Molecular Profiling Doesn't Trump Standard Pathology

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Antonio C. Wolff, M.D.
Johns Hopkins University, Baltimore

Gene expression profile testing to guide decisions regarding the use of adjuvant chemotherapy for early-stage breast cancer should not be used to override the results of standard pathologic measures, Dr. Antonio C. Wolff asserted at the annual San Antonio Breast Cancer Symposium.

Two gene expression profile tests commonly used in breast cancer are the Oncotype DX and MammaPrint assays. The excitement surrounding this whole field is such that Dr. Wolff suspects many clinicians see the molecular assays as being superior to standard clinicopathologic data in their predictive information. This actually hasn’t been shown to be the case to date, he stressed.

"One of the big messages I bring today is that one method is not necessarily better than the other. The standard pathologic tests and the molecular assays may be complementary in the information they provide," said Dr. Wolff, professor of oncology at Johns Hopkins University, Baltimore.

He added that while guideline-based use of these first-generation molecular assays as recommended by the American Society of Clinical Oncology (ASCO), the National Comprehensive Cancer Network (NCCN), and the European St. Gallen’s Expert Consensus group is appropriate, it’s important to recognize that these tests have their shortcomings. For one, they leave a lot of patients in the gray intermediate-risk zone. Also, the tests aren’t applicable to the 25%-35% of breast cancer patients with estrogen receptor–negative tumors. But a host of new technologies are in the pipeline, he observed.

A recent study of 7,375 breast cancer patients with hormone receptor–positive breast cancer diagnosed in 2006-2008 was enlightening with regard to how clinicians are using gene expression profile testing. The study, an analysis of NCCN prospective registry data conducted by investigators at Boston’s Dana-Farber Cancer Institute, showed a progressive increase in the use of the Oncotype DX test. The test was employed in 14.7% of the patients diagnosed in 2006, nearly doubling to 27.5% of those diagnosed in 2008. Meanwhile, use of chemotherapy decreased from 53.9% to 47%.

Gene expression profile testing was associated with an overall 30% reduction in the likelihood of receiving chemotherapy. What Dr. Wolff found particularly interesting was that testing of small, node-negative cancers was associated with an 11.1-fold increased likelihood of chemotherapy, while testing of lymph node–positive or large node-negative cancers was associated with an 89% reduction in chemotherapy (J. Clin. Oncol. 2012;30:2218-26).

African American women were 30% less likely to undergo testing. Women with no more than a high school education were 37% less likely to be tested than were those with more schooling. Dr. Wolff suspects these disparities are a reflection of better-educated women asking more questions of their physician about their planned treatment course, and the physician in response seeking more laboratory data to help in making treatment decisions.

Dr. Wolff said that while clinicians await studies to determine whether molecular and standard clinicopathologic information can be combined to improve prediction, one of the most important things they can do is to pay close attention to preanalytic issues.

"It’s critical to get tissue from patient to pathologist within 1 hour to avoid cold ischemia and necrosis and deterioration of the specimen that will not only adversely affect the quality of standard measures, but also of the new measures. This is a big deal!" he emphasized.

Dr. Wolff reported that he serves as an investigator for Genentech.

http://www.ncbi.nlm.nih.gov/pubmed?t...0%3A%202218-26
http://www.cancernetwork.com/confere.../10165/2117513

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The latest British study on Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing may hinder personalized medicine strategies that depend on results from single tumor biopsy samples.

A reply to a Dr. Robert Nagourney blog posting about this study, brought up the Oncotype DX assay. The woman's breast tumor was early stage 1/grade 1 NO ER/PR positive Her2 negative and her Oncotype DX showed 8% recurrence rate.

After reading her pathology report, three years ago, she raised the question as to why the whole tumor was not looked at after the lumpectomy to make sure the diangosis was the same throughout the tumor. She was told it would be to lengthy, costly and she had nothing to worry about. She was told that they only test a slice of the tumor and throw the rest away. She was taken back by the oncologist's comment. Indeed, so would I.

After she read an article on the British study, is she to assume she may have been right? Or was the pathologist correct in telling her with his assessment of her tumor coupled with the Oncotype DX that things looked good for her and she was not on the Titanic. She was very depressed for someone who was three years out and wondering whether or not the treatment she received was really what she needed.

