCL-185IG™ derived from A549 ATCC ® CCL-185™ today!

lunc cancer cells

 

Lung Cancer

Lung cancers are classified by type: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). SCLCs are associated with smoking and metastasize very early. By contrast, non-smokers usually present with NSCLC, which are further subdivided into squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. Since both SCLC and NSCLC are usually diagnosed after the disease has spread beyond the primary site, the overall survival rates for lung cancers are poor. To breathe new life into your lung cancer research, ATCC provides numerous lung cancer cell lines, a new gene-edited isogenic NSCLC cell line, human primary cells, and h-TERT-immortalized cell lines. And to increase the throughput of your lung cancer experiments, ATCC has lung cancer cell lines organized into tumor cell panels.

Find out more about ATCC Lung Cancer Resources.

Physiologically Relevant Controls

All experiments should include physiologically relevant controls. ATCC provides both primary and hTERT-immortalized bronchial epithelial cells and small airway cells that may be used side-by-side with NSCLC or SCLC cells as normal controls. The primary and hTERT-immortalized cells may also be used to create 3D cell culture models to better represent an in vivo environment, ex vivo.

Browse the ATCC Primary Cells and hTERT Immortalized Cells to find physiological models relevant for your research needs.

Add new dimension to your research, read our application note Human Bronchial/Tracheal Epithelial Cells: Improving Functional Studies to find out how primary bronchial epithelial cells differentiate into mature airway tissue using a 3-D Air-Liquid Culture Interface model.

 

Read Full Post »


 

Multiple factors related to initial trial design may predict low patient accrual for cancer clinical trials

Reporter: Stephen J. Williams, Ph.D.

A recently published paper in JCNI highlights results determining factors which may affect cancer trial patient accrual and the development of a predictive model of accrual issues based on those factors.

To hear a JCNI podcast on the paper click here

but below is a good posting from scienmag.com which describes their findings:

Factors predicting low patient accrual in cancer clinical trials

source: http://scienmag.com/factors-predicting-low-patient-accrual-in-cancer-clinical-trials/

Nearly one in four publicly sponsored cancer clinical trials fail to enroll enough participants to draw valid conclusions about treatments or techniques. Such trials represent a waste of scarce human and economic resources and contribute little to medical knowledge. Although many studies have investigated the perceived barriers to accrual from the patient or provider perspective, very few have taken a trial-level view and asked why certain trials are able to accrue patients faster than expected while others fail to attract even a fraction of the intended number of participants. According to a study published December 29 in the JNCI: Journal of the National Cancer Institute, a number of measurable trial characteristics are predictive of low patient accrual.

Caroline S. Bennette, M.P.H., Ph.D., of the Pharmaceutical Outcomes Research and Policy Program, University of Washington, Seattle, and colleagues from the University of Washington and the Fred Hutchinson Cancer Research Center analyzed information on 787 phase II/III clinical trials sponsored by the National Clinical Trials Network (NCTN; formerly the Cooperative Group Program) launched between 2000 and 2011. After excluding trials that closed because of toxicity or interim results, Bennette et al. found that 145 (18%) of NCTN trials closed with low accrual or were accruing at less than 50% of target accrual 3 years or more after opening.

The authors identified potential risk factors from the literature and interviews with clinical trial experts and found multiple trial-level factors that were associated with poor accrual to NCTN trials, such as increased competition for patients from currently ongoing trials, planning to enroll a higher proportion of the available patient population, and not evaluating a new investigational agent or targeted therapy. Bennette et al. then developed a multivariable prediction model of low accrual using 12 trial-level risk factors, which they reported had good agreement between predicted and observed risks of low accrual in a preliminary validation using 46 trials opened between 2012 and 2013.

The researchers conclude that “Systematically considering the overall influence of these factors could aid in the design and prioritization of future clinical trials…” and that this research provides a response to the recent directive from the Institute of Medicine to “improve selection, support, and completion of publicly funded cancer clinical trials.”

In an accompanying editorial, Derek Raghavan, M.D., Levine Cancer Institute, writes that the focus needs to be on getting more patients involved in trials, saying, “we should strive to improve trial enrollment, giving the associated potential for improved results. Whether the basis is incidental, because of case selection bias, or reflects the support available to trial patients has not been determined, but the fact remains that outcomes are better.”

###

Contact info:

Article: Caroline S. Bennette, M.P.H., Ph.D., cb11@u.washington.edu

Editorial: Derek Raghavan, M.D., derek.raghavan@carolinashealthcare.org

Other investigators also feel that initial trial design is of UTMOST importance for other reasons, especially in the era of “precision” or “personalized” medicine and why the “basket trial” or one size fits all trial strategy is not always feasible.

In Why the Cancer Research Paradigm Must Transition to “N-of-1” Approach

Dr. Maurie Markman, MD gives insight into why the inital setup of a trial and the multi-center basket type of  accrual can be a problematic factor in obtaining meaningful cohorts of patients with the correct mutational spectrum.

The anticancer clinical research paradigm has rapidly evolved so that subject selection is increasingly based on the presence or absence of a particular molecular biomarker in the individual patient’s malignancy. Even where eligibility does not mandate the presence of specific biological features, tumor samples are commonly collected and an attempt is subsequently made to relate a particular outcome (eg, complete or partial objective response rate; progression-free or overall survival) to the individual cancer’s molecular characteristics.

One important result of this effort has been the recognition that there are an increasing number of patient subsets within what was previously—and incorrectly—considered a much larger homogenous patient population; for example, non–small cell lung cancer (NSCLC) versus EGFR-mutation–positive NSCLC. And, while it may still be possible to conduct phase III randomized trials involving a relatively limited percentage of patients within a large malignant entity, extensive and quite expensive effort may be required to complete this task. For example, the industry-sponsored phase III trial comparing first-line crizotinib with chemotherapy (pemetrexed plus either carboplatin or cisplatin) in ALK-rearrangement–positive NSCLC, which constitutes 3% to 5% of NSCLCs, required an international multicenter effort lasting 2.5 years to accrue the required number of research subjects.1

But what if an investigator, research team, or biotech company desired to examine the clinical utility of an antineoplastic in a patient population representing an even smaller proportion of patients with NSCLC such as in the 1% of the patient population with ROS1 abnormalities,2 or in a larger percentage of patients representing 4%-6% of patients with a less common tumor type such as ovarian cancer? How realistic is it that such a randomized trial could ever be conducted?

Further, considering the resources required to initiate and successfully conduct a multicenter international phase III registration study, it is more than likely that in the near future only the largest pharmaceutical companies will be in a position to definitively test the clinical utility of an antineoplastic in a given clinical situation.

One proposal to begin to explore the benefits of targeted antineoplastics in the setting of specific molecular abnormalities has been to develop a socalled “basket trial” where patients with different types of cancers with varying treatment histories may be permitted entry, assuming a well-defined molecular target is present within their cancer. Of interest, several pharmaceutical companies have initiated such clinical research efforts.

Yet although basket trials represent an important research advance, they may not provide the answer to the molecular complexities of cancer that many investigators believe they will. The research establishment will have to take another step toward innovation to “N-of-1” designs that truly explore the unique nature of each individual’s cancer.

Trial Illustrates Weaknesses

A recent report of the results of one multicenter basket trial focused on thoracic cancers demonstrates both the strengths but also a major fundamental weakness of the basket trial approach.3

However, the investigators were forced to conclude that despite accrual of more than 600 patients onto a study conducted at two centers over a period of approximately 2 years, “this basket trial design was not feasible for many of the arms with rare mutations.”3

They concluded that they needed a larger number of participating institutions and the ability to adapt the design for different drugs and mutations. So the question to be asked is as follows: Is the basket-type approach the only alternative to evaluate the clinical relevance of a targeted antineoplastic in the presence of a specific molecular abnormality?

Of course, the correct answer to this question is surely: No!

– See more at: http://www.onclive.com/publications/Oncology-live/2015/July-2015/Why-the-Cancer-Research-Paradigm-Must-Transition-to-N-of-1-Approach#sthash.kLGwNzi3.dpuf

The following is a video on the website ClinicalTrials.gov which is a one-stop service called EveryClinicalTrial to easily register new clinical trials and streamline the process:

Other article on this Open Access Journal on Cancer Clinical Trial Design include:

Read Full Post »


Lung Cancer Therapy

Curator: Larry H. Bernstein, MD, FCAP

 

Lung Cancer Targets

http://www.oncotherapynetwork.com/lung-cancer-targets

The enzyme ephrin receptor A2 (EphA2) normally blocks KRAS mutation-driven lung adenocarcinoma tumorigenesis, but a new study shows that EphA2 deletion mutation allows aggressive tumor growth—providing “important therapeutic targets” for this deadly form of lung cancer.

Using shRNA-mediated screening of 4,700 candidate human genes with tumor suppression potential, the team subsequently identified 16 genes in animal models that inhibit or slow KRAS- or TP53-driven tumorigenesis. The loss of EphA2 enhanced KRASG12D-driven lung adenocarcinoma carcinogenesis, they found.1

“We identified several tumor suppressors, including EphA2, loss of which promotes adenocarcinoma in the context of KRASG12D mutation,” the coauthors reported.1EphA2 loss promotes cell proliferation by activating ERK MAP kinase signaling and hedgehog signaling pathways, leading to tumorigenesis.”

A KRAS mutation is associated with tumorigenesis in 300 days, in animal models, noted senior author Inder Verma, PhD, Professor of Genetics and the Salk Institute’s Irwin and Joan Jacobs Chair in Exemplary Life Science.

“But without EphA2, the KRAS mutation leads to tumors in half the time, 120 to 150 days,” Dr. Verma noted. “This molecule EphA2 is having a huge effect on restraining cancer growth when KRAS is mutated.”

Up to 20% of all cancers harbor KRAS mutations, and these aberrations are particularly common in lung and colon cancers. EphA2 gene mutations were found in 54 of 230 patients whose lung adenocarcinoma tumor genomes were sequenced in the Cancer Genome Atlas Project, the coauthors noted.

“Oddly, among human lung cancer patients with EPhA2 mutations, around 8% of patients actually have high EphA2 expression,” cautioned coauthor Yifeng Xia, PhD, also at the Salk Institute. “So, in some instances, EphA2 is not suppressing tumors and may be context-dependent. Therefore, we need to carefully evaluate the molecule’s function when designing new therapeutics.”

EphA2 activation suppresses both cell signaling and cell proliferation, the team noted.  “We believe that the enzyme might serve as a potential drug target in KRAS-dependent lung adenocarcinoma,” explained lead study author Narayana Yeddula, PhD, a Salk research associate.

Salk Institute for Biological Studies. (2015). Molecular “brake” stifles human lung cancer.

 

Molecular “brake” stifles human lung cancer  

By testing over 4,000 genes in human tumors, a Salk team uncovered an enzyme responsible for suppressing a common and deadly lung cancer

 

 

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

Carminia Maria Della Corte1Claudio Bellevicine2Giovanni Vicidomini3Donata Vitagliano1Umberto Malapelle2, et al.

Clin Cancer Res Oct 15, 2015; 21: 4686   http://dx.doi.org:/10.1158/1078-0432.CCR-14-3319   

 

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.

Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines.

Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.

In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.

Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

This article is featured in Highlights of This Issue, p. 4497

Translational Relevance

The amplification of SMO in non–small cell lung cancer (NSCLC) resistant to EGFR-TKIs opens new possibilities of treatment for those patients who failed first-line EGFR-targeted therapies. The synergistic interaction of the Hedgehog (Hh) and MET pathways further support the rationale for a combined therapy with specific inhibitors. In addition, Hh pathway activation is essential for the acquisition of mesenchymal properties and, as such, for the aggressiveness of the disease. Also, in EGFR wild-type NSCLC models, inhibition of Hh, along with inhibition of EGF receptor (EGFR), can revert the resistance to anti-EGFR targeted drugs. In addition, inhibition of the Hh pathway sensitizes EGFR wild-type NSCLC to standard chemotherapy. These data encourage further evaluation of Hh inhibitors as novel therapeutic agents to overcome tyrosine kinase inhibitor (TKI) resistance and to revert epithelial-to-mesenchymal transition (EMT) in NSCLC.

