Dennis Bittner, PhD

The US Food and Drug Administration (FDA) released new guidance on the use of alternative endpoints, in addition to overall survival (OS), for clinical trials of nonsmall cell lung cancer (NSCLC) therapies.

Pharmaceutical companies will be allowed to use several types of clinical trial endpoints to demonstrate efficacy of drugs intended to treat NSCLC.

In 2003, the FDA announced that surrogate endpoints such as progression-free survival (PFS), time-to-progression (TTP), and objective response rate (ORR) could be used to gain special approval. In its April 2015 clarification of that guidance, the FDA noted, &ldquo;Based on these recommendations from the 2003 advisory committee meeting, we have continued to recommend OS as the primary endpoint for NSCLC clinical trials. However, a clinical trial demonstrating a large improvement in PFS, with acceptable toxicity, could potentially lead to regular approval, particularly in an unmet medical need population.&rdquo;

The FDA announcement also included guidance on patient-reported outcome (PRO) measures, stating that this measure could provide direct evidence of treatment. The guidance encouraged the development of &ldquo;well-defined and reliable PRO instruments that capture the essential treatment benefit concepts in the targeted population,&rdquo; noted the FDA announcement. It added that effective interpretation of PRO data requires a complete record of all potentially confounding concomitant medications that might impact anticancer effects.

&ldquo;This is a welcome announcement. PFS has been used successfully as a primary endpoint in breast cancer for drug approval because of the number of available therapies there. The pace of development for lung cancer drugs has increased to a point where this makes sense for NSCLC, too,&rdquo; said James P. Stevenson, MD, of the Taussig Cancer Institute at the Cleveland Clinic in Ohio, in an interview with Targeted Oncology. The guidelines for approval based on extending PFS an additional 2 to 3 months at statistical significance are reasonable, according to Stevenson. The inclusion of endpoints for PRO measures in the FDA&rsquo;s announcement will be advantageous in helping select treatments for his patients based on potential toxicity, Stevenson stated. &ldquo;The more options, the better,&rdquo; he said, adding that validated quality of life (QOL) measurement tools are already available to apply to such data.

Commenting on the announcement, Chicago-area radiation oncologist Kamal Patel, MD, explained, &ldquo;OS will always be considered a gold standard measure as an endpoint. However, there are some problems with looking at OS; it does not take into account the patient&rsquo;s QOL and it requires a large number of patients and longer follow-up than other endpoints. With a big demand to get drugs to market ASAP, studies are shifting to other endpoints such as PFS, TTP, and ORR. Although PFS data can be evaluated much earlier, thus allowing drugs to come to market sooner, PFS and similar metrics may be subject to measurement error, subjectivity of measurement, or a predisposition to different types of biases. Knowing the advantages, disadvantages, and limitations of each endpoint is key in trial design.&rdquo;

Helping to clarify trends in NSCLC studies, Adrian G. Sacher, MD, and colleagues at the University of Toronto examined more than 200 phase III NSCLC clinical trials performed in the 3 decades between 1980 and 2010 in an article published last year.

Although OS remained the most common primary endpoint for these trials, the portion of trials using PFS in place of OS increased significantly, to 13% of the total in the last decade of the study. An increase in the percentage of trials reporting a positive outcome without meeting their primary endpoint also increased significantly in the last decade, to more than 50%. Other trends identified included a decreasing magnitude of survival gain in positive trials, leading the researchers to suggest that their findings raised questions about the design and interpretation of phase III trials in NSCLC.

In an accompanying editorial, David Johnson, MD, chair of Internal Medicine at University of Texas Southwestern Medical Center, Dallas, wrote that increased use of PFS in the most recent NSCLC trials was likely due to the availability of multiple agents and the 2003 guidance permitting PFS as a surrogate endpoint.

However, Johnson said that more worrisome trends are the increase in trials declared positive despite failure to meet a primary statistical endpoint and a tendency to attach clinically meaningful value to progressively lower survival advantages. &ldquo;Unabated, such a trend could lead to an ever increasing number of trials achieving so-called statistically significant results that turn out to be clinically irrelevant and costly,&rdquo; said Johnson.

Howard L.(Jack) West, MD, Swedish General Hospital, Seattle, Washington, countered Johnson&rsquo;s editorial, saying that it implied that this relaxation of standards was &ldquo;regrettable.&rdquo;

West cited trials that could not report OS because patients were surviving so long without progression, and that the improvement in survival was &ldquo;self-obvious.&rdquo; West said that the results of a single-arm, phase I/II trial of crizotinib in ALK-positive patients revealed a median PFS of 8 months to 10 months, about twice that expected from chemotherapy, yet the OS evidence for crizotinib&rsquo;s superiority was not available until years later. West asked rhetorically how many patients with ALK-positive mutations would have been denied a clearly beneficial therapy during the additional 3 years needed to reach a randomized trial data that he said would be &ldquo;&hellip;the equivalent of running a trial to confirm the value of parachutes for people falling out of an airplane.&rdquo; However, West emphasized the importance of the difference in magnitude of PFS improvements, between those offering only 1 to 2 months&rsquo; difference in PFS and those that &ldquo;profoundly change the trajectory of the disease and are much more likely to track with a clinically meaningful OS difference.&rdquo; West believes in the benefit of balancing treatment costs. He thinks that while patients might argue the value of a few extra weeks before being told that their scan looks worse, there needs to be compelling value for cancer drugs, &ldquo;when everyone ends up sharing the cost burden by paying taxes or rising insurance premiums.&rdquo;