Indeed, intratumor heterogeneity can lead to underestimation of the tumor genomic landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized medicine and genetic biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure.

The study is significant because it suggests relying on one sample could overlook important genetic biomarkers that help make tailored treatments effective, explaining perhaps why personalized cancer therapy has been less successful than expected.

It's a theoretical but overrated problem. The same problem applies to ER, Her2, EGFR mutations, KRAS, OncotypeDx. Even worse for trying to do studies on individual cells, e.g. as from circulating tumor cells. Less of a problem for cell function analysis, since they are sampling a much bigger mass of cells and are homogenizing the mass (actually homogenizing the distribution of microclusters).

It's analogous to the Gallup poll. You are projecting the behavior of a national electorate, based on a sample of 1,500 voters, who may or may not be representative of the whole. Rasmussen and Gallup have the same sized sample, but select different people for their polling ("likely voters" vs "all voters"), so their projections often disagree.

It is one of the reasons why (1) "resistance" predictions tend to be more accurate than "sensitive" predictions (of the cancer is resistant anywhere, it pretty much doesn't matter), if you use the "resistant" drug, the patient will have progressive disease and (2) the tests are more analogous to using the barometric pressure to predict for rain than they are analogous to a serum sodium level; i.e. the predictions are useful (assay "sensitive" drugs being seven times more likely to work than assay "resistant" drugs), but they aren't perfect (i.e. 100%), no diagnostic test in medicine is.

Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

http://cancerfocus.org/forum/showthread.php?t=3640

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Is Gene Array Testing To Be Considered Routine Now?

Breast. 2011 Oct 1;20(3), S Paik

Abstract

Background:

There are five multi-gene expression based prognostic tests for breast cancer offered as reference lab tests - Mammaprint, MapQuant Dx, OncotypeDx, PAM50 Breast Cancer Intrinsic Subtype Classifier, and Theros Breast Cancer Index. Each claims to provide additional prognostic information beyond conventional prognostic markers and to aid in determining who should receive systemic therapy. Evidence for their clinical utility was reviewed to determine whether any of them should be considered as routine clinical test.

Methods:

Peer reviewed publications, meeting abstracts, and information provided by company web sites have been reviewed to compile information regarding their clinical utility according to the following criteria; (1) Analytical validity and regulatory approval of the reference lab test. (2) Level of evidence for clinical utility. (3) Whether published evidences support prognostic and/or predictive claim.

Results:

While published evidences for clinical claims for OncotypeDx and Mammaprint used reference lab tests, and the supporting evidences for other tests come from academic assays before being converted to reference lab tests, results from two large randomized clinical trials testing the clinical utility of OncotypeDx and Mammaprint are still several years away and until that time none of the markers would reach level I evidence by Marker Utility Grading System. However Oncotype Dx has reached a level IB evidence according to Simon modification to Marker Utility Grading System. Therefore OncotypeDx may be considered for routine clinical use as an adjunct to clinical and pathological information and has been incorporated into clinical guidelines in USA. While Mammaprint, MapQuantDx, and PAM50 have been repeatedly demonstrated to provide robust prognostic information, evidence for its worth as a predictive marker for chemotherapy benefit is yet to come from randomize clinical trials and therefore its utility is limited to prognostication. Meta-analysis of publicly available microarray based gene expression studies demonstrated that gene expression assays provide similar information and the most important information they provide is the proliferation activity. In untreated population, the prognostic impact of proliferation genes is limited to ER+HER2− subset since HER2+ or ER−HER2− subsets are associated with high proliferation activity. Therefore the clinical utility of these gene expression based tests is mainly for ER+HER2− subset. Since they are usually treated with adjuvant anti-estrogen therapies, for their clinical utility, demonstration of the interaction between the gene expression markers and chemotherapy in anti-estrogen treated cohort in a randomized clinical trial would be required. While OncoytpeDx is the only test supported by studies in a randomized clinical trial for adjuvant chemotherapy, other gene expression based tests are expected to provide similar information. Gene expression profiling assays as more reproducible and precise surrogates for tumor grade (MapQauntDx and Theros Breast Cancer Index) are very promising assays. However, absence of definitive predefined cut-off for defining the subset that benefit from chemotherapy validated in cohorts from randomized trials limit their clinical application.

http://www.thebreastonline.com/artic...301-0/abstract

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Cary Presant, M.D.
Wilshire Oncology Medical Group

The decision of chemotherapy plus hormonal therapy versus hormonal therapy alone in patients with breast cancer, stage 1 or 2A is an important decision which we commonly make in our oncology practices. The advent of the Oncotype DX test, along with other similar tests such as MammaPrint and Mammostrat, have given us tools whose application is selectively very important. Recently, the adoption of gene expression profile testing has been reviewed by M. Hassett and coworkers (Journal of Clinical Oncology 30:2218-2226, 2012).

