Vol. 13: Spring, 1996
Molecular Cytogenetics (FISH)
Specifics of FISH Applications in Cancer Cytogenetics
FISH analysis can identify selected acquired genetic changes in nondividing (interphase) cells and can detect masked translocations on metaphase chromosomes. However, FISH is informative only for the specific probes used, while cytogenetic analysis can detect a wide range of abnormalities and provides both diagnostic and prognostic information for patients with a variety of malignancies. The two techniques are complementary, and FISH has proven a valuable adjunct to cytogenetic analysis in the following ways:
- FISH ANALYSIS OF INTERPHASE CELLS
This application requires that a probe be available for the abnormality in question and that the laboratory has established normal background ranges for that specific probe. Interphase cells may show lack of signal or may demonstrate multiple signals because of various technical artifacts. It is important to establish the range of signals observed in normal cells for each probe used, in order to determine what this background rate is. Given that these conditions have been met, FISH analysis is very useful for:
a) Identification of abnormalities in specimens with a low mitotic index.
Diseases such as the chronic lymphoproliferative disorders are characterized by a very low proportion of mitotically active abnormal cells. In these cases traditional cytogenetic analysis is more likely to identify the karyotype of the normal dividing cells, and may miss the rare abnormal cell in mitosis. FISH on interphase cells from these patients is often helpful in detecting abnormalities when there is a high suspicion of a specific abnormality associated with the disease in question - for example, trisomy 12 or a deletion of 13q14 in patients with CLL. In cases of cytogenetic culture failures, FISH may also be helpful if an abnormality, such as the t(9;22) in CML, is suspected.
b) Confirmation of clonality when a single cytogenetically abnormal cell is detected.
Because cytogenetically abnormal cells may arise as artifacts of the tissue culture or slide making processes, the determination of clonality ( i.e. that all of the abnormal cells originated from a single aberrant cell) requires that multiple cells share the same aberration. When only a single abnormal cell is observed in the course of a cytogenetic analysis, FISH on interphase cells may be used to confirm the presence of an abnormal clone, if the appropriate probe is available and if the laboratory has established normal ranges for that probe.
c) Monitoring response to treatment and detection of minimal residual disease (MRD)
If a cytogenetic abnormality has been identified at diagnosis and a FISH probe is available to detect that abnormality, FISH may be used to screen interphase cells in subsequent studies. However FISH alone is not sufficient for monitoring, because cytogenetic analysis may also detect additional acquired changes, which may have important prognostic significance, or may uncover the development of secondary disease with a different set of abnormalities.
The sensitivity of cytogenetic analysis of mitotic cells and FISH analysis of interphase cells is roughly equivalent - i.e. a population of about 5% abnormal cells can be detected by either approach- although FISH allows a greater sampling of cell types since mitotic cells are not required.
The ability of FISH to be used to screen hundreds of interphase cells may increase sensitivity for the detection of MRD, especially when samples can be enriched for the presumptive abnormal population. However, the clinical significance of abnormal cells which are not actively dividing is not established and may vary with both the disease and the therapy used to treat it.
- FISH ANALYSIS OF METAPHASE CHROMOSOMES
This technique employs the use of whole chromosome paints, which are now commercially available for every chromosome, or cosmid probes on metaphase spreads prepared in the course of cytogenetic analysis. This applications allows:
a) Detection of masked rearrangements in cells with variant translocations or in cytogenetically normal cells
The application of molecular techniques to cancer diagnosis has taught us that some patients may have apparently normal chromosomes in their neoplastic cells but nevertheless have acquired genetic changes which are detectable only by molecular methods. FISH analysis with cosmid probes is useful for detecting masked bcr/abl rearrangements in CML, or pml/rara rearrangements in suspected APL. These cosmid probes may also be used for interphase cell analysis, with sensitivity similar to southern blotting, but with results more rapidly obtained.
b) Clarifying complex rearrangements and breakpoints
Whole chromosome paints or appropriately located cosmids may be hybridized to metaphase chromosomes to identify the origins of complex marker chromosomes, or to identify cryptic translocations which may appear as simple deletions. For example, some cases of myeloid leukemias with apparent deletions of 11q23 may actually have cryptic translocations, often involving chromosome 6. Delineation of disease groups and prognosis may hinge on this information.
The selective application of FISH techniques to cancer cytogenetic analysis has the ability to increase the resolution, sensitivity and informativeness of each analysis. In order to be more widely applied, there must be a greater selection of commercially available probes, and issues of quality control and quality assurance still need to be addressed.
Contributed by Kathy Richkind, Ph.D. (NM)
- FISH FOR GENE AMPLIFICATION: HER-2/neu EXPRESSION IN BREAST CANCER
Role of Gene Amplification in Cancer
Gene amplification is a characteristic of cancer cells that allows increased production of specific proteins used for acquisition and maintenance of the malignant phenotype. Amplification of certain oncogenes has an important role in the progression of many tumors. Detection of such amplifications may be of diagnostic and prognostic importance.
The HER-2/neu or erbB-2 oncogene codes for a 185 Kd transmembrane oncoprotein (p185). Approximately 25-30% of breast and ovarian carcinomas have amplification of this gene. Studies have demonstrated that HER-2/neu is amplified and/or overexpressed in 10-30% of invasive breast cancers and in 40-60% of intraductal breast carcinomas.
Amplification and overexpression of this gene were found to be associated with rapid proliferation, low estrogen receptor content, and high grade of ductal carcinomas, which suggests that this oncogene plays an important role in the progression of breast cancer.
Amplification or overexpression of the HER-2/neu gene was shown to be an indicator of poor prognosis in node-positive breast cancer. In node negative breast cancer patients, the subgroup for which accurate prognostic factors could make a significant contribution to treatment decisions, the prognostic value of HER-2/neu amplification or overexpression has been controversial. The technical difficulties in using standard immunohstochemical staining (IHC) and Southern blot analysis may explain not only the differences in the observed clinical-genetic correlation, but also the varied frequency of HER-2/neu overexpression reported in tumors.
Conclusion
Fluorescence in situ hybridization (FISH) has been used to demonstrate gene amplification in human breast carcinoma cell lines in both interphase and metaphase cells. FISH appears to be a useful technique as it can overcome many of the inherent limitations of other techniques and can provide the most reliable and accurate data. HER-2/neu amplification appears to be a strong indicator of breast cancer recurrence in women under 40. This gradient of risk demonstrated by FISH with a DNA probe and the more consistent recent findings of the prognostic importance of amplified HER-2/neu is evidence of probable superiority of this technique compared to standard Southern blot or IHC.
Contributed by Mary Lowery, Ph. D. (CO)
The Genetic Drift Newsletter is not copyrighted. Readers are free to duplicate all or parts of its contents. The Genetic Drift Newsletter is published semiannually by the Mountain States Genetics Network for associates & those interested in Human Genetics. In accordance with accepted publication standards, we request acknowledgement in print of any article reproduced in another publication. The views expressed in the newsletter do not necessarily reflect local, state, or federal policy. For additional information, contact Carol Clericuzio, M.D., Editor, Department of Pediatrics, The University of New Mexico, Albuquerque, NM, 87131
Table Of Contents: Molecular Cytogenetics (FISH)
Introduction & Basic Techniques
Applications of FISH Technology
FISH Applications in Cancer Cytogenetics
FISH in Microdeletion Syndromes
FISHing in Unknown Waters
Regulatory Issues and FISH
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