HER-2/neu gene amplification in breast imprint cytology analyzed by fluorescence in situ hybridization: direct comparison with companion tissue sections

Fluorescence in situ hybridization (FISH) is a validated method for detection of HER-2/neu gene amplification and was recently approved by the FDA for diagnostic use in paraffin-embedded tissue. Its use in cytologic specimens, however, has not been investigated. To see whether HER-2/neu gene amplification is detectable in cytologic specimens, we examined touch imprints and corresponding tissue sections of 27 breast carcinomas (20 invasive and 7 in situ) and 3 atypical epithelial hyperplastic lesions, using the FISH technique with the HER-2/neu DNA probe kit (Vysis, Inc., Downers Grove, IL). HER-2/neu gene amplification was determined, using the ratio of HER-2/neu:CEP 17 signal counts; a ratio of 2.0 or greater was considered amplified. Successful hybridization occurred in 55/60 (92%) slides. In all cases, at least one of the paired slides was adequate for evaluation. Whole-cell imprint and tissue section slides yielded comparable HER-2/neu:CEP 17 signal counts and ratios, including one case of low-level HER-2/neu gene copy numbers where the ratio was 2.0. Our findings indicate that whole-cell imprint cytology preparations are a reliable medium for HER-2/neu gene quantification by FISH, and may substitute for or complement tissue section analysis.     

Examination of the increased speed of detection of HER-2/neu gene amplification in breast cancer by fluorescence in situ hybridization (FISH) using image analysis software

The time required to count signals in the detection of HER-2/neu gene amplification in breast cancer by fluorescence in situ hybridization (FISH) has been a problem. To assess whether the amount of time necessary for counting could be reduced, image analysis using computer software (Win ROOF) was tested. Five photographs from each FISH sample were arranged into ten composite photographs. All ten composite photographs were necessary when using the conventional method of manual counting. However, using only four of the composite photographs and the image analysis method, the 60 necessary nucleus numbers could be measured, and a constant ratio of HER-2/neu / CEP 17 was obtained. In all 58 samples used, in the presence or absence of HER-2/neu gene amplification, there was agreement in counts between the conventional and image analysis methods, and a good correlation of r=0.961 (p<0.001) was obtained. Using the image analysis method, the necessary scoring time was reduced, particularly when the HER-2/neu gene had been amplified, where it was completed in about 1/4 of the time normally required. These results indicate that this image analysis method can be applied when using FISH in other areas of research, and may increase the speed of examination.     

Aneusomy 17 in breast cancer: its role in HER-2/neu protein expression and implication for clinical assessment of HER-2/neu status.

HER-2/neu protein overexpression in breast cancer is mostly caused by HER-2/neu gene amplification. However, it is unclear whether aneusomy 17 may also play a role. Using immunohistochemistry assay (IHC) with DAKO antibody and manual quantitation, 189 specimens were selected from archival invasive breast cancer specimens, including most IHC-positive and some IHC-negative cases (n = 158 and 31, respectively). They were then analyzed by PathVysion fluorescence in situ hybridization (FISH) assay (Vysis, Inc., Downers Grove, IL) and by an image analyzer (ACIS; ChromaVision Medical Systems, Inc., San Juan Capistrano, CA)-assisted IHC quantitation. Ninety-two cases contained disomy 17 (chromosome 17 centromere, 1.76-2.25 signals per cell) whereas 97 cases had aneusomy 17, including 82 with low polysomy (2.26-3.75 signals per cell), 10 with high polysomy (> or =3.76 signals per cell), and 5 with hypodisomy (> or =1.75 signals per cell). HER-2/neu protein expression had the highest correlation with HER-2/neu gene dosage (copy number; r =.826), followed by the HER-2/neu gene to chromosome 17 ratio (r =.733). The lowest correlation was with the chromosome 17 copy number (r =.307), on which the 10 cases with high polysomy 17 had a disproportionately high impact. The FISH assay using the PathVysion criterion for HER-2/neu gene amplification (HER-2/neu gene to chromosome 17 ratio, > or = 2.00) achieved higher concordance with ACIS IHC than did an alternative FISH criterion (absolute HER-2/neu gene copy number, > or = 4.00 signals per cell). Most ACIS IHC-PathVysion FISH-discordant cases contained disomy or low polysomy 17, whereas all 10 cases with high polysomy 17 had no such discordance. However, two cases with monosomy 17 had ACIS IHC-PathVysion FISH discordance, i.e., with gene amplification, but no protein overexpression. Both cases would have had no gene amplification if the alternative FISH criterion had been used. In conclusion, aneusomy 17 is common in breast cancer. Except in a certain subset of cases, aneusomy 17 probably is not a significant factor for HER-2/neu protein expression or for clinical assessment of HER-2/neu status.     