Sacher, the lead author of the 2014 study, currently at Dana-Farber Cancer Institute, Boston, offered his view on the FDA&rsquo;s announcement. &ldquo;Any new cancer treatment is only useful if it helps patients live longer or live betterideally both,&rdquo; he said. &ldquo;This concept has been distilled into the clinically meaningful endpoints of OS and QOL.&rdquo; While acknowledging that the convenience of surrogate endpoints such as PFS and ORR allow clinical trials to be completed more quickly, expediting the development of effective therapies for aggressive malignancies, Sacher also warned that such endpoints are potentially subjective and need to be interpreted in the context of each clinical trial. He added, &ldquo;A strong PFS benefit from a new treatment increases the likelihood that this treatment may also improve OS and/or QOL, but PFS benefit alone is not itself a clinically meaningful endpoint.&rdquo;

Both Johnson and Sacher endorsed recommendations

restricting regulatory approval based on PFS to treatments considered &ldquo;paradigm changing&rdquo; for a difficult cancer like NSCLC, defined as those that would be expected to double PFS and result in at least a 50% increment in median or 2-year survival rates, at a minimum.

1. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory-Information/Guidances/UCM259421.pdf. Accessed May 1, 2015.

2. Sacher AG, Le LW, Leighl NB. Shifting patterns in the interpretation of phase III clinical trial outcomes in advanced nonsmall-cell lung cancer: The bar is dropping.

3. Johnson DH. Setting the bar for therapeutic trials in non-small-cell lung cancer: how low can we go?

4. http://cancergrace.org/lung/2015/04/25/pfs-debatable-endpoint-in-lc/#more-15935. Accessed May 6, 2015.

5. Sobrero A, Bruzzi P. Incremental advance or seismic shift? The need to raise the bar of efficacy for drug approval.

Articles

The US Food and Drug Administration (FDA) released new guidance on the use of alternative endpoints, in addition to overall survival (OS), for clinical trials of nonâ€“small cell lung cancer (NSCLC) therapies.

More Endpoints Not Necessarily Merrier in NSCLC Drug Trials

Following recent approvals by the US Food and Drug Administration (FDA) of pembrolizumab and nivolumab,

numerous programs to develop and expand use of immunotherapies have ensued. Below is an overview of some of these programs.

announced a clinical trial collaboration on March 4, 2015, to evaluate Merck&rsquo;s programmed death 1 (PD-1) inhibitor pembrolizumab in combination with several of Eisai&rsquo;s lead compounds for oncology, including the microtubule inhibitor eribulin for triple-negative breast cancer and the multi-receptor tyrosine kinase (RTK) inhibitor lenvatinib for select solid tumors.

| A programmed death ligand 1 (PD-L1) inhibitor, MEDI4736, is in phase III trials for non-small cell lung cancer (NSCLC).

Another PD-L1 inhibitor, MPDL3280A, achieved consecutive breakthrough status designations from the FDA for treatment of bladder cancer in mid-2014, then for treatment of NSCLC on February 2, 2015.

After successful combination trials of talimogene laherparepvec (TVEC), its oncolytic viral immunotherapy treatment for melanoma, with the cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor ipilimumab, Amgen is now engaged in trials combining TVEC with the PD-1 inhibitor pembrolizumab.

Amgen. Blinatumomab, an immunotherapy monoclonal targeting the CD19 antigen for treatment of acute lymphoblastic leukemia (ALL), received accelerated FDA approval in December 2014.

|The Seattle startup, after initiating several clinical trials within just a year of launching the company, raised $265 million in its December 2014 IPO, less than a month after the FDA granted breakthrough status to JCAR015, one of Juno&rsquo;s chimeric antigen receptor (CAR) therapies. In Security and Exchange Commission filings, Juno indicated that FDA approval could come as soon as 2017. Juno has 5 ongoing phase I trials testing CAR agents as treatment for ALL, non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL). Almost all of the therapies involve CARs targeting CD19, a hematologic cancer antigen, although one trial involves modified T-cell receptors (TCRs) to treat acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and NSCLC. The IPO also came shortly after Juno made a deal with National Cancer Institute (NCI) partnered with Opus Bio to license a CAR targeting CD22,

a protein common in lymphomas. This transaction immediately added a CAR clinical candidate to Juno&rsquo;s portfolio, since NCI has already begun enrollment in a phase I study of ALL and NHL using the CD22 CAR.

| Another notable mover in the CAR arena is Kite Pharma in Santa Monica, California. Kite&rsquo;s current pipeline has CARs recognizing CD19, with one candidate already in phase II/III study for B-cell malignancies and another in phase I for NHL. A Kite CAR against epidermal growth factor receptor variant III (EGFRvIII) is in phase I trials for treatment of glioblastoma. Last June, Kite obtained an exclusive, worldwide license from NCI as part of a Cooperative Research and Development Agreement (CRADA) for TCRs developed against NY-ESO-1, an antigen expressed in numerous tumor types. NCI is in phase II clinical trials with such a TCR, and Kite has 3 more TCRs via the CRADA in phase I trials on various tumors. Kite signed a deal with Amgen on January 5, 2015, to develop CAR therapies for Amgen cancer targets on Kite&rsquo;s platform, with Kite receiving $60 million up front and a potential $525 million in milestones, plus funding for R&D costs.

of Cambridge, Massachusetts, is also a player in the CAR space, although much of its pipeline involves gene therapy. Bluebird is entering the third year of a collaboration with Celgene to develop CARs, but all products are still preclinical.

| An Oxford, United Kingdom company, Immunocore has major development collaborations with Genentech, GlaxoSmithKline (GSK), and AstraZeneca to develop CARs on the Immunocore development platform. Immunocore&rsquo;s own lead product, a CAR called IMCgp100, is in a phase I/II dosing study for late-stage melanoma.

