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Old 02-14-2010, 12:52 PM   #1
Rich66
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Chemosensitivity prediction

Below are some extracts and links from posts by "GD Pawel", cell test evangelist:



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. [NOTE: As of 2010, Medicare coverage/acceptance is spotty] 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


(Additional discussion by GDPawel here)


As for as an extensive listing for some of the reputable laboratories providing cell culture assay testing, these labs will provide you and your physician with in depth information and research on the testing they provide:

The first laboratory provides a "cell growth" assay. The assay is excellent at identifying drugs most likely "not" to work (drug resistance). The assay is not as good at identifying drugs which are "more likely" to work (drug sensitivity) or to identify the disease-specific activity patterns of new targeted drugs. Used for drug de-selection and not for drug selection.

Exiqon (formally Oncotech, Inc.)
http://www.exiqon.com/dx

All the below laboratories provide a "cell death" assay. At one time, the goal of cancer treatment was to inhibit unregulated cell "growth." In the last twenty years, the goal is to induce cell "death" in order to successfully conquer concer. The much older "cell-growth" assays measure a drug's ability to inhibit cell "growth" and only succeeds in eliminating drugs that would "not" work for a patient (drug resistance). The more modern "cell-death" assays measure the ability of chemotherapy drugs to induce cell "death" (apoptosis) in a tumor biopsy from a patient (drug sensitivity). The assays are excellent at identifying drugs most likely "not" to work and identify drugs which are "more likely" to work.

I understand from Exiqon's year-end stock reports that Precision Therapeutics has a cell-growth assay to compete with Exiqon's EDR cell-growth assay.

Anticancer, Inc., San Diego, CA.
http://www.anticancer.com/

Cancer Therapeutics, Inc., Thomasville, GA.
http://www.cancer-therapeutics.com/

DiaTech Oncology, Brentwood, TN.
http://diatech-oncology.com/
[Can work with needle biopsy amount, results in a few days. Tests at 3 dose levels. Results have shown benefits of retrying "failed" drugs at lower doses. Medicare coverage being pursued but not yet approved. $3,000 but no one turned away because of $$. (2/2010) Former ASCO president directly involved.]

R. Garry Latimer
CEO

DiaTech Oncology
www.diatech-oncology.com
College of American Pathologists #7186701

CLIA #99D1030993

9208 Heritage Dr.

Brentwood, Tennessee 37027

615 377 9668 Voice

877-434-2832 Toll Free

615 221 4387 Fax



Cary Presant, M.D., F.A.C.P.
Wilshire Oncology Medical Group - California Cancer Medical Center
Professor of Clinical Medicine, University of Southern California Keck
School of Medicine
Past President, Association of Community Cancer Centers (ACCC)
Chairman of the Board, Medical Oncology Association of Southern California
(MOASC)
Past Director, American Society of Clinical Oncology
Chairman, Los Angeles Cancer Institute
Past President, American Cancer Society, California Division
Director of Medical Oncology, DiaTech Oncology Corporation

mailing address: clinical office

Cary Presant, M.D.
Wilshire Oncology Medical Group
1250 South Sunset Avenue, Suite 303
West Covina, California 91790

Phone 626 856 5858
Fax 626 796 5239


Genomic Health, Inc. Redwood City, CA.
http://www.genomichealth.com/OncotypeDX/Index.aspx

Genoptix, Inc., San Diego, CA
http://www.genoptix.com/

Precision Therapeutics, Pittsburgh, PA. ChemoFX test
http://www.precisiontherapeutics.com/ (Medicare covered, grows out cells for 3 to 4 weeks from sample as small as needle biopsy or 100cc malignant fluid, pick up to 12 drugs and combinations depending on growth of cells, results 4-5 weeks from submission, endocrine and bisphosphonates allowed during biopsy)

The ChemoFx technology grew out of work by Dr. Paul Kornblith, who described an assay system in which cells were pre-cultured in monolayers for a period of days to more than a week and then treated with drugs.

The assay endpoint is the detachment of cells from the surface of the culture plates, which correlates very well with loss of viability. The assays is "conceptually" a cell-death assay, although one which is performed on pre-cultured (amplified) cells.

It is a monolayer assay, which may have conceptual problems. In vitro (in the lab) drug activity correlated with in vivo (in the body) drug activity when tumors are tested in vitro as three dimensional (3D) clusters, but not when they are tested in two dimensional (2D) monolayers.

All published clinical correlations with fresh (live) tumor assays with cell-death endpoints have tested the tumor cells largely in the form of three dimensional (3D) clusters.