Evaluation of Her-2/neu status in carcinomas with amplified chromosome 17 centromere locus

Accurate assessment of Her-2/neu (erb-b2) status in breast carcinoma is essential for therapy planning. Clinical assays are targeted at protein overexpression (immunohistochemical analysis) or gene amplification (fluorescence in situ hybridization [FISH]). Cases with aberrant FISH signal patterns are problematic and may lead to underreporting of Her-2/neu amplification. We performed FISH with additional chromosome 17 probes, SMS (Smith-Magenis syndrome critical region) and RARA (retinoic acid receptor), on 7 cases with unusual Her-2/CEP17 (chromosome 17 centromere control probe) results to assess whether different measurements of chromosome 17 copy number might clarify the Her-2/neu amplicon status. Although the Her-2/CEP17 ratio scores were within normal range (<2.0), the Her-2/SMS or Her-2/RARA ratio revealed amplification of Her-2/neu in 5 of 7 cases. Immunohistochemical analysis demonstrated Her-2/neu protein overexpression in the same 5 cases only. We describe novel application of SMS/RARA FISH probes for assessing cases with complex Her-2/CEP17 FISH patterns. Such additional data, correlated with immunohistochemical analysis, may help guide therapy in patients with breast carcinoma.     

HER-2/neu expression in germ cell tumours

AIMS: To determine the rate of HER-2/neu positivity of germ cell tumours by immunohistochemistry (IHC) and by fluorescence in situ hybridisation (FISH). PATIENTS/METHODS: Ninety six archival, paraffin wax embedded pathology specimens were chosen from four groups of germ cell tumours. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the INFORM assay and confirmed with a centromere 17 probe. RESULTS: Twenty two of 96 specimens overexpressed the HER-2/neu protein when measured by IHC. Only three specimens showed HER-2/neu gene amplification by FISH. There was no correlation between the results obtained by IHC and FISH. CONCLUSIONS: The lack of concordance between IHC and FISH makes it unlikely that overexpression of the HER-2/neu protein in germ cell tumours is of prognostic or therapeutic relevance. Because of the low rate of HER-2/neu gene amplification in germ cell tumours, a clinical trial of trastuzumab treatment in patients with germ cell tumours is not warranted.     

HER-2/neu基因探针的制备及特异性分析

目的 筛选、制备乳腺癌标志基因--HER-2/neu的标记探针,利用间接荧光原位杂交(FISH)技术,探索其临床应用价值.方法 通过文库构建、多级筛选、PCR鉴定、序列分析等技术获得HER-2/neu基因组片段,缺口转移法制备生物素标记探针,与生物素-亲和素放大系统和多种荧光显色系统组合,针对病理标本、细胞涂片进行间接FISH检测.结果 制备的HER-2/neu基因探针可以特异性地检测乳腺癌阳性、胃癌阳性病理片,也可以特异性检测阳性、阴性细胞标本,并与不同的荧光显色系统结合使杂交信号可以选择性地呈现绿色或红色荧光.结论 高特异性的HER-2/neu基因探针和灵活的显色系统为FISH的临床应用奠定了基础.     