Adaptimmune, a sister company to Immunocore with access to the same development platform but for TCRs only, has 6 indications in phase I/II trials for its TCR against NY-ESO as part of a GSK collaboration.

Indoleamine-2,3-dioxygenase (IDO) Inhibitors

| Novartis has been developing CAR therapies with the University of Pennsylvania as part of a global research and licensing agreement struck in 2012. Ongoing clinical trials evaluating a CAR therapy known as CTL019 have yielded promising results in patients with ALL, leading to the FDA granting breakthrough status for CTL019 in July 2014. On February 18, 2015, Novartis announced the beginning of a phase I trial of a University of Pennsylvania CAR against EGFRvIII for treatment of glioblastoma on the basis of encouraging preclinical results.

Bristol-Myers Squibb/Flexus. On February 23, 2015, BMS announced a $1.25 billion deal to acquire the IDO inhibitor assets of Bay Area startup Flexus, a program that is still Pre-Investigational New Drug (IND).

Flexus management intends to start a new company with its remaining assets; these include current personnel and facilities and FLX925, an inhibitor of Fms-like tyrosine kinase 3 (FLT3) and CDK4/6 just beginning phase I trials, and a non-IDO immunotherapy focusing on regulatory T cells.

Amgen/NewLink Genetics. The BMS/Flexus agreement came just 4 months after Amgen announced a potentially billion-dollar plus deal with NewLink Genetics to license IDO pathway inhibitor NLG919 and launch a research collaboration to discover additional compounds for IDO-tryptophan-2,3-dioxygenase (TDO) therapy. The total deal could mean more than $1 billion to NewLink.

| A startup seeded in 2014 with another $35 million in backing as of January 8, 2015, this Cambridge, Massachusetts, company may define a new category, next generation immuno-oncology. Like Flexus, Surface Oncology is also looking at manipulating regulatory T cells, but wants to exploit other immune system targets to aid recognition and destruction of tumors. Next generation immuno-oncology could modify not only T cells but also antigen-presenting cells (APCs), tumor associated macrophages, and others, helping the immune system to destroy tumors by improving antigen presentation for efficient activation or by blocking the activity of various suppressor cells in the tumor microenvironment.

| A German company, immatics, is also involved in the antigen-presentation process for the activation of T cells. Combining a proteomics approach using mass spectrometry to directly identify antigens overexpressed on tumor cells, immatics prepares cancer vaccines derived from these tumor-associated antigens. Depending on the length of the peptides derived from tumor antigens, vaccines can be designed to activate either cytotoxic T cells or T-helper cells. The company&rsquo;s lead product for treatment of renal cancer is in phase III trials, with another for colorectal cancer having recently completed phase II trials; a third therapy for glioblastoma is beginning phase I trials. On the basis of a partnership with Roche established a year ago to develop immunotherapies using the immatics platform, the company also has active early-stage programs in gastric cancer, NSCLC, and prostate cancer. On March 2, 2015, the Cancer Prevention and Research Institute of Texas (CPRIT) awarded immatics a $20 million grant for the development of adoptive T-cell therapies in collaboration with the MD Anderson Cancer Center, Houston, Texas, at a new US-based subsidiary of immatics.

| This Buffalo, New York, facility received a $12 million grant on January 14, 2015, from the New York State Stem Cell Science Program to fund development of a stem-cell based treatment for ovarian cancer.

Like Kite and Adaptimmune, RPCI is focusing on the tumor antigen NY-ESO-1, and intends to express a cloned TCR with specificity for NY-ESO-1 into hematopoietic stem cells removed from patients. A portion of the stem cells infused back into the patients will develop into mature T cells with specificity for NY ESO 1.

Merck Receives FDA Breakthrough Therapy Designation for KEYTRUDA&reg; (pembrolizumab) in Advanced Non-Small Cell Lung Cancer. http://www.businesswire.com/news/home/20141027005286/en/Merck-Receives-FDA-Breakthrough-Therapy-Designation-KEYTRUDA%C2%AE#.VQB_CpU5DIU. October 27, 2014. Accessed March 15, 2015.

FDA Expands Approved Use of Opdivo (nivolumab) to Treat Lung Cancer. http://www.drugs.com/newdrugs/fda-expands-approved-opdivo-nivolumab-lung-cancer-4179.html. March 4, 2015. Accessed March 15, 2015.

Eisai and Merck Enter Collaboration to Explore Novel Combination Regimens of Anti-PD-1 Therapy With Multi-targeting RTK Inhibitor and Microtubule Dynamics Inhibitor in Multiple Types of Cancer. http://www.mercknewsroom.com/news-release/prescription-medicine-news/eisai-and-merck-enter-collaboration-explore-novel-combinatio. March 4, 2015. Accessed March13, 2015.

FDA Grants Breakthrough Therapy Designation for Genentech&rsquo;s Investigational Cancer Immunotherapy MPDL3280A (anti-PDL1) in Non-Small Cell Lung Cancer. http://www.gene.com/media/press-releases/14583/2015-02-01/fda-grants-breakthrough-therapy-designat. February 1, 2015. Accessed March 11, 2015.

New Trial Initiated Evaluating Amgen&rsquo;s Talimogene Laherparepvec in Combination With Merck&rsquo;s Anti-PD-1 Therapy KEYTRUDA&reg; (Pembrolizumab) for Advanced Melanoma. https://www.amgen.com/media/media_pr_detail.jsp?year=2014&releaseID=1995881 . December 8, 2014. Accessed March 15, 2015.

