Tumor profiling

I asked my onc about this
I asked my onc about this before I started chemo. He said that the tumor cells change once they are out of the body, and the testing results can be unreliable. My aunt had this done a few years ago and credits it with saving her life. I'll be very interested in reading other responses to this post.
Roger

Crow71 said:

I asked my onc about this
I asked my onc about this before I started chemo. He said that the tumor cells change once they are out of the body, and the testing results can be unreliable. My aunt had this done a few years ago and credits it with saving her life. I'll be very interested in reading other responses to this post.
Roger

I will also
be interested in the information gathered regarding this. I had read about an oncologist in California that had been doing something like this. I had forgotten about it until I read your post. I hope this is a viable option for your husband.
Regards,
Joanne

Wonders....


Wouldn't it be wonderful if the multi-billions of dollars were
spent on research in an attempt to fix the reason why our own
immune system can fail to remove cancer cells the way it was
intended to...... instead of trying to find some chemical to
do the killing? (The chemical action is only temporary).

You can get rid of crabgrass by spraying chemicals and killing
everything else with the crabgrass, or promoting the growth
of good grass, that will choke out the crabgrass...

Nature can be so simple, when we allow it to be.


(just another personal opinion from the peanut gallery)

Why Community Oncology Can Benefit From Cell Function Analysis
Roger is correct, critics of the cell-based assay procedure contend that cells do not necessarily react the same in the laboratory (in vitro) as they do in the body (in vivo). Established "cell-line" is not reflective of the behavior of "fresh" live tumor cells in primary culture in the lab, much less the patient. You get different results when you test passaged "cell-lines" compared to primary, fresh tumors. Cell-lines are paraffin-embedded, preserved in paraffin wax.

However, tests that are performed using intact, living cancer cells plated in 3D microclusters, is indicative of what will happen in the human body. Three-dimensional microclusters of tumor cells are isolated from fresh tumor biopsy specimens and cultured for 96 hours (polypropylene, round-bottomed, 96-well microplates) in the presence and absence of test drugs. Established cell-line is not reflective of the behavior of "fresh" live tumor cells.

Established cell-lines have been a huge disappointment over the decades, with respect to their ability to correctly model the disease-specific activity of drugs for cancers. In other words, cancer cell-lines have proved worthless as models to predict the activity of drugs in cancer patients.

Cell-lines are useful for special stains and immunohistochemistry and can give morphological details by preserving the architectural patterns. However, cell-lines are paraffin-embedded and paraffin-embedded tissue can change over time.

As increasing numbers and types of anti-cancer drugs are developed, oncologists become increasingly likely to misuse them in their practice. There is seldom a "standard" therapy which has been proven to be superior to any other therapy. When all studies are compared by meta-analysis, there is no difference. What may work for one, may not work for another.

Cancer chemotherapy could save more lives if pre-testing were incorporated into clinical medicine. The respected cancer journals are publishing articles that identify safer and more effective treatment regimens, yet few community oncologists are incorporating these synergistic methods into their clinical practice. Cancer patients suffer through chemotherapy sessions that do not integrate all possibilities.

Distinguishing between patients with a "high" or "low" risk for early recurrence after surgical resection and identifying those who may respond to correct adjuvant therapy have been topics of great interest for many years. Both genetic and functional assay analyses share a role in the development of "personalized" patient care.

A genomic test can help to find out if a cancer patient will likely have a recurrence after surgery. If a recurrence isn't likely, they don't need chemotherapy. Genetic tests have been developed for breast and lung cancers. Hopefully, there will be more tests for other types of cancer to guide physicians as to which "high" risk patient will likely have a recurrence if treated with surgery alone (1).

If the test finds a patient to be at "high" risk, it is impossible to design a single chemotherapy protocol that is effective against all types of cancer. The oncologist might need to administer several chemotherapy drugs at varying doses because tumor cells express survival factors with a wide degree of individual cell variability. A cell culture assay test, using a cell-death endpoint, 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 (2).

The chemotherapy regimen chosen by most community oncologists is based on the type of cancer being treated. However, there are factors other than the type of cancer that can be used to determine the ideal chemotherapy drugs that should be used to treat an individual patient.

It is highly desirable to know what drugs are effective against particular cancer cells before these toxic agents are systemically administered. Pre-testing on "fresh" specimens of cancer cells to determine the optimal combination of chemotherapy drugs could be highly beneficial.

Following the collection of "fresh" cancer cells obtained at the time of biopsy or surgery, 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. The treatment program developed through this approach is known as assay-directed therapy.