Fluorescence in situ hybridization and immunohistochemical assays for HER-2/neu status determination: application to node-negative breast cancer

BACKGROUND: HER-2/neu (ERBB2) gene amplification and/or overexpression is a major event in human breast tumorigenesis. HER-2/neu gene alterations have been the most frequently assessed prognostic factors during the last 10 years in breast cancer and have recently emerged as a management decision tool and a therapeutic target. There is still controversy over the best method to determine whether a tumor is HER-2/neu positive. Because of the increasing demand for HER-2/neu gene status determination in clinical practice, we compared HER-2/neu gene alterations at the DNA level (gene amplification) and the protein level (overexpression) in a panel of patients with lymph node-negative breast cancer who had received local radiotherapy alone, with no adjuvant therapy. METHODS: We tested 100 excised lymph node-negative breast tumors, using fluorescence in situ hybridization (FISH) with a biotinylated HER-2/neu DNA probe and immunohistochemical assays (IHC) with 2 different antibodies. RESULTS: The FISH frequency of HER-2/neu gene amplification was 15%, and the IHC frequency of overexpression was 21%. CONCLUSION: Although HER-2/neu amplification by FISH and HER-2/neu overexpression by IHC correlated well in this panel of lymph node-negative breast carcinomas, there were a number of discordant cases, pointing to the important need for determining HER-2/neu alteration for the future management of HER-2/neu-based clinical applications.     

Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study

Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.     

Fluorescence in situ hybridization analysis of HER-2/neu, c-myc, and p53 in endometrial cancer

Our objective was to evaluate the association between HER-2/neu, c-myc, p53, and clinicopathologic variables in endometrial cancer using fluorescence in situ hybridization (FISH) cytogenetic analysis. FISH analysis for HER-2/neu, c-myc, and p53 was performed on 47 endometrial cancer specimens. Amplification of HER-2/neu was seen in 4/47 (8.5%) cases and amplification of c-myc was seen in 7 of 47 (15%) cases; neither was associated with adverse clinicopathologic variables or survival. Deletion of p53 was seen in 31/47 (66%) cases and was associated with poor histologic grade (P = 0.008). There was no impact of genetic alterations on overall survival or disease-free interval. Grade 3 tumor was associated with poor overall survival (P = 0.032). This study found that p53 deletion is a common genetic alteration in endometrial cancer and is associated with poor-grade tumors.     

HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.

We evaluated 750 consecutive invasive breast carcinomas for HER-2/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2/neu positive and 0 and 1+ IHC reactions were considered HER-2/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH analysis was performed on the positive and negative IHC tests. One hundred thirty-eight cases (18.4%) were HER-2/neu positive by IHC and/or FISH. One hundred twenty-three of the positive cases (89%) were 3+ IHC reactions and 14 positive cases were inconclusive by IHC and amplified by FISH. There was concordance with FISH in 77 of 78 (98.7%) of the positive or negative IHC cases that were tested (95% confidence interval [CI] = 93.1 to 100%). A single IHC-negative case showed HER-2/neu amplification by FISH. Thirty-nine cases were 2+ IHC (5.2%); 14 (36%) were amplified, 24 (62%) were not amplified, and one was not interpretable. HER-2/neu positivity was observed in 34% of grade 3 ductal carcinomas, 11.4% of grade 2 ductal carcinomas, 3.2% of grade 1 ductal carcinomas, and 3.2% of lobular carcinomas. Occasional cases with discordant IHC expression of HER-2/neu within the in situ and invasive carcinoma elements were also identified. IHC reliably characterized HER-2/neu in approximately 95% of the cases studied (95% CI = 93.0 to 96.2%) and was effective as a primary method for evaluating HER-2/neu status. In this study, 2+ IHC reactions were a heterogeneous group best regarded as indeterminate or inconclusive; in this series, only 36% were amplified by FISH analysis. Our findings suggest that a combination of IHC and FISH testing with FISH analysis performed reflexly on all 2+ IHC cases can optimize HER-2/neu testing.     

Immunocytochemical evaluation of HER-2/neu on fine-needle aspirates from primary breast carcinomas

Detection of HER-2/neu alterations is increasingly used in breast cancer patients for therapeutic purposes. This study examines the reliability of HER-2/neu immunocytochemical assessment on 66 cytospin smears obtained by fine-needle aspiration biopsy from breast cancer patients. Results were compared with those obtained by both fluorescence in situ hybridization (FISH) on fine-needle aspirate (FNA) and immunohistochemistry (IHC) on matched histologic section. Concordance between immunocytochemistry (ICC) and FISH was 78% and between ICC and IHC was 84%. Discordance mainly concerned seven unamplified cases that resulted positive by ICC and four cases scored negative by IHC but positive by ICC. Simultaneous assessment of HER-2/neu by ICC, IHC, and FISH was available in 24 cases; the concordance was 75%. In this study, the false positivity of immunocytochemical technique represents the major criticism. In our experience, FISH remains the most objective and powerful technique for HER-2/neu assessment on breast cancer FNAs.     