Tyrosine kinase inhibitors (TKI) against the EGF receptor (EGFR) represent the first example of molecularly targeted agents developed in the treatment of non–small cell lung cancer (NSCLC) and are, currently, useful treatments after failure of first-line chemotherapy and, more importantly, for the first-line treatment of patients whose tumors have EGFR-activating gene mutations (1). However, after an initial response, all patients experience disease progression as a result of resistance occurrence. Recognized mechanisms of acquired resistance to anti-EGFR-TKIs in EGFR-mutated NSCLC are METgene amplification or the acquisition of secondary mutations such as the substitution of a threonine with a methionine (T790M) in exon 20 of the EGFR gene itself (2). However, these molecular changes are able to identify only a portion of patients with cancer defined as “non-responders” to EGFR-targeted agents. A number of molecular abnormalities in cancer cells may partly contribute to resistance to anti-EGFR agents (2, 3). Our group and others have shown that epithelial-to-mesenchymal transition (EMT) is a critical event in the metastatic switch and is generally associated with resistance to molecularly targeted agents in NSCLC models (4, 5). EMT is a process characterized by loss of polarity and dramatic remodeling of cell cytoskeleton through loss of epithelial cell junction proteins, such as E-cadherin, and gain of mesenchymal markers, such as vimentin (6). The clinical relevance of EMT and drug insensitivity comes from studies showing an association between epithelial markers and sensitivity to erlotinib in NSCLC cell lines, suggesting that EMT-type cells are resistant to erlotinib (7). In particular, recent data suggest that cancer cells with EMT phenotype demonstrate stem cell–like features and strategies reverting EMT could enhance the therapeutic efficacy of EGFR inhibitors (4, 5).

The Hedgehog (Hh) signaling cascade has emerged as an important mediator of cancer development and metastatic progression. The Hh signaling pathway is composed of the ligands sonic, Indian, and desert hedgehog (Shh, Ihh, Dhh, respectively) and the cell surface molecules Patched (PTCH) and Smoothened (SMO). In the absence of Hh ligands, PTCH causes suppression of SMO; however, upon ligand binding to PTCH, SMO protein leads to activation of the transcription factor GLI1, which in turn translocates into the nucleus, leading to the expression of Hh induced genes (8). The Hh signaling pathway is normally active in human embryogenesis and in tissue repair, as well as in cancer stem cell renewal and survival. This pathway is critical for lung development and its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated in bronchial epithelial cells exposed to cigarette smoke extraction. In particular, the activation of this pathway happens at an early stage of carcinogenesis when cells acquire the ability to growth in soft agar and as tumors when xenografted in immunocompromised mice. Treatment with Hh inhibitors at this stage can cause complete regression of tumors (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Reactivation of the Hh pathway with induction of EMT has been implicated in the carcinogenesis of several cancer types (15). Inhibition of the Hh pathway can reverse EMT and is associated with enhanced tumor sensitivity to cytotoxic agents (16). Recently, upregulation of the Hh pathway has been demonstrated in the NSCLC cell line A549, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype with increased cell motility and invasion (17).

The aim of the present work was to study the role of the Hh signaling pathway as mechanism of resistance to EGFR-TKIs in different models of NSCLC.

 

Activation of Hh signaling pathway in NSCLC cell lines with resistance to EGFR-TKIs

We established an in vitro model of acquired resistance to the EGFR-TKI gefitinib using the EGFR exon 19 deletion mutant (delE746-A750) HCC827 human NSCLC cell line by continuous culturing these cells in the presence of increasing doses of gefitinib. HCC827 cells, which were initially sensitive to gefitinib treatment (in vitro IC50 ∼ 80 nmol/L), became resistant (HCC827-GR cells) after 12 months of continuous treatment with IC50 > 20 μmol/L. This cell line was also cross-resistant to erlotinib and to the irreversible EGFR kinase inhibitor BIBW2992 (afatinib; data not shown). Sequencing of the EGFR gene in gefitinib-resistant HCC827-GR cells showed the absence of EGFRT790M mutation (data not shown). After the establishment of HCC827-GR cells, we characterized their resistant phenotype by protein expression analysis. While the activation of EGFR resulted efficiently inhibited by gefitinib treatment both in HCC827 and in HCC827-GR cells, phosphorylation of AKT and MAPK proteins persisted in HCC827-GR cells despite the inhibition of the upstream EGFR (Fig. 1A).

Figure 1.

Activation of Hh signaling pathway in NSCLC cell lines resistant to EGFR-TKIs. A, Western blot analysis of EGFR and of downstream signaling pathways in parental EGFR-mutated human lung adenocarcinoma HCC827 cells and in their gefitinib-resistant derivative (HCC827-GR). β-Actin was included as a loading control. B, Western blot analysis of Hh pathway, MET, and selected epithelial- and mesenchymal-related proteins in a panel of EGFR-TKI–sensitive (HCC827, H322, and Calu-3) and -resistant (HCC827-GR, H1299, Calu-3 ER, H460) NSCLC cell lines. β -Actin was included as a loading control. C, FISH analysis of gain in MET andSMO gene copy number in HCC827 and HCC827-GR. D, top, GLI-driven luciferase expression in HCC827 and HCC827-GR cells before and after depletion of GLI1 in both cell lines; bottom, evidence of GLI1 mRNA downregulation by siRNA. β-Actin was included as a loading control. E, MTT cell proliferation assays in HCC827-GR and PC9 cancer cell transfected with an empty vector or SMO expression plasmid with the indicated concentrations of gefitinib for 3 days. Bottom, Western blotting for evaluation of SMO after transfection.

HCC827-GR cells exhibited a mesenchymal phenotype with increased ability to invade, to migrate, and to grow in an anchorage-independent manner (Fig. 2A–C). Therefore, we next examined whether HCC827-GR cell line exhibits molecular changes known to occur during the EMT. Indeed, we found expression of vimentin and SLUG proteins and loss of E-cadherin protein expression in gefitinib-resistant cells as compared with gefitinib-sensitive cells (Fig. 1B). Although activation of the AXL kinase and NF-κB (20–22) have been described as known mechanisms of EGFR-TKI resistance, the analysis of their activation status resulted not significantly different among our cell lines. However, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

Figure 2.

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines. A, invasion assay. B, migration assay, C, anchorage-independent colony formation in soft agar. D, cell proliferation measured with the MTT assay in parental human lung adenocarcinoma HCC827 cells and in HCC827-GR derivative. The results are the average ± SD of 3 independent experiments, each done in triplicate.

Recently, expression of Shh and activation of the Hh pathway have been correlated to the TGFβ-induced EMT in A549 lung cancer cells (17). To investigate the expression profile of Hh signaling components in this in vitro model of acquired resistance to anti-EGFR–TKIs, we performed Western blot analysis for Shh, GLI1, 2, 3, SMO, and PTCH in HCC827-GR cells. While Shh levels did not differ between HCC827 and HCC827-GR cells, a significantly increased expression of SMO and GLI1 was found in HCC827-GR cells as compared with parental cells (Fig. 1B). No differences in the levels of GLI2 and 3 were observed (data not shown). Of interest, also PTCH protein levels resulted increased in HCC827-GR cells. This is of relevance, as PTCH is a target gene of GLI1 transcriptional activity and increased PTCH levels indicate activation of Hh signaling. We further analyzed expression and activation of MET, as a known mechanism of acquired resistance to anti-EGFR drugs in NSCLC. Indeed, MET phosphorylation resulted strongly activated in HCC827-GR cells (Fig. 1B). Analysis of the MET ligand levels, HGF, by ELISA assay, did not evidence any significant difference in conditioned media of our cells (data not shown). As previous studies have demonstrated MET gene amplification in NSCLC cell lines with acquired resistance to gefitinib (23), we evaluated MET gene copy number by FISH analysis and D-PCR in HCC827 and in HCC827-GR cell lines. The mean MET gene copy number was similar between gefitinib-sensitive and gefitinib-resistant HCC827 cell line (Fig. 1C).

Of interest, while we were working to these experiments, data on SMO gene amplification in EGFR-mutated NSCLC patients with acquired resistance to anti-EGFR targeted drugs were reported on rebiopsies performed at progression, revealing SMO amplification in 2 of 16 patients (12.5%; ref. 24). For this reason, we evaluated by FISH SMO gene copy number in HCC827-GR cells, in which the mean SMO gene copy number was 4-fold higher than that of parental HCC827 cells, indicating SMO gene amplification (Fig. 1C).

We further analyzed the expression and the activation of these molecules on a larger panel of EGFR-WT NSCLC cell lines, including NSCLC cells sensitive to EGFRTKIs, such as H322 and Calu-3 cells, NSCLC cell lines with intrinsic resistance to EGFR-TKIs, such as H1299 and H460 cells and Calu-3 ER (erlotinib-resistant) cells, which represents an in vitromodel of acquired resistance to erlotinib obtained from Calu-3 cells (refs. 4, 18; Supplementary Table S1). As shown in Fig. 1B, similarly to HCC827-GR cells, the Hh signaling pathway resulted in activation of these NSCLC models of intrinsic or acquired resistance to EGFR-TKI.

To further investigate the presence of specific mutations in the Hh pathway components, we sequenced DNA from our panel of NSCLC cell lines by Ion Torrent NGS; results indicated the absence of specific mutations in Hh-related genes (data not shown).

Because GLI1 is a transcription factor, we tested the functional significance of increased expression of this gene in the EGFR-sensitive and -resistant cell lines, using a GLI1-responsive promoter within a luciferase reporter expression vector (Fig. 1D). Analysis of luciferase activity of HCC827-GR cells revealed a 6- to 7-fold increase in GLI-responsive promoter activity as compared with HCC827 cells (P < 0.001), suggesting that transcriptional activity of GLI1 is significantly higher in gefitinib-resistant HCC827-GR cells. Furthermore, depletion of GLI1 protein expression by transfection with a GLI1-specific siRNA expression vector led to approximately 65% decrease in GLI1-driven promoter activity in HCC827-GR (P < 0.01; Fig. 1D). To determine whether SMO expression may promote resistance to gefitinib, 2 cell lines harboring the mutated EGFR gene, HCC827 and PC9 cells, and the sensitive EGFR-WT cell line Calu-3, were transiently transfected with an SMO expression plasmid. When treated with gefitinib, transfected cells exhibited a partial loss of sensitivity to the EGFR inhibition (Fig. 1E).

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines

As previously mentioned, HCC827-GR cells acquired expression of vimentin and SLUG and loss of E-cadherin when compared with gefitinib-sensitive HCC827 cancer cells along with an increased ability to invade, migrate, and form colonies in semisolid medium (Fig. 2A–C). We next evaluated whether the Hh pathway activation was necessary for gefitinib acquired resistance by genetically or by pharmacologically inhibiting Hh components in the HCC827-GR cell line. Knockdown of GLI1 by a GLI1siRNA approach had a very little effect on HCC827-GR cells. However, when gefitinib treatment (1 μmol/L) was performed in HCC827-GR cells after GLI1 blockade, invasion, migration, and colony-forming capabilities were significantly inhibited (Fig. 2A–C). Next, we evaluated the effects of 2 small-molecule inhibitors of SMO, such as LDE225 and vismodegib. Treatment with LDE225 (1 μmol/L;Fig. 2A–D) or with vismodegib (1 μmol/L; data not shown) alone did not significantly affect the viability and the invasion and migration abilities of HCC827-GR cells. Combined treatment with gefitinib and LDE225 (1 μmol/L) or vismodegib (1 μmol/L) caused inhibition of these parameters in HCC827-GR cells (Fig. 2A–C).

Taken together, these data show that Hh activation is required for acquisition of gefitinib resistance in HCC827-GR cells.

As overexpression and activation of MET was found in HCC827-GR cells, we evaluated whether inhibition of MET phosphorylation by PHA-665752 could restore gefitinib sensitivity in this model. Although abrogation of MET signaling in combination with the inhibition of EGFR signaling marginally affected gefitinib sensitivity of HCC827-GR cells, surprisingly, inhibition of MET synergistically enhanced the effects of Hh inhibition in HCC827-GR cells (Fig. 2A–D) in terms of invasion, migration, colony-forming, and proliferation abilities, indicating a significant synergism between these 2 signaling pathways. The triple inhibition of EGFR, SMO, and MET did not result in any additional antiproliferative effects (data not shown).