FDA Approves BLINCYTO&trade; (Blinatumomab) Immunotherapy for the Treatment of Relapsed or Refractory B-Cell Precursor Acute Lymphoblastic Leukemia. http://www.amgen.com/media/media_pr_detail.jsp?releaseID=1994704. December 3, 2014. Accessed March 10, 2015.

Juno Therapeutics Executes License for Phase I Car T Product Candidate Targeting CD22 for Hematological Malignancies. https://junotherapeutics.com/juno-therapeutics-executes-license-for-phase-i-car-t-product-candidate-targeting-cd22-for-hematological-malignancies/. Accessed March 12, 2015.

Amgen and Kite Pharma Announce Strategic Cancer Immunotherapy Collaboration to Advance the Application of Novel Chimeric Antigen Receptor (CAR) T Cell Therapies. http://www.amgen.com/media/media_pr_detail.jsp?releaseID=2002967. January 5, 2015. Accessed March 9, 2015.

IMCgp100 in Clinical Development. http://www.immunocore.com/pipeline/imcgp100/. Accessed March 13, 2015.

Penn Medicine Study Describes Development of Personalized Cellular Therapy for Brain Cancer. http://www.uphs.upenn.edu/news/News_Releases/2015/02/maus/. February 18, 2015. Accessed March 10, 2015.

Bristol-Myers Squibb Co Expands Cancer Treatment Pipeline With $1.25 Billion Purchase of Flexus Biosciences. http://www.ibtimes.com/bristol-myers-squibb-co-bmy-expands-cancer-treatment-pipeline-125-billion-purchase-1825010. February 23, 2015. Accessed March 13, 2015.

Genentech, NewLink Launch $1.15B Cancer Collaboration. http://www.genengnews.com/gen-news-highlights/genentech-newlink-launch-1-15b-cancer-collaboration/81250487/. October 20, 2014. Accessed March 15, 2015.

New York State Awards Grant for First Stem-Cell Immunotherapy Trial in Ovarian Cancer. http://www.targetedonc.com/articles/New-York-State-Awards-Grant-for-First-Stem-Cell-Immunotherapy-Trial-in-Ovarian-Cancer. January 15, 2015. Accessed March 14, 2015.

View the entire Special Report on Immunotherapy

Following recent approvals by the US Food and Drug Administration (FDA) of pembrolizumab and nivolumab, numerous programs to develop and expand use of immunotherapies have ensued.

Major Programs Under Way in Immuno-Oncology

The capture of whole, circulating tumor cells (CTCs) was the initial focus of liquid biopsies. However, the definition of liquid biopsy has expanded to include the collection of cell-free DNA (cfDNA), along with various species of cell-free RNA and exosomes, all of which are capable of providing information on the disease status of cancer patients.

Compared with conventional tissue biopsy of tumors, liquid biopsy offers advantages in the diagnosis and treatment of non-small cell lung cancer (NSCLC). A blood draw is minimally invasive for the patient, can be done at the point-of-care, and can be performed serially and inexpensively to monitor disease progression, an approach not possible with tissue biopsies in the lungs.

Liquid biopsy for NSCLC has potential applications in both screening and in monitoring. Survival rates for NSCLC are low, despite advances in treatment of the disease, because patients often present with advanced disease. The availability of screens to detect validated biomarkers of the disease from CTCs, cfDNA, or other elements in blood may make it practical to one day perform routine screening for lung cancer, particularly for smokers.

A key benefit to liquid biopsies is the potential for overall characterization of disease progression and genetic variation. Intratumoral heterogeneity is a common feature of primary tumors, but the particular sample collected in a tissue biopsy may not be representative of the tumor as a whole. Heterogeneity also exists between the primary tumor and metastases. Liquid biopsy can address this issue by detecting DNA that is shed by cancer cells in all parts of a tumor, as well as by secondary, metastatic tumors.

The major reason for studying heterogeneity is detection of mutations for the assignment of targeted therapies. In this way, liquid biopsy is a crucial component of the shift to personalized medicine. For NSCLC, this relates to the detection of mutations in exon 20 of the

gene, where 50% to 60% of acquired resistance to inhibitors of

&rsquo;s tyrosine kinase activity is associated with point mutation

Other key mutations consist of rearrangements of the

However, despite the widely acknowledged potential of liquid biopsy, its integration into everyday clinical practice in oncology has yet to occur. Alberto Bardelli, PhD, associate professor, Turin University School of Medicine, Italy, expressed the need for further data when summarizing a session on liquid biopsy that he chaired at the 2014 European Society for Medical Oncology (ESMO) Congress. &ldquo;I have no doubt liquid biopsy will become routine, but right now the evidence is lacking,&rdquo; he said.

CTCs are intact cancer cells that have been released into the bloodstream and have been associated with worse prognosis in several major cancers, including lung cancer, with an increase in CTCs predicting tumor progression and aggressiveness. In patients with NSCLC, the CTC count has been correlated with both progression-free survival (PFS) and overall survival (OS). Although CTCs are potentially more informative than cfDNA by their very nature as intact cells, CTCs are scarcer and therefore more difficult to obtain from a blood sample.

