HER2 Ratio Research Articles

A UK NEQAS ring study evaluating observer variation in the diagnosis of HER2 amplification using the Kreatech™ HER2 FISH probe.

Abstract Abstract #3005 Background: Whilst HER2 testing is well established as a key requirement for all newly diagnosed breast cancers the quality and consistency of HER2 testing remains a point of controversy. Most countries use FISH or ISH based testing for HER2 gene amplification in equivocal (IHC 2+) cases. Data on reproducibility and portability of novel and even established HER2 methods is sparse. We report on a multicentre evaluation of intra and inter-observer/site variation in scoring HER2 results from 45 breast cancers measured in 8 laboratories using the Kreatech™ HER2 FISH probe.
 Methods: A commercial tissue micro-array (TMA; Stretton Scientific UK) containing 2 replicate cores from 45 breast cancers was circulated to 8 laboratories with varying experience in FISH methodology. Five laboratories (1-5) were UK NEQAS reference labs, 3 further experienced routine diagnostic labs (A-C) participated in the scheme designed to test the site to site reproducibility of the Kreatech™ FISH probe. All laboratories FISHed a 90 core TMA and 7/8 reported results for all assessable cores, 1 lab reported 1 result per evaluable cases (as per the original study design). Results were collated centrally and analyzed for intra and inter-observer between both “reference” laboratories (labs 1-5) and non-reference laboratories (A-C).
 Results: Overall 81% of cores (range 70-94%) were successfully analysed by Kreatech™ FISH. Intra-observer variation was lower than expected at 4.7% (range 3.5-8.4%) for HER2 ratio. Mean intersite variation in HER2:Chr 17 ratio was significantly lower (8.5%) for reference vs non-reference (13.4%) laboratories (p=0.0013). Of 546 reported cores only 7 (1.3%) were discordant (with 4 borderline). Analysis of site to site variation highlighted possible systematic differences in scoring approaches in 3/8 laboratories including a “reference laboratory”.
 Conclusions: Intra- and inter-observer variation of absolute HER2 ratios appears to be tightly controlled across 8 laboratories using Kreatech™ HER2 FISH probes. Detailed comparisons of scoring results suggests some systematic differences between a few laboratories in the interpretation of FISH. However the majority of laboratories show extremely tight reproducibility of numeric HER2 FISH results. This study highlights the potential for extremely robust and quantitatively reproducible FISH results from both “diagnostic” and “reference” laboratories. However it also suggests continued quality assessments are essential to good performance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3005.

HER2 status in breast cancer of Chinese women: a study of 1170 cases using fluorescence in-situ hybridization

To assess the prevalence of HER2 amplification according to HER2 and chromosome 17 copy numbers and HER2 FISH (fluorescence in-situ hybridization) ratio in breast cancer occurring in Chinese women. Eleven hundreds and seventy cases of breast cancer occurring in Chinese women, who would be treated by trastuzumab and/or relevant chemotherapy based on HER2 status, were enrolled into the study. The formalin-fixed and paraffin-embedded tumor tissues were tested by FISH (PathVysion, Vysis). Among the 1170 cases of breast cancer studied, 408 cases (34.87%) were FISH-negative, whereas 762 cases (65.13%) were FISH-positive, including 87 cases (87/762, 11.42%) with highly amplified HER2 gene (signals arranged in aggregates). As for the remaining 675 FISH-positive cases, 159 cases (23.56%) showed low amplification (HER2/CEP17 ratio = 2 to 4), 422 cases (62.52%) showed moderate amplification (ratio = 4 to 10) and 94 cases (13.93%) showed high amplification (ratio > 10) for HER2 gene. Only 14 of the 1170 cases (1.20%) had indeterminate results (ratio between 1.8 and 2.2), including 1.23% (5/408) borderline FISH-negative (ratio between 1.8 and 2.0) and 1.18% (9/762) borderline FISH-positive (ratio between 2.0 and 2.2). Our data showed that 73.00% (854/1170) of cases were chromosome 17 aneusomy, including 22.65% (265/1170) hypodisomy (chromosome 17 copy number per cell < or = 1.75), 38.38% (449/1170) low polysomy (chromosome 17 copy number per cell 2.26 to 3.75) and 11.97% (140/1170) high polysomy (chromosome 17 copy number per cell > or = 3.76). The frequency of chromosome 17 polysomy was 50.34%. In the FISH-positive subgroup, 23.88% (182/762) was disomy (chromosome 17 copy number per cell between 1.76 and 2.25), 24.15% (184/762) hypodisomy, 39.37% (300/762) low polysomy and 12.60% (96/762) high polysomy. The frequency of chromosome 17 polysomy in the FISH-positive subgroup was 51.97%. In the FISH-negative subgroup, 32.84% (134/408) were disomy, 19.85% (81/408) hypodisomy, 36.52% (149/408) low polysomy and 10.78% (44/408) high polysomy. The frequency of chromosome 17 polysomy in the FISH-negative subgroup was 47.30%. On the other hand, HER2 monoallelic deletion (HER2/CEP17 < or = 0.7) was observed in 2.39% of cases. Chromosome 17 monosomy was detected in 5.00% (38/762) and 4.41% (18/408) of HER2-positive and HER2-negative groups, respectively. A HER2 ratio of < 1.5 was noted in 32.30% of all cases (including 92.65% of HER2-negative cases), compared with 9.23% (108/1170) with ratio between 1.5 and 2.2. The results show that a high amplification of HER2 gene is detected by FISH. Moderate amplification of HER2 gene and chromosome 17 polysomy are commonly seen in breast cancer patients in China Mainland. These findings may carry significant clinical and pathogenetic implication.