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKI in EGFR-dependent NSCLC cell lines

To study the role of Hh pathway in the regulation of key signaling mediators downstream of the EGFR and to explore the interaction between Hh and MET pathways, we further characterized the effects of Hh inhibition alone and in combination with EGFR or MET inhibitor on the intracellular signaling by Western blotting. As illustrated in Fig. 3A, treatment of HCC827-GR cells with the SMO inhibitor LDE225, gefitinib or with the MET inhibitor PHA-665772, for 72 hours, did not affect total MAPK and AKT protein levels and activation. A marked decrease of the activated form of both proteins was observed only when LDE225 was combined with PHA-665772, at greater level than inhibition of EGFR and MET, suggesting that the Hh pathway cooperates with MET to the activation of both MAPK and AKT signaling pathways. In addition, vimentin expression, induced during the acquisition of gefitinib resistance, was significantly decreased after Hh inhibition, suggesting that the Hh pathway represents a key mediator of EMT in this model. The combination of MET and Hh inhibitors strongly induced cleavage of the 113-kDa PARP to the 89-kDa fragment, indicating an enhanced programmed cell death.

Figure 3.

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKIs in HCC827-GR cells. A, Western blot analysis of Hh, MET, and EGFR activation and their downstream pathways activation following treatment with the indicated concentration LDE225 and PHA-556752 on HCC827-GR NSCLC cell line. β-Actin was included as a loading control. B, co-immunoprecipitation for the interaction between MET and SMO. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti- SMO (top) or anti-MET (bottom). The immunoprecipitates were subjected to Western blot analysis (WB) with indicated antibodies. Control immunoprecipitation was done using control mouse preimmune serum (PS). C, GLI-driven luciferase expression in HCC827-GR cells during treatment with gefitinib, LDE225, PHA-556752, or their combinations. D, co-immunoprecipitation for the interaction between SUFU and GLI1. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti-GLI1 (top) or anti-SUFU (bottom) antibodies. The immunoprecipitates were subjected to Western blot analysis with indicated antibodies. Control immunoprecipitation was done using control mouse PS.

Of interest, the inhibition of SMO by LDE225 also reduced the activated, phosphorylated form of MET (Fig. 3A), revealing an interaction between SMO and MET receptors. To address this issue, we hypothesized a direct interplay between both receptors. SMO immunoprecipitates from HCC827-GR cells showed greater MET binding than that from the parental HCC827 cells (Fig. 3B). As MET has been demonstrated to interact with HER3 to mediate resistance to EGFR inhibitors (25), we explored the expression of HER3 in SMO immunoprecipitates. Protein expression analysis did not show any association with HER3; similar results were obtained with EGFR protein expression analysis in the immunoprecipitates (data not shown).

The increased SMO/MET heterodimerization observed in HCC827-GR cells was partially reduced by the inhibition of SMO or MET with LDE225 or PHA-665752, respectively, and to a greater extent with the combined treatment (Fig. 3B). These results support the hypothesis that Hh and MET pathways interplay at level of their receptors.

To study whether the cooperation between these 2 pathways appears also at a downstream level, and considering that, as shown in Fig. 3A, MET inhibition partially reduces the levels of GLI1 and PTCH proteins, we analyzed luciferase expression of GLI1 reporter vector in HCC827-GR cells after treatment with LDE225, PHA-665752, or both. As shown in Fig. 3C, transcriptional activity of GLI1 resulted strongly decreased by the combined treatment. In particular, treatment with single-agent LDE225 did not abrogate the transcriptional activity of GLI1 suggesting a GLI1 noncanonical activation. In addition, single-agent PHA-665752 reduced GLI1-dependent signal, suggesting a role for MET in GLI1 regulation. To better investigate these findings, we hypothesized that MET can regulate GLI1 activity through its nuclear translocation. We, therefore, analyzed the binding ability of SUFU, a known cytoplasmic negative regulator of GLI1, following treatment of HCC827-GR cells with LDE225 and/or PHA-665752. Indeed, interaction between SUFU and GLI1 was markedly decreased in HCC827-GR cells as compared with HCC827 cells (Fig. 3D), which further confirmed the role of the activation of Hh pathway in this gefitinib-resistant NSCLC model. Furthermore, while combined treatment with LDE225 and PHA-665752 strongly increased the binding between GLI1 and SUFU, suggesting an inhibitory effect on GLI1 activity, also treatment with the MET inhibitor PHA-665752 alone favored the interaction of GLI1 with SUFU (Fig. 3D), indicating a role of MET on the activation of GLI1. This phenomenon could be a consequence of the decreased interplay between SMO and MET receptors or the effect of a direct regulation of GLI1 by MET.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts

We finally investigated the in vivo antitumor activity of Hh inhibition by LDE225, alone and in combination with gefitinib or with the MET inhibitor in nude mice bearing HCC827-GR cells. Treatment with gefitinib, as single agent, did not cause any change in tumor size as compared with control untreated mice, confirming that the in vitro model of gefitinib resistance is valid also in vivo. Treatment with LDE225 or with PHA-665752 as single agents caused a decrease in tumor size even stronger than that observed in vitro, suggesting a major role of these drugs on tumor microenvironment. However, combined treatments, such as LDE225 plus gefitinib or LDE225 plus PHA-665752, significantly suppressed HCC827-GR tumor growth with a major activity of LDE225 plus PHA-665752 combination. Indeed at 21 days from the starting of treatment, the mean tumor volumes in mice bearing HCC827-GR tumor xenografts and treated with LDE225 plus gefitinib or with LDE225 plus PHA-665752 were 24% and 2%, respectively, as compared with control untreated mice (Fig. 4A). Figure 4B shows changes in tumor size from baseline in the 6 groups of treatment. A total of eight mice for each treatment group were considered. Combined treatment of LDE225 plus gefitinib caused objective responses in 5 of 8 mice (62.5%). Of interest, the most active treatment combination was LDE225 plus PHA-665752 with complete responses in 8 of 8 mice (100%).

Figure 4.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts. A, athymic nude mice were injected subcutaneously into the dorsal flank with 107 HCC827-GR cancer cells. After 7 to 10 days (average tumor size, 75 mm3), mice were treated as indicated in Materials and Methods for 3 weeks. HCC827-GR xenografted mice received only vehicle (control group), gefitinib (100 mg/kg daily orally by gavage), LDE225 (20 mg/kg intraperitoneally three times a week), PHA-665752 (25 mg/kg intraperitoneally twice a week), or their combination. Data represent the average (±SD). The Student t test was used to compare tumor sizes among different treatment groups at day 21 following the start of treatment. B, waterfall plot representing the change in tumor size from baseline in the 6 groups of treatment. A total of 8 mice for each treatment group were evaluated. C, effects of combined LDE225 and PHA-665752 on expression of MET, PTCH, and vimentin. Tissues were stained with hematoxylin and eosin (H&E). Representative section from each condition.

We then studied the effects of gefitinib, LDE225, PHA-665752, and their combinations on the expression of PTCH, MET, and vimentin in tumor xenografts biopsies from mice of each group of treatment (Fig. 4C and Supplementary Table S2). We measured PTCH expression, as it represents a direct marker of Hh activation. While vimentin staining was particularly intense in control and gefitinib-treated tumors, treatment with LDE225 alone and in combination with PHA-665752 significantly reduced the intensity of the staining further confirming the role of Hh inhibition on the reversal of mesenchymal phenotype. Of interest, MET immunostaining resulted in a consistent nuclear positivity: this particular localization has been described as a marker of poor outcome and tendency to a mesenchymal phenotype (26). Although the combination of LDE225 and gefitinib resulted in a significant reduction of tumor growth with a concomitant reduction in staining intensity of vimentin, the combination of LDE225 and PHA-665752 was the most effective treatment, with 8 of 8 (100%) mice having a complete response in their tumors. In fact, histologic evaluations of these tumors found only fibrosis and no viable cancer cells. According to Western blot analysis of protein extracts harvested from the HCC827-GR xenograft tumors, the levels of phospho-EGFR, phospho-MET, and GLI1 resulted in a decrease after treatment with the respective inhibitor. Interestingly, the combined treatment with LDE225 and PHA-665752 resulted in a stronger inhibition of phospho-MAPK and phospho-AKT (Supplementary Fig. S1).

Role of the Hh pathway in mediating resistance to EGFR inhibitors in EGFR-WT NSCLC

As shown in Fig. 1B, although H1299, H460, and Calu-3 ER lacked SMO amplification (data not shown), these cells displayed Hh pathway activation. We further conducted luciferase expression analysis that showed a 8- to 9-fold increase in GLI1-dependent promoter activity in these lines as compared with EGFR inhibitor–sensitive H322 and Calu-3 cells, suggesting that transcriptional activity of GLI1 is higher in EGFR-TKI–resistant EGFR-WT NSCLC lines (Supplementary Fig. S2A). Similar to HCC827-GR cells, these cells showed also activation of MET. However, as reported in previous studies (4), MET inhibition alone or in combination with EGFR inhibition or with SMO inhibition resulted ineffective in inhibiting cancer cell proliferation and survival (data not shown).

We therefore tested the effects of Hh inhibition, by silencing GLI1 or by using LDE225, alone and/or in combination with erlotinib. Although knockdown of GLI1 or treatment with LDE225 (1 μmol/L) did not significantly affect NSCLC cell viability, combined treatment with erlotinib restored sensitivity to erlotinib (Supplementary Fig. S2B).

In addition, H1299, Calu-3 ER, and H460 cells exhibited significantly higher invasive and migratory abilities than H322 and Calu-3 cells and inhibition of Hh pathway significantly reduced these abilities. Collectively, these results suggest that Hh pathway activation mediates the acquisition of mesenchymal properties in EGFR-WT lung adenocarcinoma cells with erlotinib resistance (Supplementary Fig. S2B–S2D).

We next evaluated the effects of LDE225 alone and/or in combination with erlotinib on the activation of downstream pathways. Erlotinib treatment result was unable to decrease the phosphorylation levels of AKT and MAPK in H1299 and Calu-3 ER cells (Fig. 5A). However, when LDE225 was combined with erlotinib, a strong inhibition of AKT and MAPK activation was observed in these EGFR inhibitor–resistant cells (Fig. 5A). Furthermore, flow cytometric analysis revealed that combined treatment with both erlotinib and LDE225 significantly enhanced the apoptotic cell percentage to 65% and 70% (P < 0.001) in H1299 and Calu-3 ER cells, respectively (Fig. 5B), confirmed by the induction of PARP cleavage after the combined treatment (Fig. 5A). These findings suggest that Hh pathway drives proliferation and survival signals in NSCLC cells in which EGFR is blocked by erlotinib, and only the inhibition of both pathways can induce strong antiproliferative and proapoptotic effects. The in vitro synergism between EGFR and SMO was confirmed alsoin vivo. Combination of erlotinib and LDE225 significantly suppressed growth of Calu-3 ER xenografted tumors in nude mice (Supplementary Fig. S1F).

Figure 5.

Activation of Hh signaling pathway mediates resistance to EGFR-TKI in EGFR-WT NSCLC cell lines. A, Western blot analysis of EGFR and its downstream pathways activation, including PARP cleaved form, following treatment with the indicated concentration LDE225 and erlotinib on Calu-3, Calu-3 ER, and H1299 NSCLC cell line. β-Actin was included as a loading control. B, apoptosis was evaluated as described in Supplementary Materials and Methods with annexin V staining in Calu-3, Calu-3-GR, and H1299 cancer cells, which were treated with the indicated concentration LDE225 and erlotinib. Columns, mean of 3 identical wells of a single representative experiment; bars, top 95% confidence interval; ***, P < 0.001 for comparisons between cells treated with drug combination and cells treated with single agent.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy

To extend our preclinical observations, we further investigated the effects of Hh pathway inhibition on sensitivity of EGFR-WT NSCLC cells to standard chemotherapy used in this setting and mostly represented by cisplatin.