At present, medical oncologists prescribe chemotherapy according to "fixed" schedules. These schedules are standardized drug regimens that correspond to specific cancers by type or diagnosis. These regimens, developed over many years of clinical trials, assign patients to the drugs which previously worked for some percentage of patients.

However, cancer is a disease whose hallmark is heterogeneity. It is well known that drugs which work for one patient often don't work for another and patients who fail to respond to first line chemotherapy with one regimen often respond to second or third line therapy with alternative drugs. Why not identify the right regimen before ever exposing a patient to a single course of chemotherapy? A failed attempt at chemotherapy is detrimental to the physical and emotional well being of patients, is financially burdensome, and may promote the onset of clinically acquired multi-drug resistance.

A "fresh" sample tumor can be obtained from surgery or biopsy (Tru-cut needle biopsies). Tissue, blood, bone marrow, and ascites and pleural effusions are possibilities, providing tumor cells are present, and only live cells should be used. At least one gram of fresh biopsy tissue is needed to perfom the tests, and a special kit is obtained in advance from the lab. Arrangements are made with the surgeon and/or pathologist for preparation and sending of the specimen.

Upgrading clinical therapy by using drug sensitivity assays measuring "cell death" of three dimensional microclusters of live "fresh" tumor cells can improve the conventional situation by allowing more drugs to be considered. The key to improving drug sensitivity tests is related to the number and types of drugs tested. The more anti-cancer drug types there are in the selective arsenal, the more likely the system is to prove beneficial.

In order to acquire sufficient data, tumors should be tested with at least two assay endpoints, and most often three, for sensitivity tests in any one patient. On average, up to twenty drugs and combinations at two concentrations in three different assay systems, is an effective way to avoid false-positive or false-negative data.

Assays based on "cell-death" occur in the entire population of tumor cells, as opposed to only in a small fraction of the tumor cells occurring in "cell-growth" assays. Drug "sensitivity" testing is merely a point a little farther along on the very same continuum upon which "resistance" testing resides, which has been proven to be accurate and reliable, as reported in numerous peer-reviewed publications.

Good review papers exist on cell culture assays and are increasingly appreciated and applied in the private sector by European clinicians and scientists. The literature on these assays have not been understood by many NCI investigators and by NCI-funded university investigators, because their knowledge has been based largely on an assay technique (cell-growth) that hasn't been used in most private labs for over fifteen years (3).

Data show conclusively that patients benefit both in terms of response and survival from drugs and drug combinations found to be "active" in the assay 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.

Patients receiving a drug that tested "sensitive" were 1.44 times [i.e. 44%] more likely to respond compared to all patients treated in studies, while patients testing "resistant" were 0.23 as likely to respond as all patients. Patients receiving treatment with drugs testing "sensitive" enjoyed a 6-fold advantage (1.44/0.23 = 6.23) over patients treated with drugs testing "resistant."

This data includes both patients with solid tumors (e.g., breast cancer, lung cancer) and hematological (blood system) tumors (e.g. leukemia, lymphoma). In the case of solid tumors only, the advantage to receiving sensitive versus resistant drugs was 9.3 fold. In the case of breast cancer, it was more than 10-fold. Furthermore, patients receiving "sensitive" drugs were shown in many studies to enjoy significantly longer durations of survival than patients treated with "resistant" drugs.

Patients treated with a "positive" (sensitive) drug would respond 79.1% of the time, while patients treated with a "negative" (resistant) drug would respond only 12.6% of the time. Once again, there would be a huge advantage to the patient to receive a "positive/sensitive" drug, compared to a "negative/resistant" drug (4).

Profiles from DNA and RNA expression analysis can help define patients at risk for early recurrence. Cell Culture Assays with "functional profiling" have a role in eliminating ineffective agents and avoid unnecessary toxicity and in directing "correct" therapy.

An ASCO tech review of drug sensitivity and resistance assays, concluded that the use of these assays for selection of chemotherapeutic agents for individual patients is not recommended outside the clinical trial setting (5).

However, Medicare contractor National Heritage Insurance Company spent six months reviewing everything about the cell culture assay, read all of ASCO arguments, and upon reviewing all available information, made the decision to reverse trend and go on record as formally approving the service and providing coverage.

They found that even back in 1999, the Medicare Advisory Panel concluded that cell culture assays tests offered clinical utility. After listening to detailed clinical evidence, the Medicare Coverage Advisory Committee found that these assay systems can aid physicians in deciding which chemotherapies work best in battling an individual patient's form of cancer (6).