Evaluation of chromosome 17 (Chr-17) polysomy in HER2 FISH-negative metastatic breast cancer (MBC) patients enrolled in a randomized phase III study of paclitaxel and lapatinib

1006 Background: Patients with polysomy of Chr-17 have been suggested as a FISH-negative subpopulation that might benefit from HER2-directed therapy, due to an increase in HER2 gene expression without HER2 specific amplification. A recent phase III clinical trial studied lapatinib (L) in combination with paclitaxel (P) as 1st-line therapy for HER2-negative or HER2-untested MBC. We examined the hypothesis that HER2 FISH-negative patients with Chr-17 polysomy may be sensitive to HER2 inhibitors using data from this trial. Methods: Patients (n=579) with newly diagnosed MBC were randomized to P with placebo or L (P+L). Response rate (RR) using RECIST and progression-free survival (PFS), were evaluated. HER2 expression level by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and mean HER2 ratio of Chr-17 values were determined in a central laboratory using archival tissue. Tumors with a HER2:Chr-17 ratio < 2.0 were considered HER2-negative by FISH. Chr-17 centromere averages of 2.0 and 2.2 were used as thresholds for Chr-17 polysomy. Associations with PFS and RR in the HER2-negative groups were evaluated across treatment arms. There were insufficient numbers of patients in higher categories of polysomy for meaningful comparisons. Results: In this study, 406 pts were HER2 FISH-negative. Data are available for PFS in 405, of whom 44 (11%) met the definition of polysomy (Chr-17 >2.2, HER2 FISH-negative). Median PFS in the polysomy group was 20.9 vs. 24.4 wks for L+P and P respectively, while in the non-polysomy group, the median PFS times were 24.6 and 23.1 wks for L+P and P. Log-rank testing showed no treatment advantage for either polysomy group. Similar results were found for the PFS comparisons using 2.0 as the specified cutoff for Chr-17 polysomy. RR in the polysomy group were 17.4% for P+L vs. 9.5% for P, while in the non-polysomy group RR was 32.0% for P+L vs. 25.1% for P; neither comparison was statistically significant. Conclusions: We could not confirm the hypothesis that Chr-17 polysomy in HER2 FISH-negative pts is associated with improved outcome when lapatinib is added to chemotherapy as a HER2-targeted treatment. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline GlaxoSmithKline, Roche GlaxoSmithKline Genentech, GlaxoSmithKline, Roche Genentech, GlaxoSmithKline

Impact of HER2 Gene and Protein Status on the Treatment Outcome of Cisplatin-Based Chemoradiotherapy for Locally Advanced Non-small Cell Lung Cancer

It has not been fully evaluated whether both HER2 gene copy number and HER2 protein expression are related to the outcome of chemoradiotherapy in patients with locally advanced non-small cell lung cancer (LA-NSCLC). The aim of this study was to evaluate their relationships. HER2 gene copy number determined by fluorescence in situ hybridization (FISH) and HER2 protein expression determined by immunohistochemistry (IHC) were assessed in 68 patients with LA-NSCLC enrolled in our previous phase II trials of concurrent cisplatin-based chemoradiotherapy, and a multivariate analysis was conducted for response and survival. HER2-IHC-positive tumors were detected in 23 patients (34%), and the median ratio of HER2 to chromosome 17 copy number was 0.93 (range, 0.55-2.00). The HER2-FISH results were marginally correlated with the IHC results (p = 0.0715). When the median ratio in the FISH analysis was used as a cut-off level for its positivity, there was no association between either HER2-FISH or IHC status and objective response to chemoradiotherapy. Contrary, a multivariate analysis revealed HER2-FISH result but not IHC result was an independent poor prognostic factor for both overall survival and progression-free survival (hazard ratio = 2.568, 95% confidence interval [CI] = 1.117-5.903, p = 0.0264 and hazard ratio = 2.283, 95% CI = 1.005-5.189, p = 0.0487, respectively). Patients with HER2 FISH-positive LA-NSCLC had a poorer outcome even when treated with cisplatin-based chemoradiotherapy, despite the strong need for validation assessment of these observations. Development of more effective treatment for these high-risk patients is needed to improve their poor prognosis.

