Functional profiling with cell culture assays

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Functional profiling (whole cell profiling) with cell culture assays for targeted drug therapy

Recent findings presented at the American Society of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium in Orlando, Florida concluded that Functional Profiling (whole cell profiling) with cell culture assays is relevant for the study of both "conventional" and "targeted" antineoplastic drug agents.

Cell Culture Assays with "cell-death" endpoints can show disease-specific drug activity, are useful clinical and research tools for "conventional" and "targeted" drugs, and provide unique information complementary to that provided by "molecular" tests. There have been more than 25 peer-reviewed publicatons showing significant correlations between cell-death assay results and patient response and survival.

The Whole Cell Profiling technique is a cell-death endpoint assay in which drug effect upon cancer cells is visualized directly. Photomicrographs of actual tumor cells sometime show that the exact same identical individual culture well, shows some clusters have taken up vast amounts of a drug, while right next door, clusters of the same size, same appearance, same everything haven't taken up any of the drug.

So it doesn't matter if there is a "target" molecule (protein or receptor) in the cell that the targeted drug is going after, if the drug either won't "get in" in the first place or if it gets pumped out/extruded or if it gets immediately metabolized inside the cell, drug resistance is multifactorial. The advantage of the Whole Cell Profiling technique is that it can show this in the "population" of cells.

The Whole Cell Profiling technique makes the statistically significant association between prospectively reported test results and patient survival. It can correlate test results which are obtained in the lab and reported to physicians prior to patient treatment, with significantly longer or shorter overall patient survival depending upon whether the drug was found to be effective or ineffective at killing the patient's tumor cells in the laboratory.

This could help solve the problem of knowing which patients can tolerate costly, new treatments and their harmful side-effects. These "smart" drugs are a really exciting element of cancer medicine, but do not work for everyone, and a test to determine the efficacy of these drugs in a patient could be the first crucial step in personalizing treatment to the individual.

Functional profiling (FP) with cell culture assays for targeted drug therapy.
Sub-category: Translational research
Category: Colon and Rectum
Meeting: 2007 Gastrointestinal Cancers Symposium

Abstract No: 440
Author(s): L. M. Weisenthal

Abstract: Introduction: We studied the relevance of FP for standard and targeted drugs.

Methods: Drugs were tested against fresh human tumor microclusters, with 96 hr drug exposures and multiple FP endpoints (MTT, DISC, resazurin, and/or ATP).

Results: In 65 previously chemonaive stage 4 colon cancer patients, those with FP assays showing 5FU results in the most resistant tertile had inferior overall survival, compared to pts without 5FU resistance (303 days vs. 686 days, H.R. 2.1, 95% C.I. 1.2 - 5.0, P2=0.011). In subset analysis restricted only to 53 pts who subsequently died (eliminating potential surgical cures), the respective results were 292 vs 493 days, HR 1.5 - 6.9, P2=0.0021. We applied FP to test targeted agents, including gefitinib, erlotinib, sunitinib, sorafenib, and bevacizumab. Gefitinib was tested against > 700 fresh tumor specimens; we reported striking correlations between gefitinib activity and overall pt survival in non-small lung cancer (2006 ASCO Annu Mtg, Abst 17117). Gefitinib and erlotinib are moderately cross resistant (R2=0.48, n paired comparisons=190). Gefitinib/sunitinib (R2=0.20, n=46) and erlotinib/sunitinib (R2=0.12, n=44) are largely non-cross resistant. We also developed a new microvascular viability assay (MVVA) to test microvascular cells present in tumor clusters. In the MVVA, bevacizumab was tested in 81 fresh tumor specimens (including 15 GI). Bevacizumab was nontoxic to the tumor cells, but often strikingly toxic to microvascular cells present within the same tumor clusters. Grading on a 0-4 scale, there was absent (Gr 0) effect in 23 specimens, weak (Gr 1-2) effect in 28, and a strong (Gr 3-4) effect in 26. In contrast to bevacizumab, neither sunitinib (n=87) nor sorafenib (n=20) showed selective effects against microvascular cells compared to tumor cells.

Conclusions: We cannot rule out a cytostatic effect of sunitinib or sorafenib on tumor microvascular cells. However, our results imply that the antitumor effects of bevacizumab are predominately mediated through antimicrovascular effects, while effects of sunitinib and sorafenib may be mediated largely through tumor cell apoptosis. We conclude that FP is relevant for the study of both traditional and targeted antineoplastic agents.

http://www.weisenthal.org/Tokyo_Cancer_Symposium_Nov_2006_Weisenthal.pdf

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Medicare Contractor Establishes Reimbursement Coverage Policy for Cell Culture Assay Tests

National Heritage Insurance Company (NHIC), the contractor that administers Medicare programs in California, has established a positive coverage policy for Cell Culture Assay Tests known as Chemosensitivity (Resistance) Testing for a tumor specimen from a Medicare patient obtained anywhere within the United States, but submitted for testing by one of the approved laboratories located within California. Medicare bills for Chemosensitivity (Resistance) Testing are billed through NHIC because the test is conducted by the approved laboratories in California.