In over 7,000 women, 20% received gene expression profile testing. Of those who had the test, 50% received chemotherapy. The use of gene expression profiling became more common between 2006 and 2008 (increasing from 14.7% to 27.5%). Unfortunately, they did not have more up to date data in the past 4 years.

Needless to say, if an oncologist sees a clinical indication to use chemotherapy, addition of a gene expression profile test would be unnecessary. Similarly, if a patient were unwilling to consider chemotherapy, such a test would again be unnecessary. However, for women wanting the most appropriate, personalized, individualized therapy, the use of a gene expression profile test would be very important.

Surprisingly, in the Hassett analysis, small, node negative cancers were associated with a higher odds of chemotherapy use. Surprisingly, node positive and large node negative cancers were associated with a lower chance of chemotherapy use following testing.

Today, the use of this testing is extremely widespread in my community. At each tumor board, patients with stage 1 or 2 breast cancer are nearly universally recommended for such testing. The NCCN clinical practice guidelines suggests using this test to help make the clinical decision regarding the addition of chemotherapy.

However, the intermediate risk group identified in the Oncotype DX testing is still the subject of an ongoing randomized phase III clinical trial. So the application of the test may not be associated with a clear cut answer as to whether or not chemotherapy is useful. Indeed, the frequency of intermediate test results seems to be increasing (Abstracts ASCO 2012). It is important to realize that until such a trial to determine the predictive utility of an intermediate result is completed, the study may be inconclusive. Furthermore, none of the gene expression profile tests have been subjected to a phase III prospective randomized trial (so far only retrospective data has been used).

In my practice, discussing the uncertainty of this testing and applying one of the tests selectively is an important component of breast cancer adjuvant care. Therefore, such patients require several comprehensive visits to both evaluate the appropriateness of possible chemotherapy, and to evaluate the results of testing.

The advance that these tests provide in care of cancer patients must be associated with continued awareness of the results of ongoing randomized prospective trials. The application of similar tests to colorectal cancer, lung cancer and other tumors remains a challenge for clinical trials as well as for the adoption into clinical practice.

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Whole Organism Testing

The most direct way to see if a chemotherapy drug will work is to administer it to the patient and see what happens. This is how most drugs are administered. Each patient, in effect, becomes his/her own experiment. If the drug(s) does not work, the patient needlessly suffers harmful side-effects, valuable treatment time is wasted, costs are incurred and the surviving tumor cells may become more resistant to all chemotherapy drugs, including drugs which the patient has not received. This is what is called clinically-acquired multi-drug resistance. Since patients cannot endure exposure to all of the available chemotherapy drugs, many potentially effective drugs will go untested. Also, since chemotherapy drugs commonly are administered in combination, there is no way to know which drug or drugs in a particular combination may have been more effective and which were less effective or not effective at all.

Cytometric Cancer Testing (Functional Profiling)

It pinpoints the ability of each drug to kill each patient's actual cancer cells. It accurately assesses the combined effect of the fullest possible range of genetic, chemical and mechanical interactions which govern tumor cell susceptibility to drug treatment. Factors include the presence and functionality of proteins and how those proteins interact with all other proteins and with other intracellular and intercellular processes. It accounts for cellular drug uptake, exclusion, expulsion, DNA repair and other resistance mechanisms.

Protein Testing

Proteins are chemicals which govern cell behaviors, such as susceptibility or resistance to specific chemotherapy drugs. They are manufactured within the cell's cytoplasm from blueprints which are carried from the nucleus in the form of messenger RNA. Protein testing is more direct than RNA testing because it reveals whether or not a protein which theorectially was encoded for by DNA actually was produced. However, not all proteins which are involved in response to treatment have been identified, only a small number of known proteins are tested and without additional types of testing, there is no way to tell if a protein which is present actually is functional or how it interacts with other proteins of known and unknown function.