It is testing a subcultured cell population and studies by the NCI Navy medical oncology branch did not find that monolayer assays performed on pre-cultured, pre-amplified tumor cells clearly gave clinically relevant results.

However, taken in context of a growing number of studies showing correlations between the results of cell-death assays and patient survival in ovarian cancer, these results indicate that ChemoFx is capable of providing clinically relevant information and is conceptually a "cell-death" assay.

Gallion, H. H., W. A. Christopherson, et al. (Jan/Feb 2006). "Relationship between ex vivo chemosensitivity assay and progression free interval in ovarian cancer." International Journal of Gynecological Cancer

http://www.ncbi.nlm.nih.gov/pubmed/1...?dopt=Abstract





The two laboratories below, in addition to providing a "cell death" assay, provide a functional profiling assay which is the only testing that involves direct visualization of the cancer cells at endpoint. This allows for accurate assessment of drug activity, discriminates tumor from non-tumor cells, and provides a permanent archival record, which improves quality, serves as control, and assesses dose response in vitro (includes newly-emergent drug combinations). These two assay labs have about the most extensive information about the technology with knowledge spanning decades, utilize functional tumor cell profiling. Besides identifying drugs most likely "not" to work (drug resistance) and identify drugs which are "more likely" to work (drug sensitivity), these assays can identify the disease-specific activity patterns of new targeted drugs.

Rational Therapeutics Institute, Long Beach, CA.
(No Medicare coverage ($3,500), 1cm sample or malignant fluid, 8-16 drugs/combinations depending on sample size, endocrine and bisphosphonates allowed during biopsy, no Herceptin or Tykerb 2 weeks prior 9/2010 response: "If the cells have been recently treated, then they will be compromised by that treatment, and then the drugs we add will deem the results more sensitive than if just treated with one therapy. (Hopefully that makes sense). We prefer a 2-3 week treatment free period. Biologics and hormonal agents may not directly induce cell death, so a shorter timeframe may be OK, but if you are going to do this, we want the results to be as accurate as possible.")
http://www.rational-t.com/patients/extra.aspx
Physician/Patient Relations
Rational Therapeutics
750 East 29th Street, Long Beach, CA 90806-1402
Ph 562-989-6455, x103; Fx 562-989-8160






Weisenthal Cancer Group, Huntington Beach, CA.
http://weisenthalcancer.com/
(No Medicare coverage ($2-6,000 w/financing), 1-2gm sugar cube sized sample or multiple needle biopsy for 5-8 tests, 20-25 tests the norm, includes some Tamoxifen synergy combinations, includes Rational T's Disc assay as well as overlapping metabolic tests, 7-10 days for results, endocrine and bisphosphonates allowed during biopsy)


GD Pawel's commentary continued:

The most common statement about tumor testing from doctors (who still mistakenly refer to two distinct "older" types of assays, which hasn't been used in private labs in over twenty years) is that test tube reactions don't always represent real treatment reactions.

With the limitations of old cell culture assay technology, which only used two dimensions (2D) from which information is gathered, the newer cell culture technology works with three dimensions (3D), which is able to provide data on the behavior of cells towards cancer drugs when they are in a complex relationship which resembles or mimics their situation inside the human body.

Rational Therapeutics and Weisenthal Cancer Group protocol takes "fresh" patient tumor cells and floats them in newer 3D cell suspensions
. Our body is 3D, not 2D in form. This novel step better replicates that of the human body.

Traditionally, in-vitro (in lab) cell-lines have been studied in 2 dimensions which therefore had inherent limitations in applicability to real life 3D in-vivo (in body) states (3D analysis, more reflective of our bodies natural state).

The bottom line is the functional tumor cell profiling assay is able to predict the patient's response to treatment. It does this by measuring the net effect of all processes within the cancer, acting with and against each other in real-time because it tests fresh (live) cells, in their natural state, actually exposed to drugs and drug combinations of interest, before they are administered to the patient.



Paraffin embedded, fixed, minced, or frozen tissue can change over time. One gets more accurate information when using intact RNA isolated from "fresh" living tissue than from using degraded RNA, which is present in paraffin-fixed tissue (old tissue).

With paraffin embedded and cell-lines, investigators can only measure those analytes (analytical procedure) in paraffin that they "know" to measure. That is, if you are not aware of and capable of measuring a biologically relevant event, you cannot seek to detect it.

As to cell lines, these proliferating populations are biologically distinct in their behavior from the relatively quiescent (inactive) cells that comprise human tumors.

While scientists feel that certain well characterized markers like K-ras, Her2 or ERCC2 have relevance, the complexity of cell biology, the need to assess synergy and the need to assess sequence dependence can only be done in real-time cellular platforms.