HER-2/neu expression in glioblastoma multiforme.

BACKGROUND: The HER-2/neu oncogene encodes for a transmembrane glycoprotein with intracellular tyrosine kinase activity. The HER-2/neu receptor belongs to the family of epidermal growth factor receptors that are crucial in the activation of subcellular signal transduction pathways controlling epithelial cell growth and differentiation. Overexpression of HER-2/neu is observed in 20% to 40% of breast cancers and other solid tumors. Although information is limited, one study suggested that 15% of glioblastoma multiforme (GBM) express HER-2/neu by immunohistochemistry (IHC); gene amplification by fluorescence in situ hybridization (FISH) was not investigated. Studies in this area are potentially significant owing to the role of recombinant monoclonal anti-HER-2/neu antibody traztuzumab (Herceptin) in the treatment of tumors. DESIGN: A retrospective clinicopathologic review of 49 patients with GBM with HER-2/neu IHC staining and HER-2/neu gene amplification by FISH was performed. RESULTS: The study included 44 patients (17 women, 27 men; age range 20 to 79 y, mean 57.9 y). Initial surgery involved tumor debulking or subtotal resection in 34 patients. Thirty-six patients received adjuvant radiation therapy and 19 patients received adjuvant chemotherapy. At follow-up (range 1.0 to 49.5 mo, mean 10.5 mo), 40 patients died with tumor and 4 patients were lost to follow-up. All tumors were negative for HER-2/neu protein by IHC and for HER-2/neu gene amplification by FISH. CONCLUSIONS: No GBM demonstrates HER-2/neu protein by IHC or amplification of the HER-2/neu gene by FISH. The HER-2/neu oncogene does not seem to play a role in the pathogenesis of GBM.     

Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization

Breast cancer patients with HER-2/neu oncogene amplification by fluorescence in situ hybridization (FISH) have been shown to have a better response to trastuzumab (Herceptin) therapy than those showing HER-2/neu protein overexpression only. Many centers currently perform FISH only on tumors showing 2+ HER-2/neu positivity by immunohistochemistry (IHC), with the assumption that 3+ positivity virtually equates with amplification. Results of FISH performed on 102 breast cancer cases over a 12-month period were correlated with HER-2/neu IHC results. FISH was performed using a ratio of HER-2/neu and chromosome 17 centromere signal counts (PathVysion; Vysis, Downers Grove, IL). Immunohistochemical expression of HER-2/neu was evaluated according to the published scoring guidelines of the HercepTest (Dako, Carpinteria, CA). Only 22 of 45 tumors with 3+ positivity (49%) showed amplification by FISH. Only 2 of 25 cases with 2+ staining by IHC (6%) showed gene amplification, and 1 of 25 cases with negative IHC staining (4%) showed weak amplification. Of the 25 cases showing oncogene amplification, 22 (88%) showed 3+ IHC positivity, 2 (8%) showed 2+ positivity, and 1 (4%) was negative by IHC. More than 50% of breast tumors showing strong 3+ HER-2/neu staining do not show oncogene amplification by FISH. Most tumors with 2+ and negative IHC also fail to amplify. In our experience, FISH studies should be performed on all 3+ and 2+ staining tumors to avoid inappropriate and toxic treatment. The decision to perform FISH on IHC-negative tumors should be guided by additional parameters, including tumor grade and estrogen receptor status.     