Messenger RNA Testing

RNA acts as a messenger, splitting off from the DNA helix in the cell nucleus and carrying the blueprint for production of proteinis out of the nucleus and into the cytoplasm, essentially the 'factory floor' where proteins are manufactured. Proteins ultimately govern the behaviors of both normal cells and tumor cells. Tests which can detect the presence of specific types of RNA are a more direct method than testing the DNA because it shows that certain genetic information stored in the DNA actually is producing messenger RNA. However, the presence of RNA only implies but does not prove that proteins were produced. The importance of all proteins is not fully understood and so the tests look for only certain RNA sequences. Also, not fully understood is how the various proteins interact to support or interfere with drug sensitivity and resistance.

DNA Testing (Molecular Profiling)

Located in the cell nucleus, DNA contains genetic information which causes cells to behave as they do. DNA encodes for RNA, which encodes for production of proteins. DNA is the farthest upstream factor predisposing a theoretical even occurring at the other end of a highly complex process. Even if all genetic factors were understood, which they are not, the mere presence of a DNA sequence does not mean that the downstream even will occur. If a specific drug targets only a specific DNA sequence, the absence of that sequence might preclude the use of that drug. However, even if the sequence is present, it is still impossible to know if the drug will work. No DNA test can pinpoint specific drugs in the same class or detect synergy in drug combinastions. DNA tests involve tiny fragments of cells. In DNA tests, patients' tumor cells are never exposed to any anti-cancer drugs.

Source: Larry Weisenthal, M.D., PhD., Medical and Laboratory Director, Weisenthal Cancer Group, Huntington Beach, California

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Located in the cell nucleus, DNA contains genetic information which causes cells to behave as they do. DNA encodes for RNA, which encodes for production of proteins. DNA is the farthest upstream factor predisposing a theoretical event occurring at the other end of a highly complex process. This is the least direct testing method (DNA testing or molecular profiling).

Even if all genetic factors were understood, the mere presence of a DNA sequence does not mean that the downstream even will occur. If a specific drug targets only a specific DNA sequence, the absence of that sequence might preclude the use of that drug.

However, even if the sequence is present, it is still impossible to know if the drug will work. No DNA test can pinpoint specific drugs in the same class or detect synergy in drug combinastions. DNA tests involve tiny fragments of cells. In DNA tests, patients' tumor cells are never exposed to any anti-cancer drugs.

RNA acts as a messenger, splitting off from the DNA helix in the cell nucleus and carrying the blueprint for production of proteinis out of the nucleus and into the cytoplasm, essentially the 'factory floor' where proteins are manufactured.

Proteins ultimately govern the behaviors of both normal cells and tumor cells. Tests which can detect the presence of specific types of RNA are a more direct method than testing the DNA because it shows that certain genetic information stored in the DNA actually is producing messenger RNA. This is a more direct testing method (RNA testing).

However, the presence of RNA only implies but does not prove that proteins were produced. The importance of all proteins is not fully understood and so the tests look for only certain RNA sequences. Also, not fully understood is how the various proteins interact to support or interfere with drug sensitivity and resistance.

Proteins are chemicals which govern cell behaviors, such as susceptibility or resistance to specific chemotherapy drugs. They are manufactured within the cell's cytoplasm from blueprints which are carried from the nucleus in the form of messenger RNA.

Protein testing (proteomic profiling) is more direct than RNA testing because it reveals whether or not a protein which theoretically was encoded for by DNA actually was produced.

However, not all proteins which are involved in response to treatment have been identified, only a small number of known proteins are tested and without additional types of testing, there is no way to tell if a protein which is present actually is functional or how it interacts with other proteins of known and unknown function.

Then there is the most direct testing method of whole cell testing (short of administering a drug to the patient and see what happens). The ability to pinpoint each drug to kill each patient's actual cancer cells (functional profiling). To accurately assesses the combined effect of the fullest possible range of genetic, chemical and mechanical interactions which govern tumor cell susceptibility to drug treatment.

Factors include the presence and functionality of proteins and how those proteins interact with all other proteins and with other intracellular and intercellular processes. It accounts for cellular drug uptake, exclusion, expulsion, DNA repair and other resistance mechanisms.

Source: Larry Weisenthal, M.D., PhD., Medical and Laboratory Director, Weisenthal Cancer Group, Huntington Beach, California