Although Medicare had been reimbursing for cell culture drug "resistance" tests since 2000, it wasn't until the beginning of this year that they abandoned the artificial distinction between "resistance" testing and "sensitivity" testing and are providing coverage for the whole FDA-approved kit. The decision had been made that the assay is a perfectly appropriate medical service, worthy of coverage on a non-investigational basis (7).

References:

1. J Thorac Cardiovasc Surg 2007;133:352-363. Chemotherapy Resistance and Oncogene Expression in NSCLC.

2. J Clin Onco, 2006 ASCO Annual Meeting Proceedings Part 1. Vol 24, No. 18S (June 20 Supplement), 2006: 17117. Genfitinib-induced cell death in short term fresh tumor cultures predicts for long term patient survival in previously-treated NSCLC.

3. Eur J Clin Invest, Volume 37(suppl. 1):60, April 2007. Functional profiling with cell culture-based assays for kinase inhibitors and anti-angiogenic agents.

4. 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 (CCDRT) May, 2002.

5. Journal of Clinical Oncology Reviews on Chemotherapy Sensitivity and Resistance Assays, September1,2004.

6. Verbatim Transcript of Medicare Coverage Advisory Committee (MCAC) Meeting, November 15-16, 1999.

7. Centers for Medicare & Medicaid Services

gdpawel said:

Why Community Oncology Can Benefit From Cell Function Analysis
Roger is correct, critics of the cell-based assay procedure contend that cells do not necessarily react the same in the laboratory (in vitro) as they do in the body (in vivo). Established "cell-line" is not reflective of the behavior of "fresh" live tumor cells in primary culture in the lab, much less the patient. You get different results when you test passaged "cell-lines" compared to primary, fresh tumors. Cell-lines are paraffin-embedded, preserved in paraffin wax.

However, tests that are performed using intact, living cancer cells plated in 3D microclusters, is indicative of what will happen in the human body. Three-dimensional microclusters of tumor cells are isolated from fresh tumor biopsy specimens and cultured for 96 hours (polypropylene, round-bottomed, 96-well microplates) in the presence and absence of test drugs. Established cell-line is not reflective of the behavior of "fresh" live tumor cells.

Established cell-lines have been a huge disappointment over the decades, with respect to their ability to correctly model the disease-specific activity of drugs for cancers. In other words, cancer cell-lines have proved worthless as models to predict the activity of drugs in cancer patients.

Cell-lines are useful for special stains and immunohistochemistry and can give morphological details by preserving the architectural patterns. However, cell-lines are paraffin-embedded and paraffin-embedded tissue can change over time.

As increasing numbers and types of anti-cancer drugs are developed, oncologists become increasingly likely to misuse them in their practice. There is seldom a "standard" therapy which has been proven to be superior to any other therapy. When all studies are compared by meta-analysis, there is no difference. What may work for one, may not work for another.

Cancer chemotherapy could save more lives if pre-testing were incorporated into clinical medicine. The respected cancer journals are publishing articles that identify safer and more effective treatment regimens, yet few community oncologists are incorporating these synergistic methods into their clinical practice. Cancer patients suffer through chemotherapy sessions that do not integrate all possibilities.

Distinguishing between patients with a "high" or "low" risk for early recurrence after surgical resection and identifying those who may respond to correct adjuvant therapy have been topics of great interest for many years. Both genetic and functional assay analyses share a role in the development of "personalized" patient care.

A genomic test can help to find out if a cancer patient will likely have a recurrence after surgery. If a recurrence isn't likely, they don't need chemotherapy. Genetic tests have been developed for breast and lung cancers. Hopefully, there will be more tests for other types of cancer to guide physicians as to which "high" risk patient will likely have a recurrence if treated with surgery alone (1).

If the test finds a patient to be at "high" risk, it is impossible to design a single chemotherapy protocol that is effective against all types of cancer. The oncologist might need to administer several chemotherapy drugs at varying doses because tumor cells express survival factors with a wide degree of individual cell variability. A cell culture assay test, using a cell-death endpoint, 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 (2).

The chemotherapy regimen chosen by most community oncologists is based on the type of cancer being treated. However, there are factors other than the type of cancer that can be used to determine the ideal chemotherapy drugs that should be used to treat an individual patient.

It is highly desirable to know what drugs are effective against particular cancer cells before these toxic agents are systemically administered. Pre-testing on "fresh" specimens of cancer cells to determine the optimal combination of chemotherapy drugs could be highly beneficial.

Following the collection of "fresh" cancer cells obtained at the time of biopsy or surgery, 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. The treatment program developed through this approach is known as assay-directed therapy.