AQUA and FISH analysis of HER-2/neu expression and amplification in a small cell lung carcinoma tissue microarray

AIMS: Most small cell lung carcinoma (SCLC) patients have metastatic disease at the time of diagnosis and are faced with poor prognosis and limited treatment options. Reports of HER-2/neu gene amplification and overexpression in this malignancy have raised the possibility of applying targeted immunotherapy with trastuzumab, the monoclonal antibody used to treat metastatic breast cancer. However, a review of the studies measuring HER-2/neu gene amplification and protein expression in SCLC reveals discordant results. The aim of the present study was to re-examine HER-2/neu expression in SCLC in relation to gene copy number using the new, highly sensitive, immunofluorescence automated quantitative analysis (AQUA) technology. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) was used to measure HER-2/neu gene copy number and amplification status and AQUA was used to measure protein expression in a series of 23 SCLC tumours on a tissue microarray. None of the 17 SCLC specimens assessable by FISH exhibited HER-2/neu gene amplification as defined by a HER-2/neu/chromosome 17 ratio = or > 2. Twelve of 17 (70.1%) SCLC samples were polysomic for chromosome 17 with corresponding increases in HER-2/neu gene copy numbers. Intermediate levels of protein expression corresponding to AQUA scores in the range of 4-24 were detected in all 23 specimens. High protein expression levels corresponding to AQUA scores up to 83, observed previously in association with gene amplification and poor prognosis in breast cancer cases, were not detected in the present study. No statistically significant association was observed between absolute chromosome 17 or HER-2/neu gene copy numbers and protein expression levels in tumour cells (P > 0.45). CONCLUSIONS: The lack of gene amplification and robust HER-2/neu protein expression in SCLC tumour cells in this series does not suggest a prominent role for the HER-2/neu gene in SCLC tumour progression and does not support the general applicability of targeted immunotherapy with trastuzumab to this malignancy.     

HER-2/neu gene amplification by FISH predicts poor survival in Barrett's esophagus-associated adenocarcinoma

The HER-2/neu oncogene is localized to chromosome 17q and shares significant homology with the epidermal growth factor receptor. HER-2/neu protein overexpression has been associated with poor prognosis in a variety of tumors, but its significance in Barrett's esophagus-associated adenocarcinoma (BEAd) is unknown. Therefore, the aim of this study was to evaluate the prevalence and prognostic value of HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) in 63 cases of BEAd. Routinely processed tissue sections from resection specimens of 63 patients with BEAd (M/F ratio, 10:1; mean age, 63 years) were assayed for HER-2/neu gene amplification by FISH using the Ventana unique sequence probe (Ventana Medical Systems, Inc, Tuscon, AZ). FISH results were correlated with the pathological features of the tumors and with patient survival. Clinical follow-up data were available for 54 patients (mean follow-up, 31 months [range, 1 to 152 months]). The HER-2/ neu gene was amplified in 12 of 63 (19%) cases. The presence of HER-2/neu gene amplification showed a trend toward a correlation with depth of tumor invasion (P = .07), lymph node metastasis (P = .13), and pathological stage (P = .14), but did not correlate with any of the other pathological features, such as degree of differentiation or tumor size. On both univariate and multivariate analysis, HER-2/neu gene amplification was associated with shortened survival (P = .03). HER-2/neu oncogene amplification, as determined by FISH, correlates with shortened patient survival and independently predicts poor outcome in patients with BEAd.     

Overexpression/amplification of HER-2/neu is uncommon in hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent fatal cancers in the world. Despite advances in early diagnosis and improvements in surgical techniques, the survival of patients with HCC even after resection is poor because of the high incidence of recurrences. Therefore, the identification of prognostic factors may be helpful in the development of new treatment protocols. AIMS: To investigate HER-2/neu status in HCC by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and to explore the possibility of using trastuzumab in the treatment of HCC. METHODS: Eight hundred and sixty eight surgical samples from patients with primary HCC were examined for their HER-2/neu status. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the PathVysion HER-2 DNA probe kit. The correlations between HER-2/neu overexpression and clinicopathological characteristics were analysed statistically. RESULTS: HER-2/neu overexpression was detected in 21 (2.42%) of the 868 primary HCCs. Only one specimen showed HER-2/neu gene amplification by FISH. No significant associations were found between HER-2/neu overexpression and the clinicopathological parameters. CONCLUSIONS: There is a low frequency of HER-2/neu overexpression/amplification in HCC. There appears to be no role for HER-2/neu as a prognostic marker and no benefit of anti-HER-2/neu trastuzumab treatment in patients with HCC.     

Testing for HER-2/neu in breast cancer: is fluorescence in situ hybridization superior in predicting outcome?

