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Functional Biomarker For Cancer Treatment

gdpawel's picture
gdpawel
Posts: 549
Joined: May 2001

Using a cell culture assay (in vitro apoptosis) for choosing cancer drugs is no different than a marker like estrogen receptor or CD20 or a gene expression pattern. They are all markers. One is a structural marker, the other is a functional marker. A cell culture assay is a "functional" biomarker. A functional biomarker provides information about the biomarker uptake rate in tumor cells or on tumor cell surfaces through fluorescence intensity changes.

As with any other laboratory test, the determination of the efficacy of cell culture assay tests is based on comparisions of laboratory tests with patient response (clinical correlations). The hypothesis to be tested with clinical correlations is that above-average drugs effects in the assays correlate with above-average drug effects in the patient, as measured by both response rates and patient survival.

Patients with test results in the "sensitive" range were more likely to respond than the total patient population as a whole. Conversely, patients with test results in the "resistant" range were less likely to respond than the patient population as a whole. On average, patients with assays in the "sensitive" range were 3.5-fold more likely to respond than patients with assays in the "resistant" range.

Targeted treatments take advantage of the biologic differences between cancer cells and healthy cells by "targeting" faulty genes or proteins that contribute to the growth and development of cancer. Many times these drugs are combined with chemotherapy, biologic therapy (immunotherapy), or other targeted treatments.

Understanding targeted treatments begins with understanding the cancer cell. Every tissue and organ in the body is made of cells. In order for cells to grow, divide, or die, they send and receive chemical messages. These messages are transmitted along specific pathways that involve various genes and proteins in a cell.

Targeted treatments fight cancer by correcting or modifying defective pathways in a cancer cell. In healthy cells, each pathway is tightly controlled. For instance, healthy cells are allowed to divide into new cells, and damaged cells are destroyed. However, in cancerous cells, certain points in the pathway become disrupted, usually through a genetic mutation (change in form).

Serious consequences to the cell may result from these mutations, depending on which pathway is affected. For example, suppose a cell develops a mutation that causes it to continue dividing into new cells? In other words, the signal is always on. If the signal never turns off, the cells that keep growing may eventually form a tumor.

The most appealing idea behind targeted drug therapy is that cancerous cells are destroyed and healthy cells are spared, resulting in fewer side effects of treatment. In contrast, traditional chemotherapy destroys both the cancer cells and the healthy cells, and does not have any mechanism to distinguish between them.

Because many cancer cells use similar pathways, the same drug could be used to treat one person's breast cancer and another person's lung cancer, as long as each tumor contained similar targets. This is why many of these treatments are being used in a variety of cancer types. Gleevec is used to treat both leukemia and a rare stomach tumor, called gastrointestinal stromal tumor (GIST).

Although targeted therapy is appealing, it is more complex than meets the eye. Cancer cells often have many mutations in many different pathways, so even if one route is shut down by a targeted treatment, the cancer cell may be able to use other routes. In other words, cancer cells have "backup systems" that allow them to survive. The result is that the drug does not shrink the tumor as expected. One approach to this problem is to target multiple pathways in a cancer cell.

Another challenge is to identify for which patients the targeted treatment will be effective. When Iressa is used in patients with lung cancer, researchers discovered that only patients whose tumors contained specific mutations responded to this drug.

Finally, tumors can become resistant to a targeted treatment. This means that the drug no longer works, even if it has previously been effective in shrinking a tumor. To solve this problem, new drugs are being designed or combined with existing ones to target the tumor more effectively.

The introduction of targeted drugs has not been accompanied by specific "predictive tests" allowing for a rational and economical use of these drugs. However, given the technical and conceptual advantages of cell culture analysis, together with its performance and the modest efficacy of therapy prediction on analysis of genome expression, there is reason for a renewal in the interest of cell culture assays (functional biomarker) for optimized use of medical treatment of malignant disease.

There should be an immediate recognition that matchmaking between cancer and cancer treatment is one area in cancer research and treatment which is deserving of much greater attention and utilization. There should be an inclusive effort to study and utilize technologies which are based on both the sub-cellular (molecular) level and at the cellular (cell function/cell culture) level.