One of the problems with genetic tests is a large number of archival specimens are batch processed together, within a very narrow time frame, by the same research team, so all the technical variables are minimized, which makes it much easier to get good results than in a "real world" setting, where specimens are tested over a period of weeks, months, years, by different people, with different laboratory reagents (substance added in order to bring about a chemical reaction), as occurs in the "real world."

Evaluating "real world" data, requires specimens that are tested as they are logged into the lab in question, in "real time." No one is publishing "real world" studies, except private laboratories performing cell-based assays, which can only do "real world" studies, because their studies require fresh, viable specimen, which must be accessioned and tested in "real time," under "real world" conditions.

Cell-based assays test fresh "live" cells in their three dimensional, floating clusters (in their natural state), not passaged cells (cell-lines). Established cell-line is not reflective of the behavior of "fresh" tumor cells in primary culture in the lab, much less in the patient. 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, floating clusters. Real life 3D analysis makes cell-based assays indicative of what will happen in the body.
----------------------------------------------------------



Different than above and less known...
Here's a blood based test from a lab in Greece:

http://www.rgcc-genlab.com/html/services.html


Rumored serum test at German clinic here: info@leonardis-klinik.de



Caris Target Now molecular profiling test
http://www.carislifesciences.com/oncology-target-now
Tumor Sample options/specs: PDF

Caris Target Now helps patients and their treating physicians create a cancer treatment plan based on the tumor tested. By comparing the tumor's information with data from published clinical studies by thousands of the world's leading cancer researchers, Caris can help determine which treatments are likely to be most effective and, just as important, which treatments are likely to be ineffective.
The Caris Target Now test is performed after a cancer diagnosis has been established and the patient has exhausted standard of care therapies or if questions in therapeutic management exists. Using tumor samples obtained from a biopsy, the tumor is examined to identify biomarkers that may have an influence on therapy. Using this information, Caris Target Now provides valuable information on the drugs that will be more likely to produce a positive response. Caris Target Now can be used with any solid cancer such as lung cancer, breast cancer, and prostate cancer.


1: Proc Natl Acad Sci U S A. 2009 May 19. [Epub ahead of print]
Chromosomal instability determines taxane response.

Swanton C, Nicke B, Schuett M, Eklund AC, Ng C, Li Q, Hardcastle T, Lee A, Roy R, East P, Kschischo M, Endesfelder D, Wylie P, Kim SN, Chen JG, Howell M, Ried T, Habermann JK, Auer G, Brenton JD, Szallasi Z, Downward J.
Cancer Research UK, London Research Institute, London WC2A 3PX, United Kingdom;
Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these "CIN-survival" genes is associated with poor outcome in estrogen receptor-positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.
Later article on CIN:

3/1/2020


Gene test aid to cancer treatment
Scientists have developed a gene test which predicts how well chemotherapy will work in cancer patients.
Starting with 829 genes in breast cancer cells, the team whittled down the possibilities to six genes which had an impact on whether a drug worked.
They then showed that these genes could be used to predict the effectiveness of a drug called paclitaxel in patients.
It is hoped the approach, reported in The Lancet Oncology, can be replicated for other cancers and treatments.
The international project, including researchers from Cancer Research UK's London Research Institute, opens the way for breast cancer treatment to be targeted to those who will benefit the most.
To find which genes, if missing or faulty, could prevent the drug from working, they deleted them one by one from cancer cells in the laboratory.
They eventually highlighted the six genes which if absent or not working prevent paclitaxel from properly killing breast cancer cells.
Spare treatment
More than 45,500 women are diagnosed with breast cancer in the UK each year - and it is estimated that around 15% of these women will be prescribed paclitaxel.
The researchers estimate they could potentially spare half of the patients currently receiving this drug from treatment which would not be effective.
Study leader, Dr Charles Swanton, head of translational cancer therapeutics at the Institute, said one of the great challenges in cancer medicine is determining which patients will benefit from particular cancer drugs, which are in themselves toxic and carry severe side effects.


The challenge is to apply these methods to other drugs in cancer medicine
Dr Charles Swanton, study leader.
"Our research shows it is now possible to rapidly pinpoint genes which prevent cancer cells from being destroyed by anti-cancer drugs and use these same genes to predict which patients will benefit from specific types of treatment."
Further studies will now be done to see if the technique can be developed into a simple diagnostic test to be given to patients to help inform doctors about whether or not to prescribe paclitaxel.
He said the challenge will be to apply these methods to other drugs in cancer medicine.
"These could include treatments that are currently deemed too expensive to fund on the NHS - however, in the future, treating only the patients that will benefit from certain treatments will save the NHS money in the long term."
Dr Lesley Walker, Cancer Research UK's director of cancer information, said: "New techniques such as these can enable drugs to be tailored to individual patients, and this could potentially improve cancer survival in the long term.
"Health professionals may in the future be able to use this information to direct treatment to patients most likely to benefit, and avoid giving treatment that is less likely to be effective to patients with drug resistant cancers."