There is only one technology approved by the US Food and Drug Administration (FDA) for CTC detection (in metastatic cancers of the breast, prostate, and colon, but not in metastatic lung cancer). This technique was used in clinical trials to establish thresholds for patient CTC counts that could be used as prognostic markers and predictors of patient outcomes. However, the technique relies on the presence of the epithelial cell adhesion molecule (EpCAM) on the surface of the CTC. Because metastatic cells that have undergone an epithelial-mesenchymal transition (EMT) no longer express epithelial biomarkers, the EpCAM system might not detect CTCs of interest in patients with lung cancer. There are scientific publications demonstrating CTC detection in patients with lung cancer by other means, but these new methods have not yet become part of routine clinical practice. The abundance of methods available for CTC detection may be a contributing factor, because selecting the optimal method can be challenging.

Proper treatment of NSCLC requires evaluation of specific predictive biomarkers, with molecular characterization of

mutation status in patients with NSCLC via tissue biopsy firmly established in current clinical practice. However, the fine needle aspiration procedures used to collect tumor tissue can make molecular profiling difficult because of the tiny amount of material obtained.

More sensitive techniques have been developed to deal with the small sample sizes characteristic of such tissue biopsies, but analysis of circulating tumor (ct)DNA may be a better solution. Although cfDNA is released by both healthy and tumor cells, the DNA released from normal cells undergoing apoptosis is trimmed into relatively uniform fragments of 185 to 200 base pairs, while the DNA from tumor cells undergoing necrosis varies in size. Healthy individuals display only cfDNA from apoptotic cells, meaning that prevalence of longer DNA fragments could be used a marker of malignancy, and higher ctDNA levels compared with healthy controls have been demonstrated in patients with cancer.

ctDNA can be also used for mutational analysis. For example, examination of

status in ctDNA from patients with NSCLC has demonstrated the occurrence of the resistance mutation

at progression, but not at the time of treatment initiation. Evaluation of the

mutation in ctDNA from patients with NSCLC showed that those with a

mutation detectable in plasma had shorter OS and PFS compared with patients with wild-type

mutations in plasma may have a prognostic as well as a predictive value. Use of ctDNA to detect somatic mutations, particularly of

, by next generation sequencing (NGS), shows promise.

However, there is still a dearth of widely accepted and approved methods for ctDNA analysis, and detection of ctDNA in patients with lung cancer is not used by physicians in daily practice.

Exosomes represent another potential avenue for revealing mutational heterogeneity and tracking disease progression. Exosomes are small (40 nm to 100 nm diameter), membrane-derived vesicles that are released from normal and malignant cells to blood and other body fluids. Exosomes contain DNA fragments, such as signal proteins and/or peptides, micro RNAs, (mRNAs), and lipids, and they can promote cancer progression when they originate from tumor cells. Although the normal cellular function of exosomes appears to include cell-to-cell communication via direct activation of surface-expressed ligands or by transfer of molecules between cells, their diagnostic potential is clear. Not only can exosomes be subjected to NGS but also to various RNA and proteomic analyses.

Standardization of analytical processes for liquid biopsy and the development of robust commercial platforms for the analysis of biomarkers in body fluids are being actively pursued.

&ldquo;There are biomarkers with the potential to be introduced into daily clinical practice in the near future, but success depends on robust validation in sufficiently large, independent, prospectively designed studies,&rdquo; said Paul Hofman, MD, PhD, IRCAN team leader at INSERM, Nice, France. The utility of liquid biopsy in diagnosis and treatment of NSCLC will increase with discovery of additional biomarkers to differentiate and stratify various forms of the disease, supporting the global trend toward personalized medicine.

Although hurdles remain before liquid biopsy is seen in routine clinical practice for diagnosis and treatment of NSCLC, its considerable potential and the energy being applied to its development mean that every day clinical use of liquid biopsy is inevitable and relatively imminent.

Companies Developing Liquid Biopsy for NSCLC and Other Cancers

Although liquid biopsy testing has not yet entered daily clinical practice in oncology, numerous companies are vying for the opportunity to make the technology commonplace. Here are some of the players:

Received CE-IVD marking in Europe on January 21, 2015, for its ctDNA-based companion diagnostic to gefitinib for NSCLC. Test is quantitative polymerase chain reactions (QPCR)-based analysis checking

status across 21 mutations in serum. Announced codevelopment of liquid biopsy assays with Exosome a week earlier.

Next generation sequencing (NGS)-based mutation analysis of ctDNA using 68-gene panel with complete exon coverage of

. Received additional $50 million in funding February 2015.

The companies settled their IP differences December 3, 2014, and have pooled their patent portfolios, announcing plans to enter the cancer market. RUO kits to be released in 2015 while Illumina seeks FDA approval for tests. Strong position in noninvasive prenatal testing (NIPT) market using nearly identical technology.

mutations in urine as well as in blood. Pipeline includes tests for

Roche announced purchase of a majority stake in Foundation Medicine on January 12, 2015, for more than $1 billion. The company currently offers a 315-gene oncology-based test for solid tumors, and another that checks 405 genes for hematologic tumors. Roche says it will convert tests to liquid biopsy.

Thermo Fisher Scientific announced the deal on January 13, 2015, to distribute Cynvenio&rsquo;s LiquidBiopsy platform. Processes CTCs, uses NGS to check mutations in 50 genes important in breast, CRC, and prostate cancers. Mutations of additional cancers on the way, as well as analysis of ctDNA. Samples processed currently in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory but will launch RUO assay kits this year.

Uses CTCs and ctDNA from blood for CLIA-validated tests of lung, breast, and gastric cancer. Biocept announced January 7, 2015, that its tests will be offered to a 19-million member preferred provider network. Two weeks later, Biocept announced the launch of a blood test for

Focuses on advantages of exosomes to derive additional information on lung cancer beyond what is available from ctDNA. In addition to current

tests for NSCLC, company plans to launch a plasma-based cancer panel and a urine-based prostate cancer diagnostic in 2015. Announced codevelopment of liquid biopsy assays with Qiagen on January 13, 2015.

