Fluorescence in situ Hybridization (FISH)

FISH is a gene-based test used to determine the number of HER2 genes in the cells of the tumor. In HER2-positive breast cancer there are too many copies of the HER2 gene, the gene is amplified.

The FISH test is done by a pathologist in a laboratory on a sample of a tumor removed during a biopsy, a lumpectomy or a mastectomy. The steps in the most commonly used procedure (Pathvysion) are:

* breast cancer tissue is prepared for testing

* the sample of tumor (a thin slice) is exposed to fluorescent compounds (will glow under certain light)

* one fluorescent compound adheres (sticks) to the HER2 genes in a cell and another adheres to chromosome 17 in a cell

* when the sample is exposed to a special light the HER2 genes and chromosome 17 light up with different colors and can be counted; a pathologist or computer then reads the prepared slide

* the proportion or ratio of genes to chromosomes 17 in 60 cells is determined and the average number of HER2 genes/chromosome 17 per cell is then reported

* The HER2 gene/chromosome 17 ratio in a normal, nondividing cell should be 1 to 1- 1 gene on each of the 2 copies of chromosome 17; the HER2 gene/chromosome 17 ratio can increase up to 2 to 1 in cells during certain stages of normal cell division; an average HER2 gene/chromosome 17 ratio of less than 2 to 1 is reported as HER2 negative

* tumor samples with an average HER2 gene/chromosome 17 ratio of greater than or equal to 2 to 1 (2.5 to 1, 3.2 to 1, etc.) are reported as HER2 positive

* Another FISH procedure (INFORM) uses a similar procedure but measures only the average number of HER2 gene copies/cell; tumor samples with an average of greater than 4 HER2 gene copies/cell are considered HER2 positive.

Problems encountered in FISH testing:

* Scoring problems can occur when the HER2 gene/chromosome 17 ratios are in the 1.8 to 2.2 range (Pathvysion test) or when the average number of HER2 gene copies/cell are in the range of greater than 4 to less than 6 (INFORM test); these are considered borderline amplified.

* At this time no high-level evidence or agreement is available on how results in the borderline range should be interpreted or confirmed.

* Scoring difficulties found with FISH testing may be associated with the specific set of cells chosen to include in the determination or tissue processing.

* False positive or false negative HER2 test results can occur

NCCN RECOMMENDATIONS

If appropriate quality control/assurance procedures are in place for a laboratory⁵, either IHC or FISH methods may be used to determine HER2 tumor status. If the laboratory does not have control/assurance procedures in place, the sample should be sent to a reference laboratory⁶ that does meet quality control/assurance procedures.

* HER2 positive status is IHC 3+ or FISH positive

* HER2 negative status is IHC 0, 1+ or FISH negative

* A borderline IHC result of 2+ should be followed by performing a FISH test.

* A borderline FISH result of an average HER2 gene/chromosome 17 ratio of 1.8 to 2.2 (or an average of greater than 4 to less than 6 HER2 gene copies/cell) should be followed by one of the following:

* Counting additional cells in the tissue sample

* Retesting with FISH

* Performing an IHC test

Questions To Ask About Testing

* What tests will be done on the sample of my breast cancer tumor?
One test done on a sample determines whether the tumor is estrogen/progesterone positive or negative. This will help determine if treatment will include hormone therapy. Another test will determine if your tumor is HER2 positive or negative. The report from the pathologist will also contain information about how the sample looked to the naked eye and then under a microscope. ⁷ ,⁸

* Is the laboratory that will test my tumor for HER2 status accredited to perform such testing?
Your doctor may or may not know the answer to this question. If your doctor does not know, it is important that she find out because treatment decisions will be made based on the results of the HER2 tumor testing. The information about the accreditation of the laboratory can be found by contacting the head of the laboratory (often a pathologist). If the laboratory is not accredited to perform HER2 testing, your doctor should have the test done at a reference laboratory that is. If the laboratory is connected with a large hospital and does many HER2 tests, it is more likely to meet the standards for accreditation. ⁵

* Has the laboratory properly validated the HER2 test(s) it uses?
Your doctor may or may not know the answer to this question. If your doctor does not know, it is important that she find out because treatment decisions will be made based on the results of the HER2 tumor test. The information about the validation of the HER2 test(s) can be found by contacting the head of the laboratory (often a pathologist). If the laboratory has not validated the HER2 test(s) they use, your doctor should have the test done in a reference laboratory that has validated HER2 testing.. ⁵

* Which test will be used to determine my HER2 status?
Either IHC or FISH testing is acceptable.

* What will happen if the IHC test comes back 2+?
This is considered a borderline result and it should be followed by doing a FISH test.

* What will happen if the FISH test comes back with a borderline result?
There are three possible things that could be done: The pathologist can look at the original slide and count more cells to determine the gene/chromosome ratio (or the number of gene copies/cell); the sample can be retested using FISH; or an IHC test can be done.

Romond EH, Perez EA, Bryant J, et al Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005;353:1673-168 4.

Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783-792.

³ breastcancer.org. Herceptin Side Effects, 19 Sep 2006.http://www.breastcancer.org/herceptin_side_effects.html

Carlson RW, Moench SJ, Hammond MEH et al. HER2 testing in breast cancer: NCCN Task Force report and recommendations. J Natl Compr Canc Netw.. 2006;4 suppl 3:S1-S-22. (url to be added when available online)

Standards for Laboratory Accreditation . College of American Pathologists. 2000. 19 Sep 2006. http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_accreditation%2Fstandards%2Fstandards.html&_state=maximized&_pageLabel=cntvwr

⁶ �Reference Laboratory. Encyclopedia of Public Health. Ed Lester Breslow, Thomson Gale, 2002. enotes.com. 2006. 19 Sep 2006. http://health.enotes.com/public-health-encyclopedia/reference-laboratory

⁷Your Pathology Report. breastcancer.org. http://www.breastcancer.org/pathology_intro.html (Free brochure)