Source: Weisenthal Cancer Group, Huntington Beach, CA and Departments of Clinical Pharmacology and Oncology, Uppsala University, Uppsala, Sweden. Current Status of Cell Culture Drug Resistance Testing May, 2002.

gdpawel's picture
gdpawel
Posts: 549
Joined: May 2001

The AngioRx microvascular viability assay a laboratory test which identifies anti-angiogenic drug activity in live tumor microclusters. The test is capable of discriminating anti-tumor effect from anti-angiogenic effect in the same mixed-cell population. It is the only known technology which discriminates the effects of different types of anti-angiogenic drugs within the same class of drugs and within different classes of drugs, and is capable of identifying synergistic effects among different angiogenic and non-angiogenic drugs in specific drug combinations.

Drugs are tested against fresh human tumor microclusters, with 96 hour drug exposures and multiple functional profiling endpoints (MTT, DISC, resazurin and/or ATP). Functional Profiling consists of a combination of a morphologic (structure) endpoint (DISC) and two or more metabolic (cell metabolism) endpoints (MTT, resazurin, ATP) at the cell "population" level. Additional drug concentrations for the targeted/angiogenic agents, some of which have very steep dose response relationships.

A major modification of the DISC (cell death) assay allows for the study of anti-microvascular drug effects of standard and targeted agents, such as Avastin, Nexavar and Sutent. The microvascular viability assay is based upon the principle that microvascular (endothelial and associated) cells are present in tumor cell microclusters obtained from solid tumor specimens.

The assay which has a morphological endpoint, allows for visualization of both tumor and microvascular cells and direct assessment of both anti-tumor and anti-microvascular drug effect. The morphologic endpoint information is gathered by examining the state of hundreds of individual cells. The metabolic endpoints measure the combined metabolism of all cells present in the culture (whole cell population profiling). CD31 cytoplasmic staining confirms morphological identification of microcapillary cells in a tumor microcluster.

The principles and methods used in the microvascular viability assay include: 1. Obtaining a tissue, blood, bone marrow or malignant fluid specimen from an individual cancer patient. 2. Exposing viable tumor cells to anti-neoplastic drugs. 3. Measuring absolute in vitro drug effect. 4. Finding a statistical comparision of in vitro drug effect to an index standard, yielding an individualized pattern of relative drug activity. 5. Information obtained is used to aid in selecting from among otherwise qualified candidate drugs.

It is the only assay which involves direct visualization of the cancer cells at endpoint, allowing for accurate assessment of drug activity, discriminating tumor from non-tumor cells, and providing a permanent archival record, which improves quality, serves as control, and assesses dose response in vitro.

Photomicrographs of the assay can show that some clones of tumor cells don't accumulate the drug. These cells won't get killed by it. The assay measures the net effect of everything which goes on (Functional Tumor Cell Profiling methodology). Are the cells ultimately killed, or aren't they?

This kind of technique exists today and might be very valuable, especially when active chemoagents are limited in a particular disease, giving more credence to testing the tumor first. After all, cutting-edge techniques can often provide superior results over tried-and true methods that have been around for many years.

Bibliography relevant to AngioRx/Microvascular Viability (MVV) assay

1. Weisenthal, L. M. Patel,N., Rueff-Weisenthal, C. (2008). "Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood." J Intern Med 264: 275-287, 2008. doi: 10.1111/j.1365-2796.2008.01955.x

2. Weisenthal, L., Lee,DJ, and Patel,N. (2008). Antivascular activity of lapatinib and bevacizumab in primary microcluster cultures of breast cancer and other human neoplasms. ASCO 2008 Breast Cancer Symposium. Washington, D.C.: Abstract # 166.

3. Weisenthal, L. M. (2010). Antitumor and anti-microvascular effects of sorafenib in fresh human tumor culture in comparison with other putative tyrosine kinase inhibitors. J Clin Oncol 28, 2010 (suppl; abstr e13617)

4. Weisenthal, L., H. Liu, Rueff-Weisenthal, C. (2010). "Death of human tumor endothelial cells in vitro through a probable calcium-associated mechanism induced by bevacizumab and detected via a novel method." Nature Precedings 28 May 2010.