Story from BBC NEWS:
http://news.bbc.co.uk/go/pr/fr/-/2/h...th/8539502.stm

Published: 2010/03/01 00:21:24 GMT

© BBC MMX
Attached Files
File Type: pdf mick assay_ResultReport(1).pdf (441.6 KB, 295 views)
File Type: pdf mick assay_ResultReport(2).pdf (480.4 KB, 265 views)
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Old 03-05-2010, 09:03 AM   #2
Rich66
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Re: Chemosensitivity prediction

Target Now, molecular profiling:
http://www.carislifesciences.com/oncology-target-now

Quote:
Target Now helps patients and their treating physicians create a cancer treatment plan based on the tumor tested. By comparing the tumor's information with data from published clinical studies by thousands of the world's leading cancer researchers, Caris can help determine which treatments are likely to be most effective and, just as important, which treatments are likely to be ineffective.
The Target Now test is performed after a cancer diagnosis has been established and the patient has exhausted standard of care therapies or if questions in therapeutic management exists. Using tumor samples obtained from a biopsy, the tumor is examined to identify biomarkers that may have an influence on therapy. Using this information, Target Now provides valuable information on the drugs that will be more likely to produce a positive response. Target Now can be used with any solid cancer such as lung cancer, breast cancer, and prostate cancer.
Quote:
What does my doctor need from me to perform the test?
Target Now is performed on tissue that is obtained during the surgical removal or biopsy of your tumor. Even if your doctor doesn’t order Target Now testing at this time, the hospital where your biopsy was performed will typically store some of your tissue as standard procedure. Now or in the future, your doctor can request that Target Now testing be run and coordinate with the hospital to have your sample sent to us.
Quote:
Is Target Now reimbursed?
The Target Now is typically reimbursed by Medicare and other third party payers. Other than co-payments or deductibles required by the patient's plan, there are normally no out-of-pocket costs for the patient



RESEARCH:

Prediction of breast cancer sensitivity to neoadjuvant chemotherapy based on status of DNA damage repair proteins


Breast Cancer Research 2010, 12:R17 doi:10.1186/bcr2486
Hideki Asakawa (hiddie_vr4@hotmail.com)
Hirotaka Koizumi (koizumi@marianna-u.ac.jp)
Ayaka Koike (a2koike@marianna-u.ac.jp)
Makiko Takahashi (takamaki@marianna-u.ac.jp)
Wenwen Wu (wuwenwen@marianna-u.ac.jp)
Hirotaka Iwase (hiwase@kumamoto-u.ac.jp)
Mamoru Fukuda (m2fukuda@marianna-u.ac.jp)
Tomohiko Ohta (to@marianna-u.ac.jp)
ISSN 1465-5411
Article type Research article
Submission date 18 September 2009
Acceptance date 5 March 2010
Publication date 5 March 2010
Article URL http://breast-cancer-research.com/content/12/2/R17
http://breast-cancer-research.com/co...df/bcr2486.pdf

Abstract
Introduction: Various agents used in breast cancer chemotherapy provoke DNA double-strand breaks (DSBs). DSB repair competence determines the sensitivity of cells to these agents whereby aberrations in the repair machinery leads to apoptosis. Proteins required for this pathway can be detected as nuclear foci at sites of DNA damage when the pathway is intact. Here we investigate whether focus formation of repair proteins can predict chemosensitivity of breast cancer.

Methods: Core needle biopsy specimens were obtained from sixty cases of primary breast cancer before and 18-24 hours after the first cycle of neoadjuvant epirubicin plus cyclophosphamide (EC) treatment. Nuclear focus formation of DNA damage repair proteins was immunohistochemically analyzed and compared with tumor response to chemotherapy.

Results: EC treatment induced nuclear foci of H2AX, conjugated ubiquitin, and Rad51 in a substantial amount of cases. In contrast, BRCA1 foci were observed
before treatment in the majority of the cases and only decreased after EC in
thirteen cases. The presence of BRCA1-, H2AX-, or Rad51-foci before treatment
or the presence of Rad51-foci after treatment was inversely correlated with tumor response to chemotherapy. DNA damage response (DDR) competence was further evaluated by considering all four repair indicators together. A high DDR score significantly correlated with low tumor response to EC and EC + docetaxel whereas other clinicopathological factors analyzed did not.