Rolfo C, Giovannetti E, Hong DS, et al. Novel therapeutic strategies for patients with NSCLC that do not respond to treatment with EGFR inhibitors.

Couraud S, Vaca-Paniagua F, Villar S, et al. Non-invasive diagnosis of actionable mutations by deep sequencing of circulating-free DNA in non-small cell lung cancer: Findings from BioCAST/IFCT-1002.

The definition of liquid biopsy has expanded to include the collection of cfDNA, along with various species of cell-free RNA and exosomes, all of which are capable of providing information on the disease status of cancer patients.

Liquid Biopsy Use in NSCLC Clinical Practice on the Horizon

A panel of expert clinical investigators discussed details of several treatments and a dozen patient cases involving gastric, colorectal, and pancreatic cancer

at a satellite meeting of the 2015 American Society of Clinical Oncology (ASCO) Gastrointestinal (GI) Cancers Symposium.

The audience participated in the discussion via electronic tablets that allowed attendees to respond to poll questions and to present cases for review and comment by the panel of key opinion leaders. In this way, community oncologists were able to gain direct input from top clinical investigators on treatment options for their own patients.

Oncologist and moderator Neil H. Love, MD, first reviewed results of an audience poll that asked, &ldquo;In the past month, how many times would it have been helpful to have input from a clinical investigator about treatment for a patient in your practice?&rdquo;

Two-thirds of respondents reported that they would have liked such input at least twice in the past month. Each expert speaker also presented information on new and emerging GI cancer treatments and applied this information to the patient cases under review by means of numerous, interwoven group discussions.

Cancer stem cells are often able to survive chemotherapy, leading to recurrence and metastasis. In a review of selected ongoing studies of novel targeted agents for gastroesophageal (GE) cancer, Philip A. Philip, MD, PhD, Karmanos Cancer Center, Wayne State University, Detroit, Michigan, discussed BBI608, a compound for treatment of cancer stem cells. BBI608 targets STAT3, beta-catenin, and Nanog, with some preclinical evidence of synergy with paclitaxel.

BRIGHTER is an ongoing phase II trial of BBI608 and paclitaxel in gastric or GE junction cancer versus placebo and paclitaxel (NCT02178956).

Axel F. Grothey, MD, professor of oncology, Mayo Clinic, Rochester, Minnesota, said that his clinic has recognized the importance of the cancer stem cell concept by creating a development program in stem cells. Mayo investigators have learned that cancer stem cell treatment has parallels with approaches used in immunotherapy.

&ldquo;Stem cells make up a very small part of the tumor,&rdquo; said Grothey. &ldquo;Instead of using conventional Response Evaluation Criteria in Solid Tumors (RECIST) standards to control progression, we use immunotherapy-like response criteria, because you might want to allow a little progression of the already committed cells before they regress. Immunotherapy has shown us a different way of thinking about cancer therapy.&rdquo;

Asked how BBI608 exerts its effect on cancer stem cells, Grothey said that the mechanism of action would be published soon in the

Proceedings of the National Academy of Sciences

. Because it involves inhibition of a component of Wnt signaling (STAT3), BBI608 is a unique agent.

In a discussion of new treatments for advanced colorectal cancer, Howard S. Hochster, MD, associate director for clinical sciences, Yale School of Medicine, New Haven, Connecticut, reviewed TAS-102, a novel fluoropyrimidine, which has shown promise in the RECOURSE phase III trial. However, when the audience was asked in a poll, &ldquo;If TAS-102 were accessible to you, would you recommend it for your patients with metastatic colorectal cancer?&rdquo; more than 60% said, &ldquo;I am not familiar with TAS-102.&rdquo;

TAS-102 was originally formulated in 1964, but was not practical to use because its half-life was only about 12 minutes. Researchers have now combined TAS-102 with an inhibitor (tipiracil) of the enzyme that metabolizes TAS-102, thymidine phosphorylase. This modification greatly extends the half-life of TAS-102, providing oncologists with another effective fluoropyrimidine in their options for cancer treatments. In RECOURSE, patients with metastatic colorectal cancer (mCRC) who had received 2 or more prior regimens of treatment with chemotherapy and bevacizumab were given TAS 102 plus best standard care (BSC) or placebo plus BSC. The primary endpoint of overall survival (OS) was met (hazard ratio [HR], 0.68, P < .0001), and TAS-102 toxicity was similar to that seen with placebo.

Commenting on the chances for approval of this novel antimetabolite combination that could be appropriate for use in earlier CRC, or combined with irinotecan, oxaliplatin, and targeted agents, Grothey said that TAS-102 is being reviewed by the US Food and Drug Administration (FDA) with a verdict of likely approval within 6 months.

Johanna C. Bendell, MD, director of GI cancer research, Sarah Cannon Research Institute, Nashville, Tennessee, discussed improved treatments for pancreatic cancer that have resulted in average survivals of 10 to 12 months now being seen in trials. In terms of new agents for pancreatic cancer, Bendell cited JAK-STAT signaling inhibition as a novel approach. Janus kinases (JAKs) are a family of kinases that includes JAK1, JAK2, JAK3, and TYK2. JAKs mediate cytokine signaling by activating STAT transcription factors, an inflammatory pathway that is highly active in pancreatic cancer.

&ldquo;This high degree of inflammation is why pancreatic tumors are so hard to treat. It is very difficult to get the chemotherapy drugs or targeted agents to the tumor. Reducing this inflammation could result in improved drug delivery and better antitumor effect,&rdquo; Bendell said.