At present, medical oncologists prescribe chemotherapy according to "fixed" schedules. These schedules are standardized drug regimens that correspond to specific cancers by type or diagnosis. These regimens, developed over many years of clinical trials, assign patients to the drugs which previously worked for some percentage of patients.

However, cancer is a disease whose hallmark is heterogeneity. It is well known that drugs which work for one patient often don't work for another and patients who fail to respond to first line chemotherapy with one regimen often respond to second or third line therapy with alternative drugs. Why not identify the right regimen before ever exposing a patient to a single course of chemotherapy? A failed attempt at chemotherapy is detrimental to the physical and emotional well being of patients, is financially burdensome, and may promote the onset of clinically acquired multi-drug resistance.

A "fresh" sample tumor can be obtained from surgery or biopsy (Tru-cut needle biopsies). Tissue, blood, bone marrow, and ascites and pleural effusions are possibilities, providing tumor cells are present, and only live cells should be used. At least one gram of fresh biopsy tissue is needed to perfom the tests, and a special kit is obtained in advance from the lab. Arrangements are made with the surgeon and/or pathologist for preparation and sending of the specimen.

Upgrading clinical therapy by using drug sensitivity assays measuring "cell death" of three dimensional microclusters of live "fresh" tumor cells can improve the conventional situation by allowing more drugs to be considered. The key to improving drug sensitivity tests is related to the number and types of drugs tested. The more anti-cancer drug types there are in the selective arsenal, the more likely the system is to prove beneficial.

In order to acquire sufficient data, tumors should be tested with at least two assay endpoints, and most often three, for sensitivity tests in any one patient. On average, up to twenty drugs and combinations at two concentrations in three different assay systems, is an effective way to avoid false-positive or false-negative data.

Assays based on "cell-death" occur in the entire population of tumor cells, as opposed to only in a small fraction of the tumor cells occurring in "cell-growth" assays. Drug "sensitivity" testing is merely a point a little farther along on the very same continuum upon which "resistance" testing resides, which has been proven to be accurate and reliable, as reported in numerous peer-reviewed publications.

Good review papers exist on cell culture assays and are increasingly appreciated and applied in the private sector by European clinicians and scientists. The literature on these assays have not been understood by many NCI investigators and by NCI-funded university investigators, because their knowledge has been based largely on an assay technique (cell-growth) that hasn't been used in most private labs for over fifteen years (3).

Data show conclusively that patients benefit both in terms of response and survival from drugs and drug combinations found to be "active" in the assay 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.

Patients receiving a drug that tested "sensitive" were 1.44 times [i.e. 44%] more likely to respond compared to all patients treated in studies, while patients testing "resistant" were 0.23 as likely to respond as all patients. Patients receiving treatment with drugs testing "sensitive" enjoyed a 6-fold advantage (1.44/0.23 = 6.23) over patients treated with drugs testing "resistant."

This data includes both patients with solid tumors (e.g., breast cancer, lung cancer) and hematological (blood system) tumors (e.g. leukemia, lymphoma). In the case of solid tumors only, the advantage to receiving sensitive versus resistant drugs was 9.3 fold. In the case of breast cancer, it was more than 10-fold. Furthermore, patients receiving "sensitive" drugs were shown in many studies to enjoy significantly longer durations of survival than patients treated with "resistant" drugs.

Patients treated with a "positive" (sensitive) drug would respond 79.1% of the time, while patients treated with a "negative" (resistant) drug would respond only 12.6% of the time. Once again, there would be a huge advantage to the patient to receive a "positive/sensitive" drug, compared to a "negative/resistant" drug (4).

Profiles from DNA and RNA expression analysis can help define patients at risk for early recurrence. Cell Culture Assays with "functional profiling" have a role in eliminating ineffective agents and avoid unnecessary toxicity and in directing "correct" therapy.

An ASCO tech review of drug sensitivity and resistance assays, concluded that the use of these assays for selection of chemotherapeutic agents for individual patients is not recommended outside the clinical trial setting (5).

However, Medicare contractor National Heritage Insurance Company spent six months reviewing everything about the cell culture assay, read all of ASCO arguments, and upon reviewing all available information, made the decision to reverse trend and go on record as formally approving the service and providing coverage.

They found that even back in 1999, the Medicare Advisory Panel concluded that cell culture assays tests offered clinical utility. After listening to detailed clinical evidence, the Medicare Coverage Advisory Committee found that these assay systems can aid physicians in deciding which chemotherapies work best in battling an individual patient's form of cancer (6).

