FISH technology accurate in measuring the HER-2 receptor in human breast tumors

[News]

The debate on how to select patients who will respond best to costly drug treatments for aggressive breast cancer now favors fluorescence in situ hybridization (FISH) to measure the HER-2 receptor found in human breast tumors, according to a leading pathologist presenting at the Association for Molecular Pathology annual meeting.

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[gdpawel]

Recent research has shown that the best way to determine whether a tumor will respond to Herceptin is to look at whether it overexpresses the Her2/neu oncogene, not whether it is making too much Her2/neu protein. And the best way to examine the oncogenes is with FISH (Fluorescence In Situ Hybridization). Because the results of the IHC test can sometimes be ambiguous, many doctors suggest the FISH test for a second opinion.

Tumors that are 3+ positive by IHC and those that test positive by FISH are most likely to benefit from Herceptin. Tumors that test 1+ by IHC are considered Her2/neu negative and those that test 2+ are considered equivical, in which case FISH testing is done to make the determination. Tumors that test negative for Her2/neu by FISH are unlikely to benefit from Herceptin.

Previous studies demonstrated that there is poor agreement between the results from local laboratory-based Her2/neu testing and those of central testing by experienced investigators. There has been poor concordance between community and central laboratory testing, in terms of both Her2 protein expression and gene amplification. Even still, there has been poor concordance in terms of FISH testing in a central laboratory compared to local laboratories, which the prevalent notion regarding FISH is that it is 100% accurate.

However, it doesn't matter if there is a "target" molecule (gene or protein) in the cell that the targeted drug is going after, if the drug either won't "get in" in the first place or if it gets pumped out/extruded or if it gets immediately metabolized inside the cell, drug resistance is multifactorial. The advantage of cell-based functional profiling is that it can show this in the "population" of cells (entire genome).

Over the past few years, researchers have put enormous efforts into genetic profiling as a way of predicting patient response to targeted therapies. However, no gene-based test can discriminate differing levels of anti-tumor activity occurring among different targeted therapy drugs. Nor can an available gene-based test identify situations in which it is advantageous to combine a targeted drug with other types of conventional cancer drugs. So far, cell-based functional profiling has demonstrated this critical ability.

Source: Cell Function Analysis

[Rich66]

So..it's about which drug works, how to get it there and how to keep it from being pumped out or disabled before it can work. Can cell analysis simulate ability to get there..in the context of a body?

[gdpawel]

Good questions Rich.

There is a headlong rush to develop tests to identify molecular predisposing mechanisms, whose presence still does not guarantee that a drug will be effective for an individual patient. Nor can they, for any patient or even large group of patients, discriminate the potential for clinical activity among different agents of the same class.

The challenge is to identify which patients targeted treatment will be most effective. Tumors can become resistant to a targeted treatment, or the drug no longer works, even if it has previously been effective in shrinking a tumor. Drugs are combined with existing ones to target the tumor more effectively. Most cancers cannot be effectively treated with targeted drugs alone.

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.

It is established "cell-line" that is not reflective of the behavior of fresh tumor cells in primary culture in the lab, much less in the patient. You get different results when you test passaged cells compared to primary, fresh tumors.

NCI had their own personal experience. At a NCI-sponsored Ovarian Cancer State of the Science Meeting in 2005, it was brought out that NCI had a huge lab working on microarrays to look for patterns of mRNA and protein expression which are predictive of chemotherapy response.

They spent 2 years trying to find patterns which correlated using the NCI's various established ovarian "cell-lines." They thought they had something, but when they started to apply them to "fresh" tumor specimens, none of the results in the "cell-lines" was applicable to the "fresh" tumors. Everything they'd worked out in the "cell-lines" was not worth anything and they had to start over from square one.

Critics of the cell-death assay procedure contend that cells do not necessarily react the same in the laboratory (in vitro) as they do in the body (in vivo). However, tests are performed using intact, living (fresh) cancer cells plated in 3D microclusters, which 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. 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 make the assays indicative of what will happen in the body.

What is needed is to measure the net effect of all processes within the cancer, acting with and against each other in real time, and test living cells actually exposed to drugs and drug combinations of interest. The key to understanding the genome is understanding how cells work. How is the cell being killed regardless of the mechanism?

Cell culture testing methods assess the net effect of all inter-cellular and intra-cellular processes occurring in real time when cells are exposed to anti-cancer agents. Molecular testing methods detect the presence or absence of selected gene mutations which theoretically correlate with single agent drug activity. Cells are never exposed to anti-cancer agents.

Cell culture testing methods are performed using intact, living cancer cells plated in 3D microclusters. It allows for testing of different drugs within the same class and drug combinations to detect drug synergy and drug antagonism.

The core understanding is the cell, composed of hundreds of complex molecules that regulate the pathways necessary for vital cellular functions. If a targeted drug could perturb any of these pathways, it is important to examine the effects of drug combinations within the context of the cell.

Both genomics and proteomics can identify potential therapeutic targets, but these targets require the determination of cellular endpoints. You still need to measure the net effect of all processes, not just the individual molecular targets.

Literature Citation:
Eur J Clin Invest, Volume 37(suppl. 1):60, April 2007
BMJ 2007;334(suppl 1):s18 (6 January), doi:10.1136/bmj.39034.719942.94

[Rich66]

I wonder if a 3D cell cluster simulates systemic "transport" issues in getting drug from IV to target. I know there is a researcher named Cristini who has spent years on computer modeling in this regard.