Conclusions: High performing DDR focus formation resulted in tumor resistance to DNA damage-inducing chemotherapy. Our results suggested an importance of evaluation of DDR competence to predict breast cancer chemosensitivity, and merits further studying into its usefulness in exclusion of non-responder patients.

Quote:
Introduction
Recent advances in chemotherapy have significantly improved the prognosis of breast cancer patients. However, prediction of tumor sensitivity to chemotherapy
has not reached a high level of confidence, whereas determining sensitivity to hormone therapy or trastuzumab is relatively more established. Estrogen and
progesterone receptors (ER and PR) and HER2/ErbB2 are practical benchmarks
to exclude non-responding patients, and tailoring treatment based on gene status significantly optimizes the response rate of hormone therapy and trastuzumab, respectively. Prediction of chemosensitivity with equivalent accuracy is currently anticipated to further improve breast cancer prognosis.
Anthracycline-based regimen, such as epirubicin plus cyclophosphamide (EC), and taxanes represent the major chemotherapeutic agents used in the breast cancer field[1, 2]. Of these, anthracycline-based chemotherapy induces DNA double-strand breaks (DSBs)[3, 4], the most cytotoxic DNA lesion, that leads cells into apoptosis especially when relevant repair pathways (represented by homologous recombination (HR) repair) are perturbed[5]. It is important to note that DNA damage repair competence varies among individual breast tumors and closely correlates with chemosensitivity. For example, secondary mutations of BRCA1 or 2 (essential factors in the HR pathway) caused by chemotherapy using cisplatin or poly(ADP-ribose) polymerase (PARP) inhibitor in BRCA1/2-mutated cancers restore the wild-type reading frame and, therefore, the tumor acquires resistance to these drugs [6-8].
These facts indicate that chemosensitivity of BRCA-associated cancers could be
strongly affected by DNA damage repair capability. Based on this evidence it has
been suggested that HR competence could be a potential biomarker for chemosensitivity [9]. Rad51, a protein that plays a direct role in HR, especially
reflects the HR-competence of cells. Therefore, knowing its status is likely
valuable when assessing HR-competence in tumor cells in order to instruct therapeutic decisions [9].