To investigate the role of the Hh pathway in mediating resistance also to chemotherapy, we evaluated the efficacy of cisplatin and Hh inhibition treatment alone or in combination on the colony-forming ability in semisolid medium of H1299 and H460 cell lines (Fig. 6). Treatment with cisplatin alone resulted in a dose-dependent inhibition of colony formation with an IC50 value of 13 and 11 μmol/L for H1299 and H460 cells, respectively. However, when combined with LDE225, the treatment resulted in a significant synergistic antiproliferative effect in both NSCLC cell lines (Fig. 6). Together, these results indicate that treatment of EGFR-WT NSCLC cells with Hh inhibitors could improve sensitivity of NSCLCs to standard chemotherapy.

Figure 6.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy. Anchorage-independent colony formation in soft agar in human lung adenocarcinoma H1299 and H460. The results are the average ± SD of 3 independent experiments, each done in triplicate. For defining the effect of the combined drug treatments, any potentiation was estimated by multiplying the percentage of cells remaining by each individual agent. The synergistic index was calculated as previously described (19). In the following equations, A and B are the effects of each individual agent and AB is the effect of the combination. Subadditivity was defined as %AB/(%A%B) < 0.9; additivity was defined as %AB/(%A%B) = 0.9–1.0; and supra-additivity was defined as %AB/(%A%B) > 1.0.

Discussion

Resistance to currently available anticancer drugs represents a major clinical challenge for the treatment of patients with advanced NSCLC. Our previous works (4, 18) reported that whereas EGFR-TKI–sensitive NSCLC cell lines express the well-established epithelial markers, cancer cell lines with intrinsic or acquired resistance to anti-EGFR drugs express mesenchymal characteristics, including the expression of vimentin and a fibroblastic scattered morphology. This transition plays a critical role in tumor invasion, metastatic dissemination, and the acquisition of resistance to therapies such as EGFR inhibitors. Among the various molecular pathways, the Hh signaling cascade has emerged as an important mediator of cancer development and progression (8). The Hh signaling pathway is active in human embryogenesis and tissue repair in cancer stem cell renewal and survival and is critical for lung development. Its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated by cigarette smoke extraction exposure in bronchial epithelial cells (12). In particular, the activation of this pathway correlated with the ability to growth in soft agar and in mice as xenograft and treatment with Hh inhibitors showed regression of tumors at this stage (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Recently, alterations of the SMO gene (mutation, amplification, mRNA overexpression) were found in 12.2% of tumors of The Cancer Genome Atlas (TCGA) lung adenocarcinomas by whole-exome sequencing (27). The incidence of SMO mutations was 2.6% and SMO gene amplifications were found in 5% of cases. SMO mutations and amplification strongly correlated with SHH gene dysregulation (P < 0.0001). In a small case report series, 3 patients with NSCLC with Hh pathway activation had been treated with the SMO inhibitor LDE225 with a significant reduction in tumor burden, suggesting that Hh pathway alterations occur in NSCLC and could be an actionable and valuable therapeutic target (27). Recently, upregulation of Shh, both at the mRNA and at the protein levels, was demonstrated in the A549 NSCLC cell line, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype (17, 28, 29) and mediated increased cell motility, invasion, and tumor cell aggressiveness (30, 31).

In the present study, SMO gene amplification has been identified for the first time as a novel mechanism of acquired resistance to EGFR-TKI in EGFR-mutant HCC827-GR NSCLC cells. These data are in agreement with the results of a cohort of patients with EGFR-mutant NSCLC that were treated with EGFR-TKIs (24). Giannikopoulus and colleagues have demonstrated the presence of SMO gene amplification in tumor biopsies taken at occurrence of resistance to EGFR-TKIs in 2 of 16 patients (24). In both cases, theMET gene was also amplified. In this respect, although the MET gene was not amplified in HCC827-GR cells, we found a significant functional and structural interaction between MET and Hh pathways in these cells. In fact, the combined inhibition of both SMO and MET exerted a significant antiproliferative and proapoptotic effect in this model, demonstrated by tumor regressions with complete response in 100% of HCC827-GR tumors xenografted in nude mice.

Several MET inhibitors have been evaluated in phase II/III clinical studies in patients with NSCLC, with controversial results. Most probably, blocking MET receptor alone is not enough to revert the resistant phenotype, as it is implicated in several intracellular interactions, and the best way to overcome resistance to anti-EGFR-TKIs is a combined approach, with Hh pathway inhibitors.

In the context of EMT, Zhang and colleagues demonstrated that AXL activation drives resistance in erlotinib-resistant subclones derived from HCC827, independently from MET activation in the same subclone, and that its inhibition is sufficient to restore erlotinib sensitivity by inhibiting downstream signal MAPK, AKT, and NF-κB (21). In addition, Bivona and colleagues described in 3 HCC827 erlotinib-resistant subclones increased RELA phosphorylation, a marker of NF-κB activation, in the absence of MET upregulation, and demonstrated that NF-κB inhibition enhanced erlotinib sensitivity, independently from AKT or MAPK inhibition (22). Differently, we detected Hh and MET hyperactivation in our resistance model HCC827-GR without a clear increase in AXL and NF-κB activation.

Although the level of activation of AXL and NF-κB did not result in contribution to resistance in our model, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

In a preclinical model, the evolution of resistance can depend strictly from the selective activation of specific pathways, whereas different mechanisms can occur simultaneously in patients with NSCLC, due to tumor heterogeneity. Thus, all data regarding EFGFR-TKIs resistance have to be considered equally valid.

We further extended the evaluation of the Hh pathway to NSCLC cell lines harboring the wild-type EGFR gene and demonstrated that Hh is selectively activated in NSCLC cells with intrinsic or acquired resistance to EGFR inhibition and occurred in the context of EMT.

To further validate these data, we blocked SMO or downregulated GLI1 RNA expression in NSCLC cells that had undergone EMT, and this resulted in resensitization of NSCLC cells to erlotinib and loss of vimentin expression, indicating an mesenchymal-to-epithelial transition promoted by the combined inhibition of EGFR and Hh. Inhibition of the Hh pathway alone was not sufficient to reverse drug resistance but required concomitant EGFR inhibition to block AKT and MAPK activation and to restore apoptosis, indicating that the prosurvival PI3K/AKT pathway and the mitogenic RAS/RAF/MEK/MAPK pathways likely represent the level of interaction of EGFR and Hh signals.

In EGFR-WT NSCLC models, the role of MET amplification/activation is less clear, and in our experience, its inhibition did not increase the antitumor activity of SMO inhibitors.

In addition, Hh inhibition contributed to increase the response to cisplatin treatment which is the standard chemotherapeutic option used in EGFR-WT NSCLC patients and in EGFR-mutated patients after progression on first-line EGFR-TKI, thus representing a valid contribution to achieve a better disease control in those patients without oncogenic activation or after progression on molecularly targeted agents.

Collectively, the results of the present study provide experimental evidence that activation of the Hh pathway, through SMO amplification, is a potential novel mechanism of acquired resistance in EGFR-mutated NSCLC patients that occurs concomitantly with MET activation, and the combined inhibition of these 2 pathways exerts a significant antitumor activity. In light of these results, screening of SMO alteration is strongly recommended in EGFR-mutated NSCLC patients with acquired resistance to EGFR-TKIs at first progression.

 

Early-stage lung cancer patients considered to be high risk for surgery can achieve good clinical outcomes with surgical resection, according to a new study.

Pembrolizumab, a PD-1 inhibitor, demonstrated better overall survival and progression-free survival vs docetaxel in non–small-cell lung cancer patients.

A study looking at trends from 1985 to 2005 found that overall survival has increased in Medicare patients with small-cell lung cancer, and that treatment with chemotherapy is associated with improved survival.

Patients with ALK-rearranged non–small-cell lung cancer and brain metastases survive longer when treated with radiotherapy and tyrosine kinase inhibitors.

– See more at: http://www.cancernetwork.com/lung-cancer

 

Pembrolizumab Offers Survival Benefit in NSCLC

http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc

The maker of pembrolizumab, a programmed death 1 (PD-1) inhibitor (Keytruda; Merck), announced phase II/III trial results showing that the drug resulted in better overall survival (OS) and progression-free survival (PFS) compared with docetaxel in patients with non–small-cell lung cancer (NSCLC). The study included only patients who had failed prior systemic therapy and whose tumors expressed programmed death ligand 1 (PD-L1). – See more at: http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc#sthash.NUIqYKmi.dpuf

“The results from this trial provide part of a growing body of evidence supporting the potential of Keytruda in the treatment of NSCLC,” said Merck’s president, Roger M. Perlmutter, MD, PhD, in a press release.

The KEYNOTE-010 trial results have not yet been presented or published. The study compared two doses of pembrolizumab (2 mg/kg and 10 mg/kg) with docetaxel in 1,034 patients. All had progressed following treatment with platinum-containing systemic therapy, and all had tumors expressing PD-L1.

According to Merck’s release, pembrolizumab was associated with longer OS, in both the 2-mg/kg and 10-mg/kg dose groups, compared with docetaxel. This survival benefit was seen both in a subgroup of patients with PD-L1 expression tumor proportion scores of 50% or higher, as well as in all enrolled patients (all had a score of 1% or higher).

Both doses also resulted in longer PFS vs docetaxel in the 50% or higher group; this was not statistically significant in the full cohort of patients.

Pembrolizumab received an accelerated approval from the US Food and Drug Administration (FDA) in early October (at the 2-mg/kg dose level). The FDA noted in a press release that the most common side effects in a safety cohort of 550 patients included fatigue, dyspnea, and decreased appetite.

Earlier this year, results of a phase I study of pembrolizumab yielded promising survival outcomes. The median OS in that cohort of 495 patients was 12.0 months, and there was an overall response rate to the drug of 19.4%; this was higher in patients who had not received any previous treatment.

Pembrolizumab is not the first immunotherapy agent approved for the treatment of NSCLC. The FDA granted approval to nivolumab (Opdivo; Bristol-Myers Squibb) for the treatment of metastatic squamous NSCLC in March, and the indication was expanded to advanced non-squamous NSCLC in October.

– See more at: http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc#sthash.NUIqYKmi.dpuf

Read Full Post »


Pathology Insights

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

Predicting the Prognosis of Lung Cancer: The Evolution of Tumor, Node and Metastasis in the Molecular Age—Challenges and Opportunities

Ramón Rami-Porta; Hisao Asamura; Peter Goldstraw

Transl Lung Cancer Res. 2015;4(4):415-423.

http://www.medscape.com/viewarticle/852315

 

 

The tumor, node and metastasis (TNM) classification of malignant tumors was proposed by Pierre Denoit in the mid-20th century to code the anatomic extent of tumors. Soon after, it was accepted by the Union for International Cancer Control and by the American Joint Committee on Cancer, and published in their respective staging manuals. Till 2002, the revisions of the TNM classification were based on the analyses of a database that included over 5,000 patients, and that was managed by Clifton Mountain. These patients originated from North America and almost all of them had undergone surgical treatment. To overcome these limitations, the International Association for the Study of Lung Cancer proposed the creation of an international database of lung cancer patients treated with a wider range of therapeutic modalities. The changes introduced in the 7th edition of the TNM classification of lung cancer, published in 2009, derived from the analysis of an international retrospective database of 81,495 patients. The revisions for the 8th edition, to be published in 2016, will be based on a new retrospective and prospective international database of 77,156 patients, and will mainly concern tumor size, extrathoracic metastatic disease, and stage grouping. These revisions will improve our capacity to indicate prognosis and will make the TNM classification more robust. In the future the TNM classification will be combined with non-anatomic parameters to define prognostic groups to further refine personalized prognosis.

