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We compared chromogenic in situ hybridization (CISH) with fluorescence in situ hybridization (FISH) for assessing HER-2/neu gene amplification using tissue microarrays (TMAs) made from formalin-fixed, paraffin-embedded tissue blocks from 113 cases of invasive breast carcinoma. TMAs were created using 0.6-mm tissue cores with 4 sampled cores per tumor. For both assays, a HER-2/chromosome 17 signal ratio of 2.0 or more was considered positive for gene amplification. The average ratio of cores from the same tumor was used for determination of gene amplification status of that particular tumor Of 113 cases, 102 were tested successfully by both assays. The results were concordant in 100.0% of cases (63 amplified; 39 nonamplified). All 22 cases of borderline (ratio, 2.0-2.5) or low-level (ratio, 2.6-3.9) amplification by FISH also showed HER-2 gene amplification by CISH. CISH is as sensitive as FISH in detecting borderline and low-level HER-2 amplification. Reliable recognition of the invasive carcinoma area by light microscopy and preservation of the test slides are added advantages of CISH. CISH performs as well as FISH in the analysis of HER-2 gene amplification in breast cancer and might have advantages in certain situations.

Tissue microarrays (TMAs) have been commonly used to study protein expression by immunohistochemistry (IHC). However, limited data exist on the validity of using TMAs to study gene amplification. In this study, we evaluated the feasibility of using breast carcinoma TMAs to study HER-2 gene amplification by fluorescence in situ hybridization (FISH). In addition, hormonal receptor status (ER and PR) and HER-2 protein overexpression by IHC were also studied, and results were compared with whole tissue sections. FISH for HER-2 was performed on formalin-fixed paraffin-embedded tissue from 114 invasive breast carcinomas both on whole tissue sections and on TMAs containing the same tumors. The TMA was created using 0.6-mm tissue cores with four sampled cores per tumor from the same tissue block used for whole section FISH. The PathVysion HER-2 probe kit was used for the FISH analysis. A ratio of HER-2:Chromosome17 > or =2.0 was interpreted as positive for gene amplification. The ER or PR was interpreted as positive when nuclear staining was detected in more than 10% of tumor cells. The HER-2 IHC (HercepTest; DAKO Corp, Carpinteria, CA) results were interpreted as 0, 1+, 2+, and 3+ according to standard criteria. The FISH results in the TMA and whole sections were concordant in 99 out of 101 successfully analyzed cases (99%). The FISH scores were consistent among the two to four cores in the majority of the cases. ER and PR results were concordant between whole sections and TMA cores in 97% (107/110) and 89% (97/109) cases, respectively. The overall concordance for HER-2 status by IHC between whole sections and TMA cores was 86% (94 out of 109 cases). TMAs are a reliable approach to study HER-2 gene amplification in a high throughput manner.

Background: Gene amplification is the primary mechanism of HER-2/neu overexpression in breast cancer and is a strong predictor of prognosis. Currently screening for HER-2/neu gene amplification in breast cancer is done by fluorescent in-situ hybridization (FISH), which is accurate but costly and labor intensive. We have evaluated a new PCR (polymerase chain reaction)-based assay for the detection of HER-2/neu gene amplification in human breast cancer., Study Design: A total of 15 breast cancer cell lines and 14 breast cancer specimens were evaluated. HER-2/neu status of the tumors was evaluated by FISH and then assessed using a quantitative polymerase chain reaction/ligase detection reaction (PCR/LRD) technique., Results: Amplification of the HER-2/neu gene was detected in seven cell lines previously reported to have amplification and no amplification was found in any of the six that had been reported not to have amplification. In the assessment of breast specimens the PCR/LDR and FISH assays were in complete agreement. All 10 tumors with amplification by FISH were also amplified by PCR/LDR., Conclusions: The PCR/LDR technique successfully detects HER-2/neu gene amplification in clinical breast cancer specimens and shows 100% concordance with FISH. This technique is an accurate and rapid alternative to FISH with the potential for automation and high throughput analysis of HER-2/neu status in breast cancer.

Her-2/neu, a proto-oncogene located on chromosome 17, is an important biomarker in breast carcinoma. Immunohistochemistry (IHC) is currently the most widely used method for assessing Her-2/neu status. Some IHC-positive cases do not show Her-2/neu gene amplification by fluorescence in situ hybridization (FISH). It has been suggested that some of these IHC "false positive" results may in part be due to increased copy number of chromosome 17 resulting in increased Her-2/neu protein expression. We analyzed IHC and FISH data from 561 cases of invasive breast carcinoma to test this hypothesis. IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded sections of 561 invasive breast carcinomas. The IHC results were interpreted as 0, 1+, 2+, or 3+ according to the manufacturer's recommended criteria. The FISH results were expressed as a ratio of Her-2/neu/chromosome 17 and were interpreted as positive (> = 2.0) or negative (<2.0) for gene amplification according to the manufacturer's recommended scoring system. We found that in IHC 3+/FISH-negative cases (n = 15) both the average chromosome 17 copy number and the average Her-2/neu copy number were significantly higher than that in IHC (0 to 2+)/FISH-negative cases (n = 411) (2.45 vs. 1.68; P < 0.0001, and 3.19 vs. 1.95; P < 0.0001, respectively). In contrast, the IHC 2+/FISH-negative cases did not exhibit a significantly increased number of chromosome 17 compared to IHC 0 to 1+ cases. In addition, the average copy number of chromosome 17 in FISH-positive cases (n = 135) was significantly higher than that in FISH-negative cases (n = 426) (2.27 vs. 1.70; P < 0.0001), indicating a general association of increased chromosome 17 copy number with Her-2/neu gene amplification. Thus, our data suggest that IHC 3+ immunostaining without scorable gene amplification may indeed be, at least in some cases, the result of increased Her-2/neu protein expression secondary to an increased copy number of chromosome 17, associated with an increased total number of Her-2/neu gene copies per tumor cell.

HER 2/neu is an important oncogene in breast cancer, but the prevalence and significance of HER 2/neu gene amplification in colon cancer have been poorly documented. We have evaluated HER 2/neu gene amplification and protein overexpression in a series of colon cancers to assess the frequency, concordance and clinical significance of these events. HER 2/neu gene copy number was measured in 154 primary colon tumors, 15 liver metastases and matched normal tissues using a quantitative PCR/ligase detection reaction (LDR) technique developed and validated in our laboratory. HER 2/neu copy number was confirmed by fluorescent in situ hybridization (FISH) in all tumors found to have gene amplification. In an independent and blinded fashion, HER 2/neu expression was assessed in paraffin sections from 139 of the tumor specimens using the HercepTest kit. HER 2/neu gene amplification was observed in 4 (2.4%) of the 169 tumor specimens and in none of the normal tissues. There was no apparent association with stage of disease, tumor grade or patient survival. Among 139 cases evaluated by immunohistochemistry (IHC), HER 2/neu overexpression was seen in 5 cases (3.6%). There was extremely high concordance (kappa = 0.852) between gene amplification and protein overexpression. The low prevalence of HER 2/neu gene amplification and protein overexpression suggests that this oncogene plays an infrequent role in the development and progression of colon cancer. These data indicate that the primary mechanism of dysregulated HER 2/neu expression in colon cancer, as in breast cancer, is gene amplification., (Copyright 2003 Wiley-Liss, Inc.)