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Chromogenic In Situ Hybridization in Diagnosis and Prognosis of Breast and Renal Cancers


Maria J. Merino
National Cancer Institute
Bethesda, MD


CISH and Breast
Determination of HER2 amplification in tissue specimens is essential for commencing treatment with trastuzumab (Herceptin). In clinical practice, IHC (DAKO Hercep Test) and more recently??? are the most widely used methods to identify patients that may respond to this form of therapy, and both are currently approved by FDA as clinical tests for breast cancer

Chromogenic in situ Hybridization (CISH) has been introduced as a practical alternative to FISH in detecting HER2 amplification in paraffin-embedded breast carcinoma samples. Different authors have reported a 93% to 100% correlation between CISH and FISH . We found correlation of 95% in our cases. The advantages of CISH over FISH include:
  1. the methodology is much faster and easier;

  2. fluorescence microscope is not needed therefore it is a cheaper technique;

  3. signal intensity does not decrease over time;

  4. it is possible to compare signals with histology, which helps to focus the analysis of selected areas in order to avoid misinterpretations because of heterogeneous amplification;

  5. it is accessible for routine pathology laboratories,

  6. slides can be stored for further studies
The application of a chromosome 17 probe is useful in discriminating between true amplification and pseudoamplification caused by chromosome 17 polysomy . In our study, we found chromosome 17 polysomy in six cases; four of which showed low-level HER2 gene amplification. Therefore, they were considered as cases with not true amplification. One case (a lobular carcinoma) displayed chromosome 17 polysomy, no HER2 gene amplification, and no IHC staining. It is possible that chromosome 17 polysomy in itself, is insufficient to cause significant increase in HER2 protein over expression when there is no true HER-2 gene amplification. The remaining case showed high-level amplification and it was considered as true amplification.

Our data suggest that chromosome 17 analysis is needed only in those cases with low-level HER2 gene amplification, as is postulated in previous reports by Zhao (CISH) [4] and Bose (FISH) [9]. One case displaying low-level amplification and chromosome 17 diploidy was assumed that represented a true amplification.

In our study, all cases negative for HER2 over expression by IHC did not show HER2 gene amplification by CISH. Other antibodies commonly used in IHC, as CB11 or TAB250, have been reported as negative in some cases displaying HER2 gene amplification. It seems that, in terms of correlation between negative results by IHC and gene amplification by CISH, antibody election for IHC may be important in the initial screening of candidates for trastuzumab therapy (i.e., potential false negative results).

We detected HER2 gene amplification in five of twenty-five (20%) cases with HER2 over expression scored as 2+ by IHC. These data are in agreement with previous reports. Tanner et al, using CB11 antibody, did not find HER2 gene amplification by CISH in the same group of cases (2+ by IHC). Perhaps, the explanation resides in the type of antibody used as A0485 antibody has a higher sensitivity compared with other commercial antibodies [4]. Consequent false-positive cases have been reported. This is an important issue because it has been reported that patients with 2+ IHC score do not have a good response to trastuzumab therapy and it could be explained by the lack of HER2 gene amplification.

Four out of forty cases were scored as 3+ by IHC, two of which showed HER2 high-level gene amplification by CISH. The two cases with no amplification included: one case considered as no true amplification because it showed HER-2 low-level gene amplification and polysomy in chromosome 17, and another one classified as unaltered for HER2 and diploid for chromosome 17 by CISH. Although over expression of HER2 protein is considered to result from gene amplification, over expression without gene amplification has been reported. For example, single-copy over expression at the transcriptional level or amplification levels below the method detection threshold may both result in protein over expression. Moreover, it has been postulated that HER2 amplification by FISH might provide more meaningful prognostic information than HER2 over expression, because of patients who were positive by FISH but negative by IHC had a worse survival than did those who had HER2 over expression but no gene amplification. One probable explanation for this discordance could be the different approach for CISH and IHC results evaluation. While for Herceptest more than 10% of tumor cells are considered the minimum for a positive result, CISH scoring according Tanner's criteria needs more than 50% of tumor cells to be called as gene amplification. Thus, it seems to be necessary a standardization in the criteria for HER2 evaluation by CISH and IHC.

We conclude, that HER2 evaluation by CISH discriminates between a positive IHC staining due to gene amplification and amplification-unrelated expression. Therefore, a more complete analysis of the HER2 status (which combines molecular and histopathologic findings) may be possible using CISH. In conjunction with IHC, CISH can provide very important information for prognosis and clinical decisions.


Figure 1 - CISH of HER-2 in a ductal carcinoma showing no more than two gene copies in each nucleus i.e. no amplification
Figure 2 - CISH of HER-2 of a carcinoma showing copy clusters or more than 10 copies per nucleus i.e. amplification
Figure 3 - FISH performed in paraffin of HER-2 of a carcinoma showing copy clusters or more than 10 copies per nucleus i.e. amplification

CISH in Renal Tumors
The current WHO classification system for renal neoplasms is based on the interrelationship of morphologic, clinical, and genetic features, which are reflected in the biology of the cell in terms of proliferation, death, differentiation, and cellular adhesion, properties that play an important role in determining morphology and behavior.

Based on the results of those analyses, alterations in various chromosomes were observed to be associated with previously described histologic phenotypes. Initially, the loss of genetic material from the short arm of chromosome 3 and changes in chromosome 5 were observed to be associated with nonpapillary (clear cell) RCC: specifically, it was found that 90 to 95% of cases of clear cell RCC had a deletion in one of the alleles of chromosome 3p, and 70% of those had duplications of DNA sequences in 5q22. Subsequently, trisomy of chromosomes 7 and 17 and loss of the Y chromosome (+7, +17 and –Y) were associated with cases of PRCC (chromophilic RCC), in which gains in other chromosomes (3q, 8p, 12q, and 20q) were also observed. Using the PRCC classification system developed by Delahunt and Eble (1997), which was validated by later studies in which the prognostic value of the histologic subtypes was also investigated, the alterations described were found most often in type 1 PRCC, whereas the alterations in type 2 were shown to be more heterogeneous. Different techniques were used in these studies: FISH was used by Hughson (1998), who observed trisomy of chromosome 7 in 2 of 3 cases of PRCC and trisomy of chromosome 17 in 1 of 3 cases.

Chromogenic in situ hybridization can be used to evaluate numeric alterations of specific chromosomes in RCC, and our results correspond to those reported in prior studies in which different techniques were used. In sporadic type 1 PRCC, we found a lack of numeric alterations of chromosome 17, data that has not been previously reported and that may prove helpful in the differentiation of these tumors. As in Brunelli's work, no alterations were observed in the 2 metanephric adenomas studied, a finding that may prove useful in the differential diagnosis between these tumors and PRCC.

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