Introduction

Obvious as it may seem, it is important that the readers of this article keep in mind that the tumor, node and metastasis (TNM) classification of lung cancer is no more and no less than a system to code the anatomic extent of the disease. Therefore, by definition, the TNM classification does not include other elements that, while they can help improve our capacity to prognosticate the disease for a given patient, are unrelated to the anatomy of the tumor, i.e., parameters from blood analysis, tumor markers, genetic signatures, comorbidity index, environmental factors, etc. Prognostic indexes combining the TNM classification and other non-anatomic parameters are called, by consensus between the Union for International Cancer Control (UICC) and the American Joint Committee on Cancer (AJCC), prognostic groups to differentiate them from the anatomic stage groupings.

The TNM classification of lung cancer is applied to all histopathological subtypes of non-small cell carcinoma, to small cell carcinoma and to typical and atypical carcinoids. It is governed by general rules[1–3] (Table 1) that apply to all malignancies classified with this system, and by site-specific rules applicable to lung cancer exclusively.[4] There also are recommendations and requirements issued with the objective to classify tumors in a uniform way when their particular characteristics do not fit in the basic rules.[4]

The three components of the classification have several categories that are defined by different descriptors. For lung cancer, those for the T component are based on tumor size, tumor location and involved structures; those for the N, on the absence, presence and location of lymph node metastasis; and those for the M, on the absence, presence and location of distant metastasis. There are optional descriptors that add information on the local aggressiveness of the tumor (differentiation grade, perineural invasion, vascular invasion and lymphatic permeation) all of which have prognostic relevance;[5–8] assess the intensity of the investigation to determine the stage (certainty factor); and assess the residual tumor after therapy (residual tumor).

The TNM classification was developed by Pierre Denoit in a series of articles published from 1943 to 1952. It was soon adopted by the UICC that published brochures covering several anatomical sites, the lung being included in 1966. Two years later, the UICC published the first edition of the TNM Classification of Malignant Tumors and agreements were reached with the AJCC, created in 1959 as the American Joint Committee for Cancer Staging and End Results Reporting, to consult each other to avoid publication of differing classifications. Since then, the UICC and the AJCC have been responsible for updating and revising the TNM classifications of malignant tumors with the participation of national TNM committees of several countries and taking into account the published reports on the topic. The second to sixth editions of the UICC manual on the TNM Classification of Malignant Tumors and the first to sixth editions of the AJCC Staging Manual included classifications for lung cancer that had been informed by a progressively enlarging database initially collected by Mountain, Carr and Anderson, and subsequently managed by Mountain. Their database originally contained a little over 2,000 patients, but it had grown to more than 5,000 by the time the fifth edition of the TNM classification for lung cancer was published in 1997. The sixth edition was published in 2002 with no modifications.[9]

While the fifth edition of the classification was being printed, the International Workshop on Intrathoracic Staging took place in London, United Kingdom, in October 1996, sponsored by the International Association for the Study of Lung Cancer (IASLC).[10] At that meeting, in the presence of Dr. Mountain, the limitations of the database that had been used to revise the TNM classification for lung cancer were openly discussed. In essence, it was considered that, while the database consisted of a relatively large number of patients, all of them originated from the United States of America, and, therefore, the staging system could not really be called ‘international’, as it was called at that time; and, although all tumors had clinical and pathological classifications, the majority had been treated surgically. So, the database was thought not to be representative of the international community, as there were no patients from other countries; or of the current clinical practice, as there were no patients treated with other therapies. Therefore, an agreement was reached to issue a worldwide call to build a really international database of lung cancer patients treated by all therapeutic modalities. This required the constitution of an International Staging Committee that was approved and given a small amount of funding, to pump-prime, by the IASLC Board in 1998. Subsequently substantial financial support was secured by an unrestricted grant from Eli-Lilly. Cancer Research And Biostatistics (CRAB), a not-for-profit biosciences statistical center in Seattle, was appointed to collect, manage and analyze the new database. The proprietors and managers of known databases were subsequently summoned to attend a series of preparatory meetings to identify potential contributors to the IASLC international database for the purpose of revising the TNM classification of lung cancer.

The Future of the TNM Classification

The TNM classification of lung cancer is the most consistent and solid prognosticator of the disease, but it does not explain the whole prognosis because prognosis is multifactorial. In addition to the anatomic extent of the tumor, patient and environmental factors also count. Prognosis also is dynamic, as it may be different at the time of diagnosis, after treatment or at recurrence.[71] In the TNM classification, tumor resection plays an important role as it defines pathological staging and may modify the prognostic assessment based on clinical staging. Other than that, the TNM classification does not include blood analyses, tumor markers, genetic characteristic of the tumor or environmental factors that may account for the differences in survival among similar tumors in different geographic areas.

In order to make progress to indicate a more personalized prognosis, instead of a prognosis based on cohorts of patients with tumors of similar anatomic extent, the IASLC Staging and Prognosis Factors Committee decided to expand its activities to the study of non-anatomic prognostic factors. Therefore, in the third phase of the IASLC Lung Cancer Staging Project, the activities of the committee will be directed to further refine the TNM classification and to find available factors that can be combined with tumor staging to define prognostic groups. To some extent, this already was done with the analyses of the database used for the 7th edition. Prognostic groups with statistically significant differences were defined by combining anatomic tumor extent and very simple clinical variables, such as performance status, gender, and age. These prognostic groups were defined for clinically and pathologically staged tumors, and for small-cell and non-small cell lung cancers.[22,23]

The database used for the 8th edition includes several non-anatomical elements related to the patient, the tumor and the environment that may help refine prognosis at clinical and pathological staging.[69]Due to the limitations of the previous databases, future revisions of the TNM classification will need to be more balanced in terms of therapeutic modalities, and better populated with patients from underrepresented geographical areas, such as Africa, India, Indonesia, North, Central and South America, and South East Asia. The data contributed in the future will have to be complete regarding the TNM descriptors, and preferably prospective. The more robust the TNM, the more important its contribution to the prognostic groups.

To achieve all of the above, international collaboration is essential. Those interested in participating in this project should send an email expressing their interest to information@crab.org, stating ‘IASLC staging project’ in the subject of the email. The IASLC Staging and Prognostic Factors Committee has been very touched by the overwhelming generosity of colleagues around the world who have contributed cases to inform the 7th and the 8th editions of the TNM classification of lung cancer. We continue to count on their collaboration to further revise future editions and to define prognostic groups that will eventually allow a more personalized indication of prognosis.

MicroRNAs in the Pathobiology of Sarcomas

Anne E Sarver; Subbaya Subramanian

Lab Invest. 2015;95(9):987-984

http://www.medscape.com/viewarticle/852145

 

Sarcomas are a rare and heterogeneous group of tumors. The last decade has witnessed extensive efforts to understand the pathobiology of many aggressive sarcoma types. In parallel, we have also begun to unravel the complex gene regulation processes mediated by microRNAs (miRNAs) in sarcomas and other cancers, discovering that microRNAs have critical roles in the majority of both oncogenic and tumor suppressor signaling networks. Expression profiles and a greater understanding of the biologic roles of microRNAs and other noncoding RNAs have considerably expanded our current knowledge and provided key pathobiological insights into many sarcomas, and helped identify novel therapeutic targets. The limited number of sarcoma patients in each sarcoma type and their heterogeneity pose distinct challenges in translating this knowledge into the clinic. It will be critical to prioritize these novel targets and choose those that have a broad applicability. A small group of microRNAs have conserved roles across many types of sarcomas and other cancers. Therapies that target these key microRNA-gene signaling and regulatory networks, in combination with standard of care treatment, may be the pivotal component in significantly improving treatment outcomes in patients with sarcoma or other cancers.

Sarcomas are a heterogenous group of tumors that account for ~200 000 cancers worldwide each year (~1% of all human malignant tumors); however, they represent a disproportionately high 15% of all pediatric malignant tumors.[1,2] Sarcomas comprise over 50 subtypes that can broadly be classified into bone and soft-tissue sarcomas that are generally based on the cell and/or tissue type.[3] The vast majority of sarcomas fall into the soft-tissue group, primarily affecting connective tissues such as muscle (smooth and skeletal), fat, and blood vessels. Bone sarcomas are relatively rare, representing only ~20% of all diagnosed sarcomas (~0.2% of all cancers). Even within a specific subtype, sarcomas are highly heterogenous making them diagnostically and therapeutically challenging. Several sarcoma types are genetically characterized by chromosomal translocations or DNA copy number alterations, both of which are used as diagnostic markers.[2,4,5]

The four main types of bone sarcomas are defined by their histology, cell of origin (when known), clinical features, and site distribution—osteosarcoma, Ewing’s sarcoma, chondrosarcoma, and chordoma. The most common primary bone malignancy, osteosarcoma, predominantly affects children and young adults and is characterized by undifferentiated bone-forming proliferating cells.[6] Ewing’s sarcoma, another aggressive pediatric malignancy, usually arises in growing bone and is genetically characterized by a fusion of EWS–FLI1 oncoproteins that act as gain-of-function transcriptional regulators.[7] Chondrosarcoma is itself a heterogenous group of malignant bone tumors arising from the malignant transformation of cartilage-producing cells, frequently with mutations in IDH1/2 and COL2A1.[8,9] Chordoma is an aggressive, locally invasive cancer that typically arises from bones in the base of the skull and along the spine. It is characterized, in part, by its abnormal expression of transcription factor T, which is normally only expressed during embryonic development or in the testes.[10]

Soft-tissue sarcomas are also primarily defined by their histology, cell of origin, and, in some cases, by characteristic genetic translocation events. Rhabdomyosarcoma is a malignant skeletal-muscle derived tumor comprised of two main histological subtypes, embryonal and alveolar, is one of the most common childhood soft-tissue sarcomas, accounting for 6–8% of all pediatric tumors.[11] Liposarcoma is the most common soft-tissue cancer overall, accounting for 20% of adult sarcoma cases. It originates in deep-tissue fat cells and is characterized primarily by amplification of the 12q chromosomal region.[12] Other common soft-tissue sarcomas include angiosarcomas, fibrosarcomas, gastrointestinal stromal tumors, and synovial sarcomas, each with their own unique genetic signature.

Ever since the discovery of oncogenes, the primary emphasis in cancer research has been on understanding the role of proteins and protein-coding genes. However, the percent of the genome dedicated to coding genes is small compared with noncoding regions. The last decade has seen a surge of interest in these noncoding regions with small noncoding RNAs such as microRNAs (miRNAs) gaining particular prominence. These small RNAs have critical roles in tumor formation and progression. Understanding their roles in sarcoma will lead to new therapeutic targets and diagnostic biomarkers, opening the door to a greater understanding of the molecular mechanisms of all cancers.

miRNAs are evolutionarily conserved, small, noncoding RNA molecules of 18–24 nucleotides in length at maturity that can control gene function through mRNA degradation, translational inhibition, or chromatin-based silencing mechanisms.[13] Each miRNA can potentially regulate hundreds of targets via a ‘seed’ sequence of ~5–8 nucleotides at the 5′ end of the mature miRNA. miRNAs bind to complementary sequences in the 3′-untranslated regions (3′-UTRs) of target mRNA molecules, leading to either translational repression or transcriptional degradation.[14] The short seed sequence length and relatively low stringency requirement for these miRNA–3′-UTR interactions allow a single miRNA to potentially regulate hundreds of genes.[15] Small changes in the expression level of a few miRNAs can therefore have a dramatic biological impact, particularly when dysregulated. miRNA expression profiles can be used to distinguish between closely related soft-tissue sarcoma subtypes and may provide a more consistent diagnosis than histological inspection.[16–18]

miRNAs have critical roles in the majority of canonical cellular signaling networks and their dysregulation is implicated in many cancers including breast cancer, colon cancer, gastric cancer, lung cancer, and sarcomas.[19,20] Dysregulation of miRNA expression may result from a variety of factors, including abnormal cellular stimuli, genetic mutations, epigenetic alterations, copy number variations, and chromosomal fusions. Because miRNAs act as critical regulator molecules in a variety of signaling pathways and regulatory networks, their dysregulation can be amplified across the entire signaling network.[21–24] Selected miRNAs and targets that have critical regulatory roles in sarcoma and other cancers are summarized in Table 1 .
The p53 signaling pathway is one of the most highly studied cellular signaling networks. It actively induces apoptosis in response to DNA damage and oncogene activation and is therefore a key tumor suppressor pathway.[25] Germline mutations in TP53 are strongly associated with the development of soft-tissue sarcomas, osteosarcoma, and are the underlying cause of Li–Fraumeni Syndrome, a familial clustering of early-onset tumors including sarcomas.[26,27] It is estimated that over 50% of human tumors harbor a TP53 mutation but over 80% of tumors have dysfunctional p53 signaling.[28,29] It is only within the last 10 years that researchers have started uncovering the roles of miRNAs in mediating p53’s activity and resulting pro-apoptotic signals (Figure 1). miRNA dysregulation could be a key factor in the ~30% of tumors with dysfunctional p53 signaling that lack an apparent TP53 mutation.