5. Eur J Clin Invest, Volume 37 (suppl. 1):60, 2007

6. Nagourney, R.A. Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)

7. Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings Part I. Vol 24, No. 18S (June 20 Supplement), 2006: 17117

gdpawel's picture
gdpawel
Posts: 549
Joined: May 2001

There are a number of new classes of drugs that target VEGF, at the protein level (Avastin), at the tyrosine kinase level (Nexavar, Sutent) and at the intracellular metabolic pathway mTOR (Afinitor, Torisel).

However, responses to any individual mechanism occurs in the miniority of patients. It is unclear why some patients repond to these interventions while others fail. In cell function analysis, it has found unexpectedly good response to conventional cytotoxic drugs following a failure to respond to these targeted agents.

This reinforces the need for cancer therapies to be individualized. It remines us that it is the good outcome of the patient not the therapy applied that constitute successful therapy.

The FDA does not have the legal authority to regulate the practice of the medicine and the physician may prescribe a drug off-label. Some drugs are used more frequently off-label than for their original, FDA-approved indications. Frequently, the standard of care for a particular type or stage of cancer involves the off-label use of one or more drugs.

The FDA wants to alter rules for cancer drug cocktails. Rather than mixing and matching approved drugs, scientists want to develop combinations designed to work in tandem to block cancer. Some have suggested to use assays to identify a potential targeted population of ovarian cancer patients that it thinks will benefit from any of the above drugs, singularly or in combination.

One can't remember phase I-II trials of combinations of drugs which had not received prior FDA approval. Cocktails tha mix drugs still in development wouldn't have been possible just five years ago.

Among the most sought after attributes of chemotherapy drug combinations is drug synergy. Synergy, defined as supra-additivity wherein the whole is greater than the sum of the parts, reflects an elegant interaction between drugs predicated on their modes of action. While some synergistic interactions can be predicted based upon the pharmacology of the agents, others are more obscure.

The application of synergy analyses may represent one of the most important applications of the functional profiling platform; enabling clinicians to explore both anticipated and unanticipated favorable interactions. Equally important may be the platform's capacity to study drug antagonism wherein two effective drugs counteract each others’ benefits. This phenomenon, characterized by the whole being less than the sum of the parts, represents a major pitfall for clinical trialists who simply combine drugs because they can.

These analyses are revolutionizing the way cell-based functional profiling applies newer classes of drugs and has the potential to accelerate drug development and clinical therapeutics. Good outcomes require good drugs, but better outcomes require good combinations. Intelligent combinations are a principle focus of functional tumor cell profiling.

Cell-based functional profiling assay labs have always tested new drugs in combination with each other, simultaneously measuring direct antitumor activity and antivascular activity.

Cocktails have become standard treatment in many oncological protocols: concoctions of two or more powerful cytotoxic agents which supposedly will attack the tumor in different ways. The ability of various agents to kill tumor and/or microvascular cells (anti-angiogenesis) in the same tumor specimen is highly variable among the different agents. There are so many agents out there now, doctors have a confusing array of choices. They don't know how to mix them together in the right order.

Data show conclusively that patients benefit both in terms of response and survival from drugs and drug combinations found to be 'active' in functional profiling assays even after treatment failure with several other drugs, many of which are in the same class, and even with combinations of drugs found to have low or no activity as single agents, but which are found in the assay to produce a synergistic and not merely an additive anti-tumor effect.

Source: Weisenthal Cancer Group, Huntington Beach, CA and Departments of Clinical Pharmacology and Oncology, Uppsala University, Uppsala, Sweden. Current Status of Cell Culture Drug Resistance Testing May, 2002.

Drbrianbennett
Posts: 1
Joined: Jun 2011

see youtube, I have invented a very powerful CSRA while at UMMS and the U of UT - I tried to bring it to the public but greedy VC's University's and others tried to make it something it was never intended to be, exclusive and unaffordable. If you know a scientist tell them to look at it - it works and it is free - I will not enforce the patent -

http://www.youtube.com/user/TheDrbrianbennett?feature=mhee

Brian

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