The Chemosensitivity (Resistance) Test can help see what treatments have the best opportunity of being successful for "high" risk cancer patients. The test measures the response of "live" tumor cells to drug exposure. Following this exposure, the assays measure both cell metabolism and cell morphology (Functional Profiling). The integrated effect of the drugs on the whole cell, resulting in a cellular response to the drug, measuring the interaction of the entire genome. Assays based on "cell-death" occur in the entire population of tumor cells.

This cell culture assay technology has been clinically validated for the selection of optimal chemotherapy regimens for individual patients. It is a laboratory analysis based on tumor tissue profiling that uses "fresh" human tumor biopsy or surgical specimen to determine which drugs or combinations of chemotherapeutic agents have the highest likelihood of response for individual cancer patients.

Following the collection of "fresh" tumor cells obtained from surgery or tru-cut needle biopsies, a cell culture assay is performed on the tumor sample to measure drug activity (sensitivity and resistance). This will pinpoint which drug(s) are most effective. Tissue, blood, bone marrow, and ascites and pleural effusions are possibilities, providing tumor cells are present. At least one gram of fresh tissue is needed to perform the tests, and a special kit is obtained in advance from the lab.The treatment program developed through this approach is known as assay-directed therapy.

Individualized assay-directed therapy is based on the premise that each patient's cancer cells are unique and therefore will respond differently to a given treatment. This is in stark contrast to standard or empiric therapy, which chemotherapy for a specific patient is based on average population studies from prior clinical trials.

The decision had been made that the assay is a perfectly appropriate medical service, worthy of coverage on a non-investigational basis. What is of particular significance is that they abandoned the artificial distinction between "resistance" testing and "sensitivity" testing and are providing coverage for the whole FDA-approved kit. Drug "sensitivity" testing is merely a point a little farther along on the very same continuum which "resistance" testing resides.

Cell cuture assay tests based on "cell-death" have proven very effective in identifying novel treatment combinations for a variety of cancers. The value of cell-death assays is that they can and do accurately predict clinical outcomes and define novel chemotherapeutic synergies. It can help see what treatments will not have the best opportunity of being successful (resistant) and identify drugs that have the best opportunity of being successful (sensitive).

The current clinical applications of in vitro chemosensitivity testing is ever more important with the influx of new "targeted" therapies. Given the technical and conceptual advantages of "functional profiling" of cell culture assays together with their performance and the modest efficacy for therapy prediction on analysis of genome expression, there is reason for renewed interest in these assays for optimized use of medical treatment of malignant disease.

The payment provided will be sufficiently realistic that all Medicare patients for whom this testing is indicated will be able to get it with only the routine 20% co-payment, as Medi-gap insurance secondaries are mandated to provide payment for co-pays for Medicare-approved services.

The coverage became effective for claims for services performed on or after February 28, 2007. The decision is posted at:

http://www.medicarenhic.com/cal_prov/policies.shtml

http://www.medicarenhic.com/cal_prov/articles/chemoassaytest_0107.htm

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Ex-Vivo Analysis Recreates Human Tumor Cell Microenvironment

Long Beach, Calif. and San Diego (AACR Meeting) -- April 12, 2008 – Investigators at Rational Therapeutics and the Nagourney Institute (Long Beach, Calif.) report that ex-vivo analysis of the chemotherapeutic response of human tumors can provide clinically relevant predictive information, so long as the tumors remain within their natural micro-environment or “micro-ecosystem.” Their findings will be reported during a poster session presentation on Sunday, April 13th at the American Association for Cancer Research (AACR) Annual Meeting in San Diego.

As outlined in the presentation titled, “Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection,” (AACR: 08-AB-1546), ex-vivo analysis (EVA™) of human tumors provides a novel, real-time view of how such tumors act within their natural microenvironment. This information can, in turn, accelerate the drug development process and improve clinical therapy.

Led by Robert Nagourney, MD, medical director of Rational Therapeutics and the Todd Cancer Institute at Long Beach Memorial Medical Center, the investigators have applied a human tumor micro-spheroid platform that measures both apoptotic and non-apoptotic cell death events and other cellular responses following exposure to a variety of agents.