The HR pathway for DSB repair is executed by sequential recruitment of repair proteins to chromatin around DNA lesions. Accumulation of the proteins is regulated by complex mechanisms that utilize phosphorylation and ubiquitination
modifications mediated by kinases, including ATM, and at least three ubiquitin
E3 ligases, RNF8, RNF168, Rad18, and BRCA1 [10-17]. The Mre11-Rad50-Nbs1 complex first recognizes DSBs and recruits ATM. ATM then phosphorylates the histone variant H2AX (H2AX) [18, 19] that triggers accumulation of the downstream E3 ligases RNF8 [11-13, 20] and RNF168 [14,15]. Lysine 63 (K63)–linked polyubiquitin chains built at the sites of DNA damage by these E3 ligases next recruits the BRCA1-Abraxas-RAP80 complex through the RAP80 component, a protein that contains UIM (ubiquitin interacting motif) domains [21-23]. BRCA1 is then essential to recruit repair effector proteins, including Rad51, that perform HR through sister chromatid exchange [24, 25].
Depletion of any one of these proteins results in HR deficiency accompanied by
loss of Rad51 focus formation, causing cells to become hypersensitive to
DSB-inducing agents.
In this study we attempt to clarify the value of HR-competence for prediction of breast cancer chemosensitivity. One contention is that nuclear focus formation of repair proteins in baseline breast cancer tissues is a response to spontaneous DNA damage during cell proliferation and, in turn, may represent a marker of HR-competence of cells to exogenous DNA damage. Therefore, it may predict tumor response to DNA damage-inducing chemotherapy such as EC. Also, the focus formation after chemotherapy could provide us additional information regarding the DNA damage response capacity. To verify in vivo whether focus formation of repair proteins actually occurs in response to DNA damage-inducing chemotherapy and whether it correlates with tumor fates after chemotherapy, we analyzed foci in core needle biopsy specimens from breast cancer before and after neoadjuvant EC treatment.
Materials and methods
Patients and tumors: Sixty patients with primary breast cancer (2 cm or larger) who
consecutively underwent neoadjuvant chemotherapy with epirubicin plus cyclophosphamide (EC) followed by docetaxel (DOC) at the Division of Breast
and Endocrine Surgery, St. Marianna University School of Medicine, Japan, were enrolled in the present study from August 2005 to July 2007. Tumor specimens were obtained by core needle biopsy prior to starting therapy and 18 to 24 hours after the first cycle of EC treatment.
Quote:
We found that foci of BRCA1, H2AX and Rad51 prior to treatment and EC-induced foci of Rad51 correlated with tumor response when compared either with the mean tumor volume reduction or the tumor response rate. Upon incorporating these four factors into one DDR score, a significant correlation was observed with mean tumor volume reduction after EC, whereas no other factors correlated with the mean tumor volume reduction (Table 4, and Figure 3a). Although it was not statistically significant the similar correlation was also observed between DDR score and the tumor response rate (Table 3). These correlations became more significant after EC+DOC treatment (Table 3, 4, 5, and Figure 3b) and the DDR score was independent predictive factor of other factors including tumor subtype when evaluated with volume reduction using 50% of the PR/SD border (Table 6). Recent studies suggested that luminal tumors have low response rate to neoadjuvant chemotherapy, while basal-like and HER2+ tumors have higher response rates. For example it has been reported that clinical response rate (CR and PR) to anthracyclin-based chemotherapy of luminal A was 39% whereas that of basal-like, which has been implicated with BRCA1 dysfunction[44, 45], was 85%[46]. The response rates to EC treatment of luminal A (15/37 cases, 40.5%) and basal-like (4/6 cases, 66.7%) subtypes in the current study were not very different from the previous report. However, we could not find any correlation between subtype and DDR sore while DDR score independently predicted the chemosensitivity. The result may reflect the fact that luminal A tumors also include DNA damage-sensitive tumors with defective HR pathway that can be counted by the DDR score.
Supporting this it has been shown that tumors caused by BRCA2 deficiency mainly become luminal A tumor[45, 47, 48].
The reason why the correlation between the DDR score and tumor response after EC+DOC treatment became more significant than that after EC is not clear at present. Because DOC does not induce DNA double-strand breaks, the observed effect is not likely due to the sensitivity to DNA damage in those tumors. DOC might be more toxic for the cells with gross genomic aberration caused by the pretreatment with EC under the condition with less HR competent. Alternatively it is possible that time length after EC treatment enhanced the difference of the outcome.
Interestingly, DDR score group 4 consisted of cases with poor tumor responses to chemotherapy when evaluated for both mean tumor volume reduction (Figure 3) and tumor response rate (Table 3). This result may lead to the possibility of using DDR status in the clinic to predict and exclude non-responders to EC treatment. It is noteworthy to point out that the HR repair cascade for DSB contains many essential proteins other than that tested in this study. By including select subsets of proteins for analysis, it may be possible to identify non-responders in order to avoid unnecessary chemotherapy. Ideally in such cases, the levels of baseline foci present prior to treatment would provide enough information to determine appropriate treatment, preventing the need for additional core needle biopsy after chemotherapy.
Conclusions
In conclusion, our results suggest the importance of evaluating DDR competence to predict breast cancer chemosensitivity and warrant further investigation into its effectiveness as a way to exclude non-responding patients.

9/2010

24-hour Test Predicts Breast Cancer's Likely Response To Chemotherapy

A new test has been developed which can predict whether a breast cancer patient will respond to chemotherapy [adriamycin]within 24-hours of starting treatment, thus sparing her unnecessary treatment and side effects, according to a study published in the medical journal Clinical Cancer Research...

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Abstract

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Low RAD51 score was strongly predictive of pathological complete response to chemotherapy, with 33% low RAD51 score cancers achieving pathological complete response compared to 3% of other cancers (p=0.011).
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Old 06-27-2010, 08:20 AM   #3
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Studying Cells in 3-D Could Reveal New Cancer Targets

Showing movies in 3-D has produced a box-office bonanza in recent months. Could viewing cell behavior in three dimensions lead to important advances in cancer research? A new study led by Johns Hopkins University engineers indicates it may happen. Looking at cells in 3-D, the team members concluded, yields more accurate information that could help develop drugs to prevent cancer's spread.

The study, a collaboration with researchers at Washington University in St. Louis, appears in the June issue of Nature Cell Biology.

"Finding out how cells move and stick to surfaces is critical to our understanding of cancer and other diseases. But most of what we know about these behaviors has been learned in the 2-D environment of Petri dishes," said Denis Wirtz, director of the Johns Hopkins Engineering in Oncology Center and principal investigator of the study. "Our study demonstrates for the first time that the way cells move inside a three-dimensional environment, such as the human body, is fundamentally different from the behavior we've seen in conventional flat lab dishes. It's both qualitatively and quantitatively different."