Bendell described RECAP, a recent phase II study of patients with metastatic pancreatic cancer who were randomized to receive second-line treatment of ruxolitinib with or without capecitabine. Ruxolitinib, a JAK1/JAK2 inhibitor, blocks signaling mediated by many proinflammatory cytokines. The calculated hazard ratio (0.79) for the primary endpoint (OS) was not statistically significant. However, when patients were stratified by the modified Glasgow Prognostic Score (mGPS), a well-characterized clinical measure of inflammation in cancer that combines levels of C reactive protein (CRP) and serum albumin, a significant hazard ratio of 0.49 was achieved by the high inflammation group. On the basis of this result, there are now 2 phase III studies involving ruxolitinib under way, JANUS-1 and JANUS-2, in patients prescreened for moderate or high mGPS score.

Another emerging agent for treatment of pancreatic cancer that focuses on improved drug delivery is MM-398, a nanoliposomal form of irinotecan. Recent positive results from NAPOLI-1, a phase III study in patients with metastatic pancreatic cancer, showed improved OS (HR, 0.67, P = .0122) of MM-398 plus 5-FU/leucovorin compared with 5-FU/leucovorin alone. Approval of MM-398 is anticipated by the end of this year, according to Bendell.

Pembrolizumab, an antiprogrammed cell death-1 (PD-1) antibody recently approved for treatment of metastatic melanoma, is being investigated for efficacy and safety in many other tumor types. Tanios S. Bekaii-Saab, MD, assistant professor of medical oncology, Ohio State University, Columbus, discussed the use of immunotherapy via PD-1 receptor blockade in GI cancer, referring to KEYNOTE-012, a phase IB study of pembrolizumab as a single agent in patients with advanced gastric cancer. Tumor cells can evade response of the immune system to malignancy by overexpressing the PD 1 ligand, PD-L1, on their surfaces to bind the negative co-stimulatory PD-1 receptor expressed on activated T cells, thereby shutting off signals directing the T cells to attack cancer cells. Bekaii-Saab described blocking of PD-1 or PD-L1 as a way to make tumors more detectable by T cells, thereby boosting immunogenic activity and tumor clearance. Patients were screened for PD-L1 expression before enrollment to provide a basis for studying the relationship between PD-L1 expression and clinical outcomes.

&ldquo;The response rate of 33% in mostly refractory patients is quite interesting. PD-L1 levels seemed to be associated with response rate, although the statistical significance is currently borderline,&rdquo; Bekaii-Saab said. &emsp;

Cases from the Community: Clinical Investigators Provide Perspectives on Actual Patients with Metastatic Colorectal, Gastric, and Pancreatic Cancer. Research To Practice.

ASCO Gastrointestinal Cancers Symposium, San Francisco, CA, January 15-17, 2015.

A panel of expert clinical investigators discussed details of several treatments and a dozen patient cases involving gastric, colorectal, and pancreatic cancer at a satellite meeting of the 2015 ASCO GI Cancers Symposium.

Expert Investigators Discuss Emerging Therapies for GI Cancers

Comparable test results were shown with both urine and plasma molecular assays in a blinded, retrospective study that evaluated the potential of quantitating

circulating tumor (ct)DNA in patients with minimal residual disease (MRD) surgical colorectal cancer (CRC) who have liver metastases.

mutation was identified in 95% of archival plasma specimens by the assay and in 92% of archival urine specimens from patients with CRC and known

mutational status in tumor tissue, according to data presented at the 2015 Gastrointestinal Cancers Symposium by Marek Minarik, PhD, director at Genomac International and assistant professor at Charles University, Prague, Czech Republic.

Samples were collected for a collaborative study by Trovagene with Genomac International, Prague, Czech Republic, from 20 patients with stage 4 CRC and a

-positive primary tumor. All 20 patients were undergoing surgical treatment in combination with systemic therapies that included neoadjuvant radio/chemotherapy, adjuvant targeted therapy, and adjuvant chemotherapy.

Archived, matched longitudinal plasma and urine samples were collected and evaluated both prior to surgery to provide a baseline, and at time intervals throughout the course of treatment. Concordance between

detection in archived tissue, plasma, and urine samples was established at baseline, and urine and plasma

mutation concordant with tumor tissue was detected in 19 of 20 evaluable baseline plasma samples (95%), while a

mutation concordant with tissue was detected in 11 of 12 evaluable baseline urine samples (92%). In a single patient, a

mutation was detected consistently across all serial plasma and urine samples, but not in tissue biopsy.

A total of 190 plasma and urine samples that had been archived for 3 to 5 years were tested, with 101 of 101 plasma samples (100%) and 79 of 92 urine samples (86%) having sufficient DNA concentration and deemed evaluable. The degree of DNA degradation in these archival urine samples was elevated compared with typical clinical samples.

The assay (Trovagene, San Diego, CA) uses a selective enrichment step for mutated DNA fragments, while suppressing wild-type sequence PCR amplification with a blocker, followed by deep next-generation sequencing. Post-sequencing analysis algorithms quantitate the amount of mutant DNA in samples. The company says that its assay has a lower limit of detection of 0.0055% and provides single-copy detection, with 7 mutant DNA copies reliably detected in 60 ng of wild-type DNA (over 18,000 genomic copies; LOD = 0.04%) or in 360 ng wild-type DNA (over 100,000 genomic copies; LOD = 0.006%).