“With its capacity to measure genetic and epigenetic events, this platform provides a functional, real-time adjunct to static genomic and proteomic platforms,” said Dr. Nagourney. “By examining small clusters of cancer cells [microspheroids] in their native state, we provide a snapshot of the response of tumor cells to drugs, combinations and targeted therapies.”

He explains that what makes his research team’s analysis unique is the fact that each micro-spheroid examined contains all the complex elements of tumor bio-systems found in the human body and that have a major impact on clinical response.

“Our system for ex-vivo analysis is a conduit that connects novel drugs to clinicians and patients in need,” Dr. Nagourney added. “Appropriate use of this platform has the potential to save the pharmaceutical industry millions of dollars, shave years off the drug development cycle and improve clinical therapy.”

In recent years, Dr. Nagourney’s EVA/PCD™ platform has proven capable of identifying activity for signal transduction inhibitors and other classes of “targeted” agents, including a 100% response rate reported in previously untreated non-small cell lung cancer patients selected for 1st line treatment with the EGFr-TKIs.

About Rational Therapeutics Founded in 1995, Rational Therapeutics is a pioneering oncology research and testing center specializing in individualized therapy based on ex-vivo analysis of human tumors

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Correlation of long term survival in stage 4 colorectal cancer with results of fluorouracil acitivity in MTT cell culture assay

ASCO Symposium
Gastrointestinal Cancers
January 15-17, 2009
San Francisco, California
Abstract #367

Introduction:

Fluorouracil (5FU) putatively acts most importantly via inhibition of thymidylate synthase. Various measures of tumor thymidylate synthase gene expression, protein content, or enzyme activity correlate inversely with 5FU activity. It is a paradox that 5FU is active in slowly-proliferating colorectal cancer and is not an important drug in rapidly proliferating actue leukemia and lymphoma.

Holleman, et al (NEJM 2002 Aug 5;352:533 used a 96 hour suspension culture assay with a cell death (MTT) endpoint to define cut-offs for "sensitivity" vs "resistance." They then used these assay data to define gene expression patterns associated with sensitivity and resistance to each of 4 drugs commonly used in the treatment of childhood leukemia. They were then able to show that the gene expression definitions of sensitivity and resistance were significantly and independently associated with treatment outcome.

We thought to determine if a similar MTT assay would be predictive of clinical outcomes in colorectal cancer and could therefore be used to identify gene expression profiles predicting for drug sensitivity and resistance. We further sought to determine if the MTT assay data for colorectal cancer provided clues to the paradox of a putative proliferation-specific drug (5FU) being almost uniquely active in a slowly proliferating solid tumor (colorectal cancer).

Methods:

Fresh tumor biopsies were scissor-minced and digested with collagenase-DNAse. Three dimensional cell clusters were selectively isolated by differential centrifugation, aliquotted in 96 well polypropene microplates, and cultured for 96 hours in the continuous presence of 5FU, 20 and 40 ug/ml. Viability (relative to vehicle and positive controls) was determined with the MTT endpoint and expressed as percent cell death (100 minus percent of vehicle control minus positive control). Post culture Cytospins were prepared to ensure that postculture composition of tumor cells was greater than 70% of total viable cells. Photomicrographs of representative pre- and postculture Cytospin preparations are recorded.

Results:

We are a free-standing clinical laboratory and the only reliable follow-up data available to us are dates of patient death. We do not have data on actual subsequent treatments received by patients. However, it is extremely likely that virtually all stage 4 patients received some form of fluoropyrimidine therapy. 5FU activity in the MTT assay remained predictive of long term survival, even with the introduction of newer agents in successive 5 year time cohorts.

Conclusion:

1. Tumor cell death in response to 5FU in a 96 hour continuous incubation MTT assay clearly correlated with long term overall patient survival in stage 4 colon cancer in chemotherapy naive patients, but not in patients who received prior chemotherapy.

2. The MTT signal is generated by mitochondrial Krebs cycle activity and also via cytosolic NADH. It is not a signal for chromosomal DNA replication or for nuclear enzyme or nuclear cofactor activity. This implies that if the clinical effect of 5FU is to inhibit DNA synthase, mitochondrial DNA synthase may be a more important target than chromosomal DNA synthase. This could explain the paradox of 5FU being a more important drug for slowly proliferating colorectal cancer than for rapidly proliferating acute leukemia and lymphoma.

3. As in the case of childhood leukemia, the MTT assay in colorectal cancer may be used to generate gold standards for predictive gene expression profiles.

4. Cell culture assays were inappropriately abandoned as a core technology for the personalization of cancer chemotherapy.