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Applications of FISH In Solid Tumors

The clinical utility of fluorescence in situ hybridization assays in lung cancer, gliomas and breast cancer are identified.

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Fluorescence in situ hybridization (FISH) assays were once considered a cytogenetic technique that could only be performed on metaphase chromosome spreads obtained from cultured cells. Various new detection chemistries have been identified that now allow FISH to be performed on non-cultured interphase cells, including those found in formalin-fixed, paraffin-embedded tissue sections.

FISH is now frequently used in molecular and cytogenetic laboratories in the diagnosis, prognosis and treatment stratification of solid tumors. FISH assays are able to identify specific translocation breakpoints, chromosomal deletions and gene amplification, all of which have clinical applications in solid tumors.

Figures/Courtesy Laura J. Tafe, MD

FIGURE 1: ALK rearrangement in a lung adenocarcinoma. Separation of the orange and green probes signifies rearrangement. The two probes are closely appropriated in the non-rearranged state and the signal overlap gives a yellowish signal.
FIGURE 2: FISH of an oligodendroglioma with chromosome 1p deletion. Chromosome 19q deletion gives an identical FISH result. In panel A, two orange and two green signals are seen in a cells, while in panel B, one of the orange signals is lost indicating deletion of 1p.
FIGURE 3: HER2 amplification by FISH in a breast cancer. Panel A illustrates a breast cancer that is non-amplified and has two orange (HER2) and two green (CEP17) signals per cell. Panel B illustrates a case of HER2 amplification (numerous orange signals per cell and only two green signals).

Gene Rearrangements

Unlike several types of hematopoietic malignancies, relatively few solid tumors have yet been shown to be associated with recurrent chromosomal translocations, with the exception of sarcomas. Two carcinomas associated with chromosomal rearrangements that are very prevalent are prostate cancer and the adenocarcinoma subtype of non-small cell lung cancer. In prostate cancer, the Transmembrane protease serine2 (TMPRSS2)-ERG (ETS-related gene) gene fusion is present in a majority of cancers and thought to be an early event that drives tumor development.

Adenocarcinomas of the lung are associated with somatic mutations in the EGFR and KRAS genes in some patient populations. An additional subpopulation (2-5 percent) of tumors do not have these mutations; rather, they have a novel recurrent gene fusion on chromosome 2 involving the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) genes. This fusion gene can be detected in lung adenocarcinoma with an ALK break-apart FISH assay. The assay is designed so that two FISH probes (labeled with spectrum orange and spectrum green) flank the breakpoint.

Close apposition of the probes indicate wild-type (not rearranged) ALK, which has a yellow signal, while separation of the orange and green signals or deletion of the green signal indicate ALK rearrangement (Fig. 1). While this assay does not determine the specific rearrangement partner gene, it is highly sensitive for the EML4-ALK rearrangement in lung adenocarcinoma. Patients with the rearrangement are eligible for targeted therapy with the novel ALK inhibitor crizotinib (Xalkori).1

Identification of Chromosomal Deletions

Gliomas are primary brain tumors classified into four grades based on histology. Treatment and prognosis varies for the different tumor grades. May of these tumors can be diagnosed by histology alone but occasional cases are morphologically ambiguous and additional molecular studies are needed to reach a diagnosis. Loss of chromosome 1p and 19q is commonly seen in oligodendrogliomas but is rare in other tumor types, such as astrocytomas.

FISH evaluation of 1p and 19q deletions requires two identical assays-one for chromosome 1 and the other for chromosome 19. The assays are designed with one FISH probe for the long arm of the chromosome (q) and a second probe for the short arm (p). A normal, undeleted case will show two orange and two green signals per cell while a deletion will have only one orange signal and two green signals (Fig. 2). Detecting 1p and 19q deletions are both diagnostic and predict a favorable response to therapy.2

Identification of Gene Amplification

Breast cancer is the classic example of how gene amplification plays a role in tumor prognosis and treatment. Human epidermal growth factor receptor 2 (HER2) overexpression is associated with a worse prognosis. However, with the development of targeted therapeutics, HER2 status is a strong predictor of response to trastuzumab (Herceptin), a monoclonal anti-HER2 antibody directed to the extracellular receptor domain and lapatinib (Tykerb), a small molecule inhibitor of the intracellular tyrosine kinase domain.

Various methodologies are available to test for HER2 overexpression, including immunohistochemistry (IHC) and in situ techniques. Many labs will test by IHC followed by reflexive FISH for IHC equivocal cases. Others prefer to test all cases by FISH.

HER2 FISH assays are designed to detect gene amplification (Fig. 3). Most are dual-colored FISH but a single color assay is also available. In dual-colored FISH, two probes are used, one to detect HER2 (spectrum orange) and the other to detect the centromere of chromosome 17 (CEP17-spectrum green), the chromosome on which HER2 is localized.3 The results of the FISH assay are reported as the ratio of HER2:CEP17 signals according to the ASCO/CAP guidelines.4

Dr. Tafe is assistant director, Molecular Pathology, assistant professor, Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, NH.


 

The question then becomes; what CPT code to use for formalin fixed paraffin embeded tissue sections for interphase FISH? 88271 or 88367

pam October 29, 2012




     

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