Although Medicare had been reimbursing for cell culture drug "resistance" tests since 2000, it wasn't until the beginning of this year that they abandoned the artificial distinction between "resistance" testing and "sensitivity" testing and are providing coverage for the whole FDA-approved kit. The decision had been made that the assay is a perfectly appropriate medical service, worthy of coverage on a non-investigational basis (7).

References:

1. J Thorac Cardiovasc Surg 2007;133:352-363. Chemotherapy Resistance and Oncogene Expression in NSCLC.

2. J Clin Onco, 2006 ASCO Annual Meeting Proceedings Part 1. Vol 24, No. 18S (June 20 Supplement), 2006: 17117. Genfitinib-induced cell death in short term fresh tumor cultures predicts for long term patient survival in previously-treated NSCLC.

3. Eur J Clin Invest, Volume 37(suppl. 1):60, April 2007. Functional profiling with cell culture-based assays for kinase inhibitors and anti-angiogenic agents.

4. 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 (CCDRT) May, 2002.

5. Journal of Clinical Oncology Reviews on Chemotherapy Sensitivity and Resistance Assays, September1,2004.

6. Verbatim Transcript of Medicare Coverage Advisory Committee (MCAC) Meeting, November 15-16, 1999.

7. Centers for Medicare & Medicaid Services

Cell Death Assays (Apoptosis)
Scientists know that cancer is not a disease in which cells grow too abundantly, but the failure of cells to expire at their appointed time. Cancer does not grow too much, it dies too little. Most basic biologists acknowledge that cancer is characterized by a failure of programmed cell death (apoptosis).

Apoptosis is shown to be an orderly series of bio-chemical events leading to a variety of morphological changes. These chenages include loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation.

In the average human adult, between 50 and 70 billion cells die each day due to programmed cell death. In a course of a year, this adds up to cells that equal the weight of the individual. Programmed cell death is a process with a strict genetic programme. The cell death machinery has deep evolutionary roots.

The scientific basis of the programmed cell death endpoint in assay testing comes from published observation that the most robust cell-death endpoint is delayed loss of membrane integrity. This endpoint has been shown to correlate with both response and survival in human cancers. Detecting this loss of membrane integrity is the principle underlying all newer cell-death assays.

Cell-death assays utilizing functional profiling, use apoptotic endpoints and also a number of other indicators of programmed cell death. The advantage of this is that they are more reflective of chemotherapy's actual effects in the human body.

It uses a combination of the morphologic endpoint (DISC) and one or more of the metabolic endpoints (MTT, ATP, resazurin) to test the targeted molecular drugs. The combination of measuring morphologic (structural) effects and metabolic (cell metabolism) effects constitutes the measuring of a "profile" at the whole cell level.

The DISC assay, and a variant of it called EVA, the entire contents of the cell culture are cytocentrifuged onto permanent microscope slides (for direct visualization of tumor cells and permanent archival record) and differentially stained to allow discrimination of normal and neoplastic cells and living and dead cells. The endpoint for cell-death is delayed loss of membrane integrity, which has been found to be a surrogate for apoptosis (cell-death). The DISC assay was the first of the new-generation functional tumor cell profiling methods to feature the "cell-death" endpoint, upon which nearly all new-generation functional profiling assays are based.

The MTT assay measures mitochondrial metabolism in the entire cell culture. In the assay, yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form purple formazan. The color can then be quantified by spectrophotometry, enabling an accurate measurement of metabolic activity.

The ATP (Adenosine Triphosphate) assay measures cellular ATP content by luminometry, based on the luciferin/luciferase reaction. Cells maintain a critical ATP thresholds whose measurement reflects cell viability, specifically indicating, in functional tumor cell profiling, whether apoptotic cell death has occurred during drug exposure.

The redox (resazurin) assay measures total metabolic activity in the entire cell culture, using the Alamar Blue reagent.

The caspase 3/7 assay measures the activation of caspases 3 and 7 using luminometry.

All of the above are cell-death endpoints. Some of the different types of cell-death assays:

The TCR Assay
ChemoFX Assay
The MiCK Assay
HDRA Assay
EVA Assay
DISC Assay
MTT Assay
ATP Assay
Fluorescein Diacetate Assay

Real life 3D analysis makes functional profiling indicative of what will happen in the body. It tests fresh "live" cells in their three dimensional (3D), floating clusters (in their natural state). Solid tumor specimens are cultured in concical polypropylene microwells for 96 hours to increase the proportion of tumor cells, relative to normal cells.

Polypropylene is a slippery material which prevents the attachment of fibroblasts and epithelial cells and encourages the tumor cells to remain in the form of three dimensional (3D), floating clusters. Our body is 3D, not 2D in form, undoubtedly, making this novel step better replicate that of the human body.