Biomarker of Antiangiogenic Therapy

[gpawelski]

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

[gpawelski]

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.

[gpawelski]

Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood.

2008 Sep;264(3):275-87.

Weisenthal LM, Patel N, Rueff-Weisenthal C.

Weisenthal Cancer Group, Huntington Beach, CA 92647, USA. mail@weisenthal.org

BACKGROUND:

Angiogenesis studies are limited by the clinical relevance of laboratory model systems. We developed a new method for measuring dead microvascular (MV) cells in clinical tissue, fluid and blood specimens, and applied this system to make several potentially novel observations relating to cancer pharmacology.

METHODS:

Dead MV cells tend to have a hyperchromatic, refractile quality, further enhanced during the process of staining with Fast Green and counterstaining with either haematoxylin-eosin or Wright-Giemsa. We used this system to quantify the relative degree of direct antitumour versus anti-MV effects of Cisplatin, erlotinib (Tarceva), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), gefitinib (Iressa) and bevacizumab (Avastin).

RESULTS:

Bevacizumab (Avastin) had striking anti-MV effects and minimal antitumour effects; Cisplatin had striking antitumour effects and minimal anti-MV effects. The 'nib' drugs had mixed antitumour and anti-MV effects. Anti-MV effects of erlotinib (Tarceva) and gefitinib (Iressa) were equal to those of sunitinib (Sutent) and sorafenib (Nexavar). There was no detectable VEGF in culture medium without cells; tumour cells secreted copious VEGF, reduced to undetectable levels by bevacizumab (Avastin), greatly reduced by cytotoxic levels of cisplatin + anguidine, and variably reduced by DMSO and/or ethanol. We observed anti-MV additivity between bevacizumab (Avastin) and other drugs on an individual patient basis. Peripheral blood specimens had numerous MV cells which were strikingly visualized for quantification with public domain image analysis software using bevacizumab (Avastin) essentially as an imaging reagent.

CONCLUSIONS:

This system could be adapted for simple, inexpensive and sensitive/specific detection of tissue and circulating MV cells in a variety of neoplastic and non-neoplastic conditions, and for drug development and individualized cancer treatment.

http://onlinelibrary.wiley.com/doi/10.1 ... 955.x/full

[gpawelski]

THE PROMISE

One of the most promising areas of cancer treatment today is also among the most perplexing. A new class of anti-cancer drugs works by interfering with the formation of capillaries which deliver blood to the tumor mass. Eliminating the blood supply to the tumor starves cancer cells of oxygen and nutrients, interferes with the elimination of cellular wastes, shuts-down routes of tumor metastasis, and potentially aids in the delivery of other types of anti-cancer drugs to the tumor mass.

THE PROBLEM

The problem is that the new drugs – called anti-angiogenesis drugs – work for only a small percentage of patients. Moreover, they can cause serious side effects in some patients and they are extremely expensive – well over $100,000 per year of treatment. Anti-angiogenesis drugs are being used more and more frequently in a widening range of cancer types and so the cost to the healthcare system and to individual patients who must pay for insurance co-payments threatens to be staggering. In fact, several new drugs have now shown anti-angiogenesis activity and these are being combined with standard drugs and with other targeted drugs to produce the maximum therapeutic benefit. A critical but previously-elusive challenge, therefore, has been to develop methods to identify exactly which patients could benefit from anti-angiogenesis therapy, which anti-angiogenesis drugs are best for these patients, and precisely which additional drugs, if any, should be administered concurrently in order to achieve the best result for each patient.

THE SOLUTION

Using a laboratory test which he invented, Dr. Weisenthal made the discovery that endothelial cells are present in disaggregated tumor micro-clusters. Previously, this was unknown in the medical literature. Endothelial cells form the capillaries which supply blood to tumor cells. Building on this discovery, Dr. Weisenthal developed the AngioRx microvascular viability assay. To date, the AngioRx assay is the only laboratory test which appears to be capable of identifying anti-angiogenic drug activity in live tumor micro-clusters. Other capabilities exclusive to Dr. Weisenthal’s AngioRx assay are:

•Discriminates anti-tumor effect from anti-angiogenic effect within mixed-populations of tumor, endothelial, and other cell types. It is extremely important to understand the mechanism by which treatment works for each patient.

•Measures the effects of different anti-angiogenic drugs within the same drug class. This is essential for pin-pointing which specific anti-angiogenic drug will work best for each patient - several such drugs are available.

•Measures the single-agent activity of specific anti-angiogenic drugs, standard cytotoxic drugs, and EGFR-targeted drugs. This can be the key to identifying potentially-beneficial drug combinations for each patient.

•Identifies synergistic effects which can occur among specific anti-angiogenic and non-anti-angiogenic drugs in rationally-selected drug combinations. This exclusive capability becomes particularly critical when combining cytotoxic drugs with anti-vascular drugs

Weisenthal Cancer Group