One implication of this discovery is that the results produced by a common high-speed method of screening drugs to prevent cell migration on flat substrates are, at best, misleading, said Wirtz, who also is the Theophilus H. Smoot Professor of Chemical and Biomolecular Engineering at Johns Hopkins. This is important because cell movement is related to the spread of cancer, Wirtz said. "Our study identified possible targets to dramatically slow down cell invasion in a three-dimensional matrix."

When cells are grown in two dimensions, Wirtz said, certain proteins help to form long-lived attachments called focal adhesions on surfaces. Under these 2-D conditions, these adhesions can last several seconds to several minutes. The cell also develops a broad, fan-shaped protrusion called a lamella along its leading edges, which helps move it forward. "In 3-D, the shape is completely different," Wirtz said. "It is more spindlelike with two pointed protrusions at opposite ends. Focal adhesions, if they exist at all, are so tiny and so short-lived they cannot be resolved with microscopy."

The study's lead author, Stephanie Fraley, a Johns Hopkins doctoral student in Chemical and Biomolecular Engineering, said that the shape and mode of movement for cells in 2-D are merely an "artifact of their environment," which could produce misleading results when testing the effect of different drugs. "It is much more difficult to do 3-D cell culture than it is to do 2-D cell culture," Fraley said. "Typically, any kind of drug study that you do is conducted in 2D cell cultures before it is carried over into animal models. Sometimes, drug study results don't resemble the outcomes of clinical studies. This may be one of the keys to understanding why things don't always match up."

Fraley's faculty supervisor, Wirtz, suggested that part of the reason for the disconnect could be that even in studies that are called 3-D, the top of the cells are still located above the matrix. "Most of the work has been for cells only partially embedded in a matrix, which we call 2.5-D," he said. "Our paper shows the fundamental difference between 3-D and 2.5-D: Focal adhesions disappear, and the role of focal adhesion proteins in regulating cell motility becomes different."

Wirtz added that "because loss of adhesion and enhanced cell movement are hallmarks of cancer," his team's findings should radically alter the way cells are cultured for drug studies. For example, the team found that in a 3-D environment, cells possessing the protein zyxin would move in a random way, exploring their local environment. But when the gene for zyxin was disabled, the cells traveled in a rapid and persistent, almost one-dimensional pathway far from their place of origin.

Fraley said such cells might even travel back down the same pathways they had already explored. "It turns out that zyxin is misregulated in many cancers," Fraley said. Therefore, she added, an understanding of the function of proteins like zyxin in a 3-D cell culture is critical to understanding how cancer spreads, or metastasizes. "Of course tumor growth is important, but what kills most cancer patients is metastasis," she said.

To study cells in 3-D, the team coated a glass slide with layers of collagen-enriched gel several millimeters thick. Collagen, the most abundant protein in the body, forms a network in the gel of cross-linked fibers similar to the natural extracellular matrix scaffold upon which cells grow in the body. The researchers then mixed cells into the gel before it set. Next, they used an inverted confocal microscope to view from below the cells traveling within the gel matrix. The displacement of tiny beads embedded in the gel was used to show movement of the collagen fibers as the cells extended protrusions in both directions and then pulled inward before releasing one fiber and propelling themselves forward.

Fraley compared the movement of the cells to a person trying to maneuver through an obstacle course crisscrossed with bungee cords. "Cells move by extending one protrusion forward and another backward, contracting inward, and then releasing one of the contacts before releasing the other," she said. Ultimately, the cell moves in the direction of the contact released last.

When a cell moves along on a 2-D surface, the underside of the cell is in constant contact with a surface, where it can form many large and long-lasting focal adhesions. Cells moving in 3-D environments, however, only make brief contacts with the network of collagen fibers surrounding them - "We think the same focal adhesion proteins identified in 2-D situations play a role in 3-D motility, but their role in 3-D is completely different and unknown," Wirtz said. "There is more we need to discover."

Fraley said her future research will be focused specifically on the role of mechanosensory proteins like zyxin on motility, as well as how factors such as gel matrix pore size and stiffness affect cell migration in 3-D.

Notes:

Co-investigators on this research from Washington University in St. Louis were Gregory D. Longmore, a professor of medicine, and his postdoctoral fellow Yunfeng Feng, both of whom are affiliated with the university's BRIGHT Institute. Longmore and Wirtz lead one of three core projects that are the focus of the Johns Hopkins Engineering in Oncology Center, a National Cancer Institute-funded Physical Sciences in Oncology Center. Additional Johns Hopkins authors, all from the Department of Chemical and Biomolecular Engineering, were Alfredo Celedon, a recent doctoral recipient; Ranjini Krishnamurthy, a recent bachelor's degree recipient; and Dong-Hwee Kim, a current doctoral student.