Figure 1.

http://img.medscape.com/article/852/145/852145-fig1.jpg

p53–miRNA interaction network. p53 interacts with the Drosha complex and promotes the processing of pri-miRNA to pre-miRNA. Although p53 directly or indirectly regulates hundreds of miRNAs, for clarity, only selected cancer-relevant miRNAs are shown. miRNAs and proteins in red are upregulated by p53. miRNAs and proteins in green are downregulated by p53. miRNAs in gray are not known to be directly regulated by p53, they are included because they target p53 regulators MDM2 and/or MDM4. miRNA, microRNA.

Like other transcription factors, p53 exerts its function primarily through transcriptional regulation of target genes that contain p53 response elements in their promoters. p53 also regulates the post-transcriptional maturation of miRNAs by interacting with the Drosha processing complex, promoting the processing of primary miRNAs to precursor miRNAs.[30] In addition to protein-coding genes, many miRNA genes also contain p53 regulatory sites in their promoter regions. Large-scale screens have revealed many different miRNAs directly regulated by p53 including miR-22-3p, miR-34a, miR-125a/b, miR-182, and miR-199a-3p.[31] Some of these miRNAs, such as miR-34a and miR-199a-3p, function themselves as tumor suppressors via the regulation of genes involved in cell cycle, cell proliferation, and even of itself.[32–34] Although some p53-targeted miRNAs form a feedback loop, translationally and transcriptionally inhibiting the TP53 gene (e.g., miR-22-3p, miR-34a, and miR-125b), others target, or are predicted to target, p53 repressors such as MDM2 and/or MDM4 (miR-199a-3p, miR-661).[31,33,35,36] It is impossible to fully understand the regulation of the p53 signaling network without considering the role of these miRNAs.

miR-34a has emerged as a critical and conserved member of the p53 signaling pathway. miR-34a is downregulated in osteosarcoma tumor samples and, in conjunction with other miRNAs, regulates p53-mediated apoptosis in human osteosarcoma cell lines.[32,33,37] The gene encoding miR-34a contains a conserved p53-binding site and is upregulated in response to cellular damage in a p53-dependent manner.[37,38] Protein-coding members of the p53 signaling pathway are well-liked targets for anticancer therapeutic development efforts and miRNAs may prove equally effective. In a preclinical model of lung cancer, therapeutic delivery of a miR-34a mimic specifically downregulated miR-34a-target genes and resulted in slower tumor growth. When combined with a siRNA targeting Kras, this small RNA combination therapy resulted in tumor regression.[39] miRNAs such as miR-34a, miR-125b, and miR-199a-3p also mediate p53’s regulation of other key signaling pathways such as the IGF-1/PI3K/AKT/mTOR signaling network. Activation of the AKT network due to downregulation of PTEN (a negative regulator of AKT) by miR-21 or miR-221 or by alternate activation of AKT is a common mechanism underlying many different types of cancer.[40–43] The induction of cell growth, migration, invasion, and metastasis resulting from the upregulation of either miR-21 or miR-221 is seen across different tumor types.[41,44–50] Dysregulation of these miRNAs is a common factor in sarcomas and other tumors. Understanding their mechanisms of action in sarcoma could lead to broadly useful cancer therapeutics.

In prospective analyses that could be models for other sarcoma studies with sufficient numbers of patient samples, Thayanithy et al[19] and Maire et al[23] each analyzed collections of osteosarcoma tissues and compared them with either normal bone or osteoblasts. They each found a set of consistently downregulated miRNAs localized to the 14q32 region.[19,23] Targeting predictions performed by Thayanithy et al[19] identified a subset of four miRNAs as potential regulators of cMYC. One of the many roles of cMYC is to promote the expression of the miR-17–92 family, a known oncogenic cluster that has been observed to be highly expressed in many cancer types including osteosarcoma, leiomyosarcoma, and alveolar rhabdomyosarcoma.[51–57] Restoring the expression of the four 14q32 miRNAs increased apoptosis of SAOS-2 cells, an effect that was attenuated either by overexpression of a cMYC construct lacking the 3′UTR or by ectopic expression of the miR-17–92 cluster.[19] Although the 14q32 region is dysregulated across many different cancer types, this pattern of dysregulation appears to be a hallmark of osteosarcoma, which is particularly interesting due to the heterogenous nature of osteosarcomas and provides an extremely attractive common therapeutic target.

One particular challenge with these types of expression profiling studies is that the cell-of-origin for a particular sarcoma subtype may not be definitely established. Another challenge is the scarcity of patient samples, particularly for the rare sarcoma subtypes. As a result, there have only been a limited number of studies designed to comprehensively profile miRNA expression in various sarcoma subtypes and to compare those expression profiles with the corresponding normal tissues or cell lines. These studies were reviewed recently in Drury et al[20] and Subramanian and Kartha.[58]

Owing to the scarcity of frozen sarcoma tissue samples, it is tempting to study sarcoma cells in vitro, using either primary or immortalized cell cultures. Studies performed in culture are less expensive and more accessible; however, the cell lines used must be chosen with care and may not truly represent the tumors. Any results derived from cultured cells must be interpreted with caution and validated in vivo when possible. A tumor cell’s microenvironment has a profound effect on gene expression and cell metabolism and culturing for even short periods of time can result in large changes in gene/miRNA expression.[59] Three-dimensional cultures can provide more physiological relevant in vitro models of individual tumors (eg, spheroid cultures) or multi-layered epithelial tissues (eg, organotypic cultures using extracellular matrix proteins, fibroblasts, and/or artificial matrix components) vs the previous standard two dimensional culture model.[60,61]

Complicating the analysis of these miRNA expression changes is the fact that many miRNAs showing differential expression in multiple different studies do not have a consistent direction of change and/or a consistent role (tumor suppressor vs tumor promoter). This likely reflects both random chance observational differences and different tissue biology reflected in different regulatory networks. Elucidation of the regulatory roles played by miRNAs in these networks in their appropriate biological contexts may provide suitable upstream targets for more effective treatment of sarcomas. Recent advances in sequencing and downstream bioinformatics techniques provide the tools to efficiently examine these questions.

For two decades, microarray gene chips containing synthetic oligonucleotides whose sequences are designed to be representative of thousands of genes have allowed researchers to perform simultaneous expression analysis of thousands of RNA transcripts in a single reaction.[62–65] Gene expression profiling has been used to characterize and classify a wide range of sarcomas, in some cases providing a diagnostic resolution more accurate than histological examination.[66–72] With the advent of high-throughput RNA-Seq, sarcoma researchers are now able to prospectively analyze the differential expression of small RNAs, such as miRNAs, without prior knowledge of their sequence.[73,74] RNA-Seq also allows for the prospective identification of novel genomic rearrangements resulting from gene fusions or premature truncations that may be of particular interest to cancer researchers.[75,76] These data are highly quantitative and digital in nature, allowing for a dynamic range that is theoretically only limited by the sequencing depth and approaches the estimated range within the cell itself.[77] Marguerat and Bähler[78] provide a basic overview of the different RNA-Seq technologies and their differences from array-based technologies.[78]

Several groups have taken advantage of these technologies to create miRNA expression profiles for a number of different sarcomas in an effort to find both common sarcoma oncomirs and to discover unique miRNA signatures that could be used in diagnosis, prognosis, and novel therapeutic development. Renner et al[18] used a microarray-based miRNA screen, followed by qRT-PCR verification, to analyze the expression of 1146 known miRNAs across a collection of 76 primary soft-tissue sarcoma samples representing eight different subtypes and across a panel of 15 sarcoma cell lines. In addition to identifying overrepresented miRNAs synovial sarcomas (miR-200 family) and liposarcomas (miR-9) compared with other sarcomas and adipose tissue, respectively, their results revealed a high degree of co-expression of 63 miRNAs clustering in the chromosomal region 14q32.[18] The most comprehensive sarcoma miRNA data set has been published by Sarver et al[79] who profiled miRNA expression in over 300 sarcoma primary tumor tissue samples representing 22 different sarcoma types. These data form the basis for the web-accessible comprehensive Sarcoma microRNA Expression Database (SMED) database, which has tools that allows users to query specific sarcoma types and/or specific miRNAs.[79]

Integrative miRNA–mRNA analysis using a tool such as Ingenuity Pathway Analysis (Qiagen) or GeneSpring (Agilent) allows for more biologically relevant results by highlighting miRNA–mRNA pairs that are linked not only by predicted targeting interactions but whose expression levels are inversely correlated (i.e., as miRNA expression increases one would expect the target mRNA levels to decrease). For example, out of 177 differentially expressed miRNAs in osteosarcoma cell lines vs normal bone, an integrated miRNA–mRNA analysis highlighted two particularly interesting miRNA/mRNA pairs (miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K) that were dysregulated.[44]

It is important to note that the general consensus is that there is often no single ‘correct’ method to analyze miRNA expression data. Different experimental and bioinformatics techniques may reveal different aspects in the data that can be further investigated and experimentally validated. All of these experiments, whether performed at the bench or systems biology, contribute to our greater understanding of sarcoma biology and the central role of dysregulated miRNA–gene networks as drivers of tumor formation and progression.

miRNAs are part of a larger family of noncoding RNAs including long noncoding RNAs (lncRNAs) and competing endogenous RNAs (ceRNAs) that deserve to be evaluated for therapeutic potential in sarcomas with broader applicability to other cancer types. Just like miRNAs, lncRNAs are widely expressed in tissue-specific patterns that are highly disrupted in cancer.[80] As their name implies, ceRNAs compete for their common miRNA targets and influence their expression, which has an indirect effect on the protein-coding genes, such as PTEN, regulated by those miRNAs.[81,82] We have just begun to unravel the role of lncRNAs and ceRNAs in cancer development and progression but recent results hint at yet another layer of complexity and genetic control in tumor biology.

The lessons learned from carcinomas, leukemias, and lymphomas will be helpful in understanding the pathobiology of sarcomas and the insights gained from sarcoma biology may form the foundation for therapeutics to treat a wide range of other cancers. Recent studies have shown miRNAs are very stable in blood serum and plasma, and extensive efforts are underway to develop circulating miRNA-based diagnostic and prognostic markers. Major technical challenges in developing circulating miRNA-based markers still need to be addressed, including standardization of pre-analytical, analytical, and post-analytical methods for effective reproducibility. For example, miR-16, which is used in the normalization of miRNA expression in serum/plasma is also found in red blood cells; thus, any hemolysis during sample collection could significantly affect the downstream expression data analysis.

Cancers do not exist in isolation inside the body and extensive research has been performed on how tumor-derived proteins adapt their microenvironment to provide more favorable conditions for tumor growth and development. Recent studies have shown that miRNAs also have a major role in modulating tumor microenvironment. Although most miRNAs are found inside the cell, a significant number of miRNAs are encapsulated in exosomes that can be used as a delivery system to send miRNAs from one cell to another, allowing tumor cells to modulate gene expression in surrounding tissues.[83,84] Exosome and miRNA-mediated cross talk between sarcoma tumor cells and the surrounding stromal cells is a new and exciting avenue of research and the potential for novel therapeutics is high.