In a study using the assay to follow individual cases of patients with metastatic colorectal cancer (mCRC), ctDNA

signal remained detectable or increased after surgery in 10 of 11 patients who had incomplete, palliative surgery. This was contrasted by a study of patients with mCRC who underwent intent-to-cure surgery, whose ctDNA

levels were undetectable after surgery in urine or plasma at all time-points measured in 4 of the 5 patients, and whose

levels became undetectable in urine or plasma in 5 out of 5 patients at 7 days following surgery. The time course levels of

signal in urine showed overlap with those in plasma in both the palliative surgery and intent-to-cure surgery groups.

Over half of patients with CRC will develop liver metastases. Surgical resection, in combination with systemic therapies, greatly improves long-term outcomes. About 40% of patients with resected liver-limited disease are alive, 5 years after diagnosis. While tumor staging and surgery are commonly used for prognostic assessment, better noninvasive methods are needed for monitoring response to therapy, following MRD, and guiding complex treatment decisions in these patients.

&ldquo;We demonstrated the clinical applicability of assessing minimal residual disease after surgery in CRC patients with liver metastases by quantitative monitoring of urinary ctDNA

with single molecule sensitivity,&rdquo; said Minarik. &ldquo;The prognostic significance of post surgical

levels and the overall survival in stage 4 CRC patients with liver metastases is being evaluated in a larger cohort.&rdquo;

Poole JC, Vibat CR, Benesova L, et al. Highly sensitive quantitative detection of circulating tumor DNA in urine and plasma from advanced colorectal cancer patients in aid of early diagnosis of clinically relevant

Comparable test results were shown with both urine and plasma molecular assays in a blinded, retrospective study.

KRAS Mutant ctDNA Assay Performance Comparable in Urine and Plasma for Advanced CRC

A post-hoc analysis of the CORRECT trial showed that patients with metastatic colorectal cancer (mCRC) who had progression-free survival (PFS) >4 months, experienced AEs at rates broadly similar to those in the overall trial population, despite exposure to regorafenib (Stivarga) for over twice as long as the overall group.

&ldquo;The results of this exploratory subgroup analysis support the clinical benefit and tolerability of regorafenib as a treatment option for patients with metastatic colorectal cancer,&rdquo; said lead investigator Axel Grothey, MD, Mayo Clinic, Rochester, Minnesota, at the 2015 Gastrointestinal Symposium. &ldquo;While adverse events across both populations were broadly similar, some did occur more frequently in patients with longer exposure, an observation that is possibly related to the longer duration in this subgroup. Analyses to identify clinical and molecular markers in these patients are ongoing.&rdquo;

Approximately 50% to 60% of patients diagnosed with CRC develop metastases, and the majority of these patients have unresectable disease. The standard of care for unresectable mCRC is chemotherapy with or without the VEGF inhibitor bevacizumab (Avastin), but few options are available for patients whose disease progresses after standard therapies. Regorafenib is an oral multikinase inhibitor that blocks the activity of multiple protein kinases involved in oncogenesis (KIT, RET, RAF-1, BRAF, and BRAF V600E), tumor angiogenesis, (VEGFR 1-3 and TIE2), and the tumor microenvironment (PDGFR and FGFR).

CORRECT was a randomized, placebo-controlled, phase III trial involving 760 patients in 114 centers across 16 countries in North America, Europe, Asia, and Australia. Key inclusion criteria were an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1, plus previous treatment with chemotherapy and bevacizumab. Patients were randomized 2:1 to receive 160 mg oral regorafenib (n = 505) or placebo (n = 255) once daily, in combination with best supportive care (BSC) and a radiologic evaluation every 8 weeks. Treatment continued until progression, death, unacceptable toxicity, or patient/investigator decision to withdraw.

The primary endpoint was overall survival (OS), defined as the time from randomization to death from any cause. Secondary endpoint was PFS, defined as the time to disease progression (radiological or clinical) or death from any cause. Patients were permitted dose interruptions or dose reductions for management of treatment-related AEs. Regorafenib was approved after meeting the primary (OS: HR = 0.77 [95% CI, 0.64-0.94; one-sided P = .0052]) and secondary (PFS: HR = 0.49 [95% CI, 0.42-0.58; one-sided P <.0001]) endpoints of the trial.

Median duration of PFS in CORRECT was 1.9 months (interquartile range [IQR] 1.6-3.9) in the regorafenib group and 1.7 months (IQR 1.4-1.9) in the placebo group. A subset of 98 (19.4%) patients treated with regorafenib had a PFS >4 months. These patients with longer PFS received a median of 6 cycles of regorafenib, with 92% receiving &ge;5 cycles and 20% receiving >8 cycles of the drug. Overall, 34% of patients had their regorafenib dose reduced and 87% underwent dose interruptions. The resulting mean daily dose was 139 mg, or 81% of the planned dose.

All patients with longer PFS experienced AEs; the most common grade &ge;3 AEs were hand-foot skin reaction (20%), hypertension (17%), diarrhea (17%), and fatigue (16%). Although the AE profile of regorafenib was broadly similar in both populations, some AEs did occur more frequently in patients treated with regorafenib for >4 months. Rates of all-grade diarrhea, hand-foot skin reaction, and weight loss were &ge;15% higher in the subgroup with longer PFS than in the overall CORRECT population. Rates of grade &ge;3 diarrhea and hypertension (17% each) in the longer PFS population were twice as high as in the overall population. According to Grothey, prospective validation of these findings in conjunction with biomarker analysis from real-life clinical experience is needed.

Grothey A, Van Cutsem E, Sobrero A, et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial.

A post-hoc analysis of the CORRECT trial showed that patients with mCRC who had PFS >4 months, experienced AEs at rates broadly similar to those in the overall trial population.