Testing for alterations in HER-2/neu in breast cancer has become increasingly popular in recent years, particularly with the recent development of a humanized antiHER-2/neu monoclonal antibody, trastuzumab, which is currently being employed in conjunction with cytotoxic chemotherapy to treat metastatic breast cancer in patients whose tumors exhibit this HER-2/neu alteration. Controversy exists not only on the optimal method of laboratory testing for this HER-2/neu alteration (i.e., fluorescence in situ hybridization (FISH) versus immunohistochemistry (IHC) versus others), but also on the type of reagents used for a given method. A plethora of published studies on tissue-based HER-2/neu testing has recently appeared in many peer-reviewed journals; many have concluded that IHC could be used as a first-line screening test, with the recommendation of FISH to confirm indeterminate results. In contrast to these studies, a recent study by Pauletti et al. showed that HER-2/neu testing by IHC does not predict clinical outcome as accurately as does FISH. This commentary discusses the findings of this study, within a broader review of critical issues relating to HER-2/neu testing in breast cancer.     

Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridization. Correlation with fluorescence in situ hybridization, HER-2/neu, p53 and Ki-67 protein expression, and outcome.

Fluorescence in situ hybridization (FISH) is the most widely used technique to detect HER-2/neu gene amplification; however, it is only available in some institutions. In contrast, chromogenic in situ hybridization (CISH) can be evaluated by routine light microscopy. In endometrial carcinoma there are few data concerning HER-2/neu status and prognosis. Therefore, we determined HER-2/neu gene status by CISH using a digoxigenin-labelled probe on 60 formalin-fixed paraffin-embedded endometrial carcinomas. The data were compared with the immunohistochemistry of HER-2/neu (A0485, TAB250), p53, Ki-67, clinicopathological factors, and survival. By conventional light microscopy, HER-2/neu amplification (>/=6 copies >50% cancer cells) was detected in 14% (8/59) tumours, HER-2/neu overexpression (>10% cells moderate/strong complete membrane staining) in 22% (13/60) for A0485, and 18% (11/60) for TAB250, p53 (>10% +cells) in 61% (36/59), and Ki-67 (>50% +cells) in 50% (30/60). Discordant cases for CISH and immunohistochemistry, as well as all (2+) were further analysed by FISH (Vysis). Among 10 cases (2+) and not amplified by CISH, two showed low-level amplification by FISH. Significant correlation was found between amplification and protein overexpression (P相似文献   

Assessment of HER‐2/neu overexpression and/or gene amplification in breast carcinomas: should in situ hybridization be the method of choice?

AIMS: Since the release of Herceptin, pathology laboratories have been requested to test breast carcinomas for HER-2/neu overexpression and/or gene amplification. Standardized IHC and FISH are mandatory in order to get reliable results, but there are problems even with standardized procedures. We decided to evaluate the two methods to determine which, or possibly if both, should be the primary investigation method(s). METHODS AND RESULTS: The material consisted of 215 primary invasive breast carcinomas with complete clinical follow-up of 15 years. HER-2/neu protein expression was determined for all specimens, whereas FISH for assessing HER-2/neu gene signal number was done in 165 cases. IHC was double-checked with two or three different antibodies in 35 tumours, including all cases with discrepancies between IHC and FISH. Among these, there were discrepancies in a third. IHC overexpression of HER-2/neu was found in 13% and gene amplification in 18%. Discordance between IHC and FISH was found in 11 cases (8%). Five tumours were IHC+/FISH- and six were IHC-/FISH+. IHC and FISH positive cases as well as FISH only positive tumours had the same prognosis respecting survival. Tumours with >2 but 4 gene signals per nucleus. In contrast, cases with IHC overexpression without gene amplification had a prognosis similar to that of IHC-/FISH- tumours. CONCLUSIONS: From our data, it seems to be more important to assess HER-2/neu gene amplification than IHC overexpression. Failure to detect FISH-amplified (IHC-negative) cases would have an adverse effect on the survival of these patients. On the other hand, IHC overexpression tumours without gene amplification appear to belong to a better prognostic group, and failure to detect them would probably not have a negative effect on the survival of these women. Even though FISH is a more complex and expensive procedure, it should be considered the method of choice for primary assessment of HER-2/neu status in breast cancer patients.