Sarcomas are a diverse collection of rare cancers with proportionally limited resources for research and development of novel treatments. It is therefore crucial that potential therapeutic targets are prioritized and novel therapeutic agents carefully selected for clinical trials to succeed. Extensive studies in preclinical models will be required; however, there are also challenges in the development of appropriate in vitro and in vivo model systems that accurately reflect the different sarcoma types. Sarcomas, such as osteosarcoma, leiomyosarcoma, and angiosarcoma are very heterogeneous in nature, making it unlikely that therapies targeting specific genomic mutations will be successful. Even if specific targets were to be identified it would still be a challenge to develop clinical trials based on the small number of patients harboring those specific mutations. Coordinated efforts such as the Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/) and its associated preclinical and clinical trial consortiums will help unravel novel miRNA–mRNA interactions and their significance as potential therapeutic targets.

Targeting common miRNA–gene oncogenic or tumor suppressor networks goes after the common denominator underlying many of these cancers. Key regulatory molecules in sarcoma are highly likely to have similar roles in leukemias and lymphomas, for instance, and vice versa. For example, oncogenic activation of STAT3 strongly promotes the expression of miR-135b in lymphoma, resulting in increased angiogenesis and tumor growth.[85] miR-135b is widely overexpressed in sarcomas and STAT3 may be having a similar transcriptional regulatory role, indicating that STAT3 inhibitors could be an effective supplemental therapy in sarcomas.[86] Interestingly, p53 promotes the transcription of miR-125b, which can directly target both STAT3 and p53 transcription. This finely balanced regulatory network is frequently dysregulated in osteosarcoma and Ewing’s sarcoma.[87,88] In retinoblastoma, STAT3 activation is associated with upregulation of the miR-17-92 cluster via a positive feedback loop and inhibition of STAT3-suppressed retinoblastoma proliferation, providing further evidence that STAT3 may be an attractive therapeutic target in many cancers.[89] The dysregulation of key signaling molecules such as the p53 and STAT3 along with their associated signaling networks are a common feature across most cancer types implying that advances in understanding of sarcoma biology may be highly impactful in more frequently occurring solid tumors and lymphomas.

Certain miRNAs appear to be common players across many types of sarcomas and other cancers and their dysregulation contributes to the development of the hallmarks of cancer (Figure 2). miR-210, a key modulator of many downstream pathways involved in the hypoxic response, is upregulated under hypoxic conditions in most solid tumors, including soft-tissue sarcomas, osteosarcoma, renal cancer, and breast cancer.[90] A recent meta-analysis demonstrated that the elevated expression of miR-210 is a prognostic indicator for disease-free, progression-free, and relapse-free survival in a variety of cancer patients.[91] Perhaps the most consistently upregulated miRNA across all tumor types is the anti-apoptotic miR-21, which directly targets the tumor suppressor PDCD4.[92] Levels of miR-21 correlate with cancer progression and patient prognosis.[93]
Figure 2.

http://img.medscape.com/article/852/145/852145-thumb2.png

Conserved miRNA-tumor suppressor signaling networks in cancer. These miRNAs and tumor suppressors are involved in other network and signaling pathway interactions, such as the p53 signaling network; this figure highlights selected critical conserved pathways.

 

Human Papillomavirus Oncogenic mRNA Testing for Cervical Cancer Screening

Jennifer L. Reid, PhD; Thomas C. Wright Jr, MD; Mark H. Stoler, MD; Jack Cuzick, PhD; Philip E. Castle, PhD; Janel Dockter; Damon Getman, PhD; Cristina Giachetti, PhD

Am J Clin Pathol. 2015;144(3):473-483.

http://www.medscape.com/viewarticle/850740

 

Objectives: This study determined the longitudinal clinical performance of a high-risk human papillomavirus (HR-HPV) E6/E7 RNA assay (Aptima HPV [AHPV]; Hologic, San Diego, CA) compared with an HR-HPV DNA assay (Hybrid Capture 2 [HC2]; Qiagen, Gaithersburg, MD) as an adjunctive method for cervical cancer screening.

Methods: Women 30 years or older with a negative result for intraepithelial lesions or malignancy cytology (n = 10,860) positive by AHPV and/or HC2 assays and randomly selected women negative by both assays were referred to colposcopy at baseline. Women without baseline cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+) continued into the 3-year follow-up.

Results: The specificity of AHPV for CIN2 or lower was significantly greater at 96.3% compared with HC2 specificity of 94.8% (P < .001). Estimated sensitivities and risks for detection of CIN2+ were similar between the two assays. After 3 years of follow-up, women negative by either human papillomavirus test had a very low risk of CIN2+ (<0.3%) compared with CIN2+ risk in women with positive AHPV results (6.3%) or positive HC2 results (5.1%).

Conclusions: These results support the use of AHPV as a safe and effective adjunctive cervical cancer screening method.

Introduction

Cervical cancer is one of the most frequent cancers in women worldwide, accounting for approximately 530,000 new cases and 275,000 deaths annually.[1] Countries with well-organized screening programs using conventional Papanicolaou (Pap) stain cytology have experienced substantially reduced mortality from the disease in the past 5 decades.[2–4] Despite this advance, the relatively low sensitivity and reproducibility of both conventional Pap smear and liquid-based cytology screening methods have prompted investigation into identifying adjunctive methods with Pap cytology for improving detection of cervical neoplasia.[5–9]

Infection with 14 high-risk human papillomavirus (HR-HPV) genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) is associated with almost all cases of cervical precancer, defined as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2), grade 3 (CIN3), and cancer.[10] Addition of HR-HPV nucleic acid testing to a cervical cytology screening regimen offers higher sensitivity and negative predictive value (NPV) for detection of cervical precancer and cancer compared with cytology alone, especially in older women.[11–15] For this reason, HR-HPV nucleic acid testing is recommended as an adjunctive test to cytology to assess the presence of HR-HPV types in women 30 years of age or older.[16] In this context, HR-HPV testing guides patient management by identifying women at elevated risk for CIN2 or higher (CIN2+) but, importantly, also reassures women who are negative for HR-HPV of their extremely low cancer risk.[17–19]
First-generation HR-HPV molecular tests used for adjunctive cervical cancer screening function by detecting viral genomic DNA in cellular samples from the uterine cervix. However, because the presence of HR-HPV in the female genital tract is common and often transient in nature,[20,21] and most cervical HPV infections resolve without becoming cancerous,[22,23] HR-HPV DNA-based test methods yield only moderate specificity for detection of high-grade cervical disease.[12,24] This leads to unnecessary follow-up and referral of patients to colposcopy, increasing the physical and emotional burdens on patients and elevating health care costs.

A test approved by the US Food and Drug Administration (FDA) for detection of HR-HPV E6/E7 messenger RNA (mRNA) (Aptima HPV [AHPV]; Hologic, San Diego, CA) has shown higher specificity with similar sensitivity for detection of CIN2+ compared with HPV DNA-based tests in patients referred for colposcopy due to an abnormal Pap smear result as well as in a screening setting.[25–30] Expression of mRNA from viral E6 and E7 oncogenes is highly associated with the development of CIN,[31,32] and extensive investigation into the role of E6 and E7 oncoproteins in the human papillomavirus (HPV) life cycle has revealed that the expression of the corresponding oncogenes is necessary and sufficient for cell immortalization, neoplastic transformation, and the development of invasive cancer.[33–35]

To confirm and extend the previous evidence on the clinical utility of HR-HPV oncogenic mRNA testing in a US population-based setting, the clinical performance of AHPV was evaluated as an adjunctive method for cervical cancer screening in women aged 30 years or older with negative for intraepithelial lesions or malignancy (NILM) cytology results from routine Pap testing in a pivotal, prospective, multicenter US clinical study including 3 years of follow-up (the Clinical Evaluation of Aptima mRNA [CLEAR] study). We report herein the results from this study.

1 of 4

Figure 1.

Clinical evaluation of Aptima mRNA study participant disposition. aReasons for withdrawal: did not meet eligibility criteria (70); Pap volume insufficient for AHPV testing (117); specimen expired or unsuitable for testing (190); specimen lost (58); noncompliant site (320); other reasons (26). bReasons for withdrawal: collection site did not participate in follow-up (243); subject terminated participation (37); participant had hysterectomy (22); participant not eligible (17); participant treated prior to CIN2+ diagnosis (8); other reasons (4). AHPV, Aptima HPV (Hologic, San Diego, CA); ASC-US, atypical squamous cells of unknown significance; CIN2+, cervical intraepithelial neoplasia grade 2 or higher; HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD); HPV, human papillomavirus; NILM, negative for intraepithelial lesions or malignancy; Pap, Papanicolaou test.

HPV Testing

Baseline PreservCyt specimens (1-mL aliquot) were tested with the AHPV (Hologic) on both the automated Tigris DTS System and Panther System. Results from the two systems were similar; Panther System results are presented here. AHPV is a target amplification assay that uses transcription-mediated amplification to detect the E6/E7 oncogene mRNA of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).

 

HPV and Disease Prevalence

Cervical disease and HPV status are shown in Table 2 for the baseline evaluation and cumulatively after 3 years of follow-up. Of the 10,860 evaluable participants with NILM cytology at baseline, 512 were positive for AHPV, yielding a prevalence of 4.7% for HR-HPV E6/E7 oncogenic mRNA, whereas prevalence of HR-HPV DNA was 6.5% among 10,229 women with HC2 results. A total of 845 HPV RNA-positive or DNA-positive women and 556 randomly selected HPV-negative women were referred to colposcopy at baseline (Figure 1).

At baseline, the percentage of colposcopy attendance was similar between HPV-positive (62%, n = 526) and randomly selected HPV-negative (61%, n = 339) women with 29 cases of CIN1, nine cases of CIN2, eight cases of CIN3, and three cases of adenocarcinoma in situ (AIS) identified (Table 2). Four of the CIN2 cases and two of the AIS cases were identified based on an ECC biopsy specimen only.

In total, 6,271 women completed the 3-year follow-up with a known disease status (Table 2). Of these, 6,098 (97.2%) women had normal (negative) disease status, and 56 (0.9%) had low-grade lesions (CIN1). In addition to the 20 women with CIN2+ identified at baseline, 15 (0.2%) women had CIN2 and 12 (0.2%) women had CIN3 identified during follow-up, with two cases identified from an ECC biopsy specimen only.

Of the 27 women with CIN2+ identified during follow-up, two had CIN1 at baseline, with CIN3 identified during year 1. Ten women had no disease found at baseline, with five cases of CIN2+ identified during year 1, one case of CIN2+ identified during year 2, and four cases of CIN2+ identified during year 3. The remaining 15 women with CIN2+ identified during follow-up did not have a baseline colposcopy; among them, two cases of CIN2+ were identified during year 1, six cases of CIN2+ during year 2, and seven cases of CIN2+ during year 3.

AHPV Assay Performance

Baseline risk and prevalence estimates adjusted for verification bias are provided in Table 3. The prevalence of CIN2+ was 0.9% in the overall population. CIN2+ occurred in 4.5% (95% CI, 2.7%-7.4%) of women with positive AHPV results and in 0.6% (95% CI, 0.2%–1.9%) of women with negative AHPV results, yielding a relative risk of 7.5 (95% CI, 2.1–26.3). This indicates that women with a positive AHPV result are at significantly greater risk of CIN2+ than women with a negative AHPV result. The CIN2+ relative risk obtained for the HC2 test at baseline was similar (7.3; 95% CI, 1.6–33.5). For CIN3+ diagnosis, the overall prevalence was 0.4%. The AHPV relative risk was 24.9 (95% CI, 2.0–307.0), again with a similar relative risk for HC2 (21.0; 95% CI, 1.0–423.8).

Cumulative absolute and relative risks for AHPV and HC2 over the 3-year follow-up period for HPV-positive and HPV-negative women are shown in Table 4. Women with an HPV-negative result with either test had very low cervical disease risk after 3 years of follow-up (<0.3%). Comparatively, 5% to 6% of women with an HPV-positive result had CIN2+ and 3% to 4% had CIN3+, with overall cumulative absolute and relative risks slightly higher for the AHPV assay than for HC2. Younger women aged 30 to 39 years who were HPV positive had twice the prevalence of disease but a similar increase in relative risk of cervical disease compared with HPV-positive women 40 years and older (Table 4). Risk of cervical disease in HPV-negative women did not vary by age group.

Figure 2 and Figure 3 show the cumulative absolute risk of CIN2+ and CIN3+, respectively, by year according to AHPV or HC2 positivity status at baseline. Both assays show a similar trend, with consistent slightly higher risk for the AHPV assay each year.