Funding for the research was provided by the National Cancer Institute.

Source: Johns Hopkins University

The whole concept of proper genetic markers (molecular profiling) is not to put patients in the position of having to receive toxic cancer drugs if they're not going to do any good. However, genomics is far too limited in scope to encompass the vagaries and complexities of human cancer biology.

Trying to find tumor mutations to predict chemo success is still a "trial-and-error" approach to therapy. Testing for the EGFR mutation may be able to tell you whether or not your cells are "potentially" susceptible to this mechanism of attack. It cannot tell you if a "targeted" drug will work for "your" individual cancer cells. They don't even test your tumor cells against the EGFR-inhibitor drug.

The situation with Erbitux and Vectibix for colon cancer, Iressa and Tarceva for lung cancer, and Herceptin for breast cancer is that all the mutation or amplication studies can tell us is whether or not the cancer cells are potentially susceptible to this mechanism of attack.

They don't tell you if one drug or the other is worse or better than some other drug which may target this. The cell is a system, an integrated, interacting network of genes, proteins and other cellular constituents that produce functions.

No genetic profile can discriminate differing levels of anti-tumor activity occurring among different targeted therapy drugs. Nor can it identify situations in which it is advantageous to combine a targeted drug with other types of conventional cancer drugs.

"Targeted" drugs are poorly-predicted by measuring the ostansible "target," but can be well-predicted by measuring the effect of a drug on the function of live cells, the net effect of all processes, not just the individual molecular targets.

The benefits of newer targeted therapies are marginal. These targeted therapies may impart a clinical benefit by stabilizing tumors, rather than shrinking them (substituting shrinkage for stabilization).

I would not want to be denied treatment with any targeted therapy because of a gene mutation or amplication. Genetic testing (molecular profiling) is not a clear predictor of a lack of benefit.

BTW. The validaton standard that private insurance companies are accepting from molecular profiling tests is accuracy and not efficacy. The "bar" had been instantly lowered. No longer will it be essential to prove that the use of a diagnostic test improves clinical outcomes, all they have to do for these molecular profiling tests is prove that the test has a useful degree of accuarcy. However, the validation standard wanted for functional tumor cell profiling is efficacy. What's good for the goose is good for the gander.
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Old 06-27-2010, 08:21 AM   #4
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Cells In 3-D Do Reveal Cancer Targets

Cell-based chemoresponse assays test fresh "live" cells in their three dimensional (3D), floating clusters (in their natural state), not passaged cells (cell-lines). Established cell-line is not reflective of the behavior of "fresh" tumor cells in primary culture in the lab, much less in the patient. 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. Real life 3D analysis makes chemoresponse assays indicative of what will happen in the body.

One of the problems with genetic tests is in evaluating the data which exists to validate the predictive accuracy of them. Generally, a large number of archival specimens are batch processed together, within a very narrow time frame, by the same research team, so all the technical variables are minimized, which makes it much easier to get good results than in a "real world" setting, where specimens are tested over a period of weeks, months, years, by different people, with different laboratory reagents, as occurs in the "real world."

Evaluating "real world" data, requires specimens that are tested as they are logged into the lab in question, in "real time." No one is publishing "real world" studies, except private laboratories performing cell-based chemoresponse assays, which can only do "real world" studies, because their studies require fresh, viable specimen, which must be accessioned and tested in "real time," under "real world" conditions.

The "cell-death" assays are not growing anything. They are testing a drug or combinations of drugs with cells that are in their natural state (live or fresh). Three dimensional tumor cell clusters. Clusters maintain natural cell-cell interactions. This makes the assays indicative of what will happen in the body. The protocol takes "fresh" patient tumor cells and floats them in newer 3D cell suspensions.

As the researchers at Johns Hopkins and Washington University have found out, our body is 3D, not 2D in form, undoubtedly, this novel step better replicates that of the human body. Traditionally, in-vitro (in lab) cell-lines have been studied in 2 dimensions (2D) which has inherent limitations in applicability to real life 3D in-vivo (in body) states. Recently, other researchers have pointed to the limitations of 2D cell line study and chemotherapy to more correctly reflect the human body.

Literature Citation:
Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007
Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)
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Old 09-15-2010, 09:35 PM   #5
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Re: Chemosensitivity prediction

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Old 10-05-2010, 09:33 AM   #6
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Re: Chemosensitivity prediction

http://her2support.org/vbulletin/showthread.php?t=46200

Clinical trial of molecularly-personalized chemotherapy

http://cancerfocus.org/forum/showthread.php?t=3167
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