Figure 2.

Cumulative absolute risk of cervical intraepithelial neoplasia grade 2 or higher (CIN2+) by year. AHPV, Aptima HPV (Hologic, San Diego, CA); HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD).

Figure 3.

Cumulative absolute risk of cervical intraepithelial neoplasia grade 3 or higher (CIN3+) by year. AHPV, Aptima HPV (Hologic, San Diego, CA); HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD).

After 3 years of follow-up, the specificity of AHPV for CIN2 or lower was 96.3% (95% CI, 95.8%-96.7%), significantly greater (P < .001) compared with HC2 specificity of 94.8% (95% CI, 94.3%-95.4%) Table 5. AHPV specificity for CIN3 or lower (96.2%; 95% CI, 95.5%–96.5%) was also significantly greater (P < .001) than HC2 specificity (94.7%; 95% CI, 94.1%-95.2%). Estimated sensitivities for detection of CIN2+ and CIN3+ were similar between the two assays (P = .219 and P = 1.0, respectively). For detection of CIN2+, AHPV sensitivity was 55.3% (95% CI, 41.2%-68.6%), and HC2 sensitivity was 63.6% (95% CI, 48.9%-76.2%). For CIN3+ detection, AHPV sensitivity was 78.3% (95% CI, 58.1%-90.3%), and HC2 sensitivity was 81.8% (95% CI, 61.5%-92.7%) (Table 5).

Discussion

This study presents the results of a 3-year longitudinal evaluation of the AHPV assay as an adjunctive method for screening women 30 years and older who have NILM Pap cytology results. Consistent with previously published data,[28,29] these results demonstrate that HR-HPV oncogenic E6/E7 mRNA testing has a sensitivity similar to an HR-HPV DNA-based test for detection of CIN2+ and CIN3+ and slightly, but significantly, improved specificity compared with HR-HPV DNA testing for both end points. We found that use of AHPV as an adjunctive method for HPV-induced cervical disease screening provided disease detection capability similar to HC2 while reducing the false-positive rate (from 5.2% to 3.7%) relative to the HPV DNA-based test. Reduction of HPV detection in women without cervical disease minimizes the anxiety and burden associated with spurious positive HPV molecular test results in women with NILM cytology, decreases health care costs, and reduces unnecessary follow-up procedures, thereby improving the safety of cervical cancer screening (unnecessary colposcopy is considered a significant “harm” in the recent American Cancer Society guidelines[16]).

Importantly, we show that women with a NILM cytology result who also had a positive AHPV result are approximately 24 times more likely to have CIN2+ disease after 3 years than women with a negative AHPV result. This risk increased to approximately 68-fold for detection of CIN3+ disease. Similar but slightly lower risk estimates were obtained with HC2, demonstrating comparable accuracy of the AHPV and HC2 for identifying participants with CIN2+ and CIN3+ in this respect.

After 3 years of follow-up, women in this study who were HPV negative at baseline using any test method had very low risk for CIN2+ (<0.3%), a result similar to previously published studies with HC2.[42,43] These findings reinforce evidence from previous studies showing that HR-HPV nucleic acid testing should be performed as an adjunctive test to routine Pap for cervical cancer screening of women 30 years or older to increase sensitivity of disease detection.[28] Correspondingly, compared with annual cytology-only screening, this study supports longer screening intervals for women negative for both abnormal cytology and HPV E6/E7 mRNA, due to the high NPV and low risk of disease afforded by this screening algorithm for 3 years following a test-negative baseline visit. Extension of cervical cancer screening intervals following negative HPV and cytology test results in women 30 years or older is a key recommendation of current US screening guidelines from both the American Cancer Society and the US Preventive Services Task Force.[16]

Conversely, since the positive predictive value of any HPV test in women with NILM cytology is low, additional AHPV testing to detect persistent HR-HPV infection during follow-up care in women with an initial AHPV-positive result is likely a better option than direct referral to colposcopy. Alternatively, genotyping with referral for HPV 16– or HPV 18–positive women can optimize referral and minimize loss to follow-up.[44]

Read Full Post »

Imaging-guided cancer treatment


Imaging-guided cancer treatment

Writer & reporter: Dror Nir, PhD

It is estimated that the medical imaging market will exceed $30 billion in 2014 (FierceMedicalImaging). To put this amount in perspective; the global pharmaceutical market size for the same year is expected to be ~$1 trillion (IMS) while the global health care spending as a percentage of Gross Domestic Product (GDP) will average 10.5% globally in 2014 (Deloitte); it will reach ~$3 trillion in the USA.

Recent technology-advances, mainly miniaturization and improvement in electronic-processing components is driving increased introduction of innovative medical-imaging devices into critical nodes of major-diseases’ management pathways. Consequently, in contrast to it’s very small contribution to global health costs, medical imaging bears outstanding potential to reduce the future growth in spending on major segments in this market mainly: Drugs development and regulation (e.g. companion diagnostics and imaging surrogate markers); Disease management (e.g. non-invasive diagnosis, guided treatment and non-invasive follow-ups); and Monitoring aging-population (e.g. Imaging-based domestic sensors).

In; The Role of Medical Imaging in Personalized Medicine I discussed in length the role medical imaging assumes in drugs development.  Integrating imaging into drug development processes, specifically at the early stages of drug discovery, as well as for monitoring drug delivery and the response of targeted processes to the therapy is a growing trend. A nice (and short) review highlighting the processes, opportunities, and challenges of medical imaging in new drug development is: Medical imaging in new drug clinical development.

The following is dedicated to the role of imaging in guiding treatment.

Precise treatment is a major pillar of modern medicine. An important aspect to enable accurate administration of treatment is complementing the accurate identification of the organ location that needs to be treated with a system and methods that ensure application of treatment only, or mainly to, that location. Imaging is off-course, a major component in such composite systems. Amongst the available solution, functional-imaging modalities are gaining traction. Specifically, molecular imaging (e.g. PET, MRS) allows the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In oncology, it can be used to depict the abnormal molecules as well as the aberrant interactions of altered molecules on which cancers depend. Being able to detect such fundamental finger-prints of cancer is key to improved matching between drugs-based treatment and disease. Moreover, imaging-based quantified monitoring of changes in tumor metabolism and its microenvironment could provide real-time non-invasive tool to predict the evolution and progression of primary tumors, as well as the development of tumor metastases.

A recent review-paper: Image-guided interventional therapy for cancer with radiotherapeutic nanoparticles nicely illustrates the role of imaging in treatment guidance through a comprehensive discussion of; Image-guided radiotherapeutic using intravenous nanoparticles for the delivery of localized radiation to solid cancer tumors.

 Graphical abstract

 Abstract

One of the major limitations of current cancer therapy is the inability to deliver tumoricidal agents throughout the entire tumor mass using traditional intravenous administration. Nanoparticles carrying beta-emitting therapeutic radionuclides [DN: radioactive isotops that emits electrons as part of the decay process a list of β-emitting radionuclides used in radiotherapeutic nanoparticle preparation is given in table1 of this paper.) that are delivered using advanced image-guidance have significant potential to improve solid tumor therapy. The use of image-guidance in combination with nanoparticle carriers can improve the delivery of localized radiation to tumors. Nanoparticles labeled with certain beta-emitting radionuclides are intrinsically theranostic agents that can provide information regarding distribution and regional dosimetry within the tumor and the body. Image-guided thermal therapy results in increased uptake of intravenous nanoparticles within tumors, improving therapy. In addition, nanoparticles are ideal carriers for direct intratumoral infusion of beta-emitting radionuclides by convection enhanced delivery, permitting the delivery of localized therapeutic radiation without the requirement of the radionuclide exiting from the nanoparticle. With this approach, very high doses of radiation can be delivered to solid tumors while sparing normal organs. Recent technological developments in image-guidance, convection enhanced delivery and newly developed nanoparticles carrying beta-emitting radionuclides will be reviewed. Examples will be shown describing how this new approach has promise for the treatment of brain, head and neck, and other types of solid tumors.

The challenges this review discusses

  • intravenously administered drugs are inhibited in their intratumoral penetration by high interstitial pressures which prevent diffusion of drugs from the blood circulation into the tumor tissue [1–5].
  • relatively rapid clearance of intravenously administered drugs from the blood circulation by kidneys and liver.
  • drugs that do reach the solid tumor by diffusion are inhomogeneously distributed at the micro-scale – This cannot be overcome by simply administering larger systemic doses as toxicity to normal organs is generally the dose limiting factor.
  • even nanoparticulate drugs have poor penetration from the vascular compartment into the tumor and the nanoparticles that do penetrate are most often heterogeneously distributed

How imaging could mitigate the above mentioned challenges

  • The inclusion of an imaging probe during drug development can aid in determining the clearance kinetics and tissue distribution of the drug non-invasively. Such probe can also be used to determine the likelihood of the drug reaching the tumor and to what extent.

Note: Drugs that have increased accumulation within the targeted site are likely to be more effective as compared with others. In that respect, Nanoparticle-based drugs have an additional advantage over free drugs with their potential to be multifunctional carriers capable of carrying both therapeutic and diagnostic imaging probes (theranostic) in the same nanocarrier. These multifunctional nanoparticles can serve as theranostic agents and facilitate personalized treatment planning.

  • Imaging can also be used for localization of the tumor to improve the placement of a catheter or external device within tumors to cause cell death through thermal ablation or oxidative stress secondary to reactive oxygen species.

See the example of Vintfolide in The Role of Medical Imaging in Personalized Medicine

vinta

Note: Image guided thermal ablation methods include radiofrequency (RF) ablation, microwave ablation or high intensity focused ultrasound (HIFU). Photodynamic therapy methods using external light devices to activate photosensitizing agents can also be used to treat superficial tumors or deeper tumors when used with endoscopic catheters.

  • Quality control during and post treatment

For example: The use of high intensity focused ultrasound (HIFU) combined with nanoparticle therapeutics: HIFU is applied to improve drug delivery and to trigger drug release from nanoparticles. Gas-bubbles are playing the role of the drug’s nano-carrier. These are used both to increase the drug transport into the cell and as ultrasound-imaging contrast material. The ultrasound is also used for processes of drug-release and ablation.

 HIFU

Additional example; Multifunctional nanoparticles for tracking CED (convection enhanced delivery)  distribution within tumors: Nanoparticle that could serve as a carrier not only for the therapeutic radionuclides but simultaneously also for a therapeutic drug and 4 different types of imaging contrast agents including an MRI contrast agent, PET and SPECT nuclear diagnostic imaging agents and optical contrast agents as shown below. The ability to perform multiple types of imaging on the same nanoparticles will allow studies investigating the distribution and retention of nanoparticles initially in vivo using non-invasive imaging and later at the histological level using optical imaging.

 multi

Conclusions

Image-guided radiotherapeutic nanoparticles have significant potential for solid tumor cancer therapy. The current success of this therapy in animals is most likely due to the improved accumulation, retention and dispersion of nanoparticles within solid tumor following image-guided therapies as well as the micro-field of the β-particle which reduces the requirement of perfectly homogeneous tumor coverage. It is also possible that the intratumoral distribution of nanoparticles may benefit from their uptake by intratumoral macrophages although more research is required to determine the importance of this aspect of intratumoral radionuclide nanoparticle therapy. This new approach to cancer therapy is a fertile ground for many new technological developments as well as for new understandings in the basic biology of cancer therapy. The clinical success of this approach will depend on progress in many areas of interdisciplinary research including imaging technology, nanoparticle technology, computer and robot assisted image-guided application of therapies, radiation physics and oncology. Close collaboration of a wide variety of scientists and physicians including chemists, nanotechnologists, drug delivery experts, radiation physicists, robotics and software experts, toxicologists, surgeons, imaging physicians, and oncologists will best facilitate the implementation of this novel approach to the treatment of cancer in the clinical environment. Image-guided nanoparticle therapies including those with β-emission radionuclide nanoparticles have excellent promise to significantly impact clinical cancer therapy and advance the field of drug delivery.

Read Full Post »

Older Posts »