New cutpoints to identify increased HER2 copy number: analysis of a large, population-based cohort with long-term follow-up

HER2 gene amplification and/or protein overexpression in breast cancer is associated with a poor prognosis and predicts response to anti-HER2 therapy. We examine the natural history of breast cancers in relationship to increased HER2 copy numbers in a large population-based study. HER2 status was measured by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in approximately 1,400 breast cancer cases with greater than 15 years of follow-up. Protein expression was evaluated with two different commercially-available antibodies. We looked for subgroups of breast cancer with different clinical outcomes, based on HER2 FISH amplification ratio. The current HER2 ratio cut point for classifying HER2 positive and negative cases is 2.2. However, we found an increased risk of disease-specific death associated with FISH ratios of >1.5. An 'intermediate' group of cases with HER2 ratios between 1.5 and 2.2 was found to have a significantly better outcome than the conventional 'amplified' group (HER2 ratio >2.2) but a significantly worse outcome than groups with FISH ratios less than 1.5. Breast cancers with increased HER2 copy numbers (low level HER2 amplification), below the currently accepted positive threshold ratio of 2.2, showed a distinct, intermediate outcome when compared to HER2 unamplified tumors and tumors with HER2 ratios greater than 2.2. These findings suggest that a new cut point to determine HER2 positivity, at a ratio of 1.5 (well below the current recommended cut point of 2.2), should be evaluated.

Relationship between the signal ratios of HER-2/CEP17 and c-MYC/CEP17 and the pathological response of neoadjuvant therapy using docetaxel and trastuzumab in breast cancer

The purpose of this study was to assess the efficacy and predictive biomarkers of combination docetaxel-trastuzumab in a neoadjuvant setting by means of a phase II trial. Women with histologically-confirmed advanced invasive breast cancer whose tumours overexpressed HER-2 received 4 cycles of docetaxel (70 mg/m2 every 3 weeks) and trastuzumab (4 mg/kg loading dose, 2 mg/kg weekly thereafter). Twenty-one patients were enrolled, and all completed 4 cycles of treatment. Two patients were later found to be inoperable, and neither pathological nor clinical response was assessed. The pathological complete response rate was 21% (4/19; 95% CI, 6-46%) and the overall clinical response rate 89% (17/19; 95% CI, 67-99%). The relationship between the expression of biomarkers (HER-2, c-MYC, BRCA1 and Ki-67) and pathological response was assessed. The results suggested the possibility that tumours showing a high signal ratio of HER-2/CEP17 or c-MYC/CEP17 might be more sensitive to this combination therapy. Based on these results, it can be speculated that approximately 30% pCR might be obtained in cases with a high signal ratio of HER2/CEP17 or c-MYC/CEP17. Further trials are needed.

The expression level and clinical signification of soluble HER-2/neu in breast cancer

目的 分析乳腺癌血清可溶性HER-2/neu水平并探讨其临床意义.方法用ELISA方法分析105例乳腺癌患者、24例乳腺良性疾病及30名健康体检者血清可溶性HER-2/neu水平,并用免疫组化(IHC)方法分析乳腺癌组织切片中癌细胞的着色情况.结果乳腺癌患者血清中可溶性HER-2/neu水平与正常对照和良性乳腺疾病相比,差异有统计学意义(P<0.05).随病程进展乳腺癌患者可溶性HER-2/neu的阳性率逐步升高,Ⅲ+Ⅳ期的阳性率(63.3 %) 与Ⅰ期(7.7 %)或Ⅱ期阳性率(14.5 %)相比,差异有统计学意义(P<0.05);发生转移的乳腺癌患者可溶性HER-2/neu的阳性率(37.5 %)与未转移患者(6.1 %)相比,差异有统计学意义(P<0.05).血清HER-2/neu水平与IHC结果有显著正相关(P<0.05).结论检测血清可溶性HER-2/neu水平可弥补免疫组化的不足,在乳腺癌监测随访、疗效观察尤其是用Herceptin治疗后的疗效观察中发挥其独到的作用。

Association of HER2/neu gene amplification and copy numbers of HER2/neu and chromosome 17 with tumor characteristics in resected colorectal cancer (CRC)

3592 Background: HER2/neu is an oncogene on chromosome (chr) 17 that encodes a type I tyrosine kinase growth factor receptor. In breast cancer, HER2/neu status, as assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), reliably identifies aggressive tumors and is routinely used for therapeutic decisions. In CRC, HER2/neu protein overexpression has been studied mostly by IHC, but its significance is still unclear. Our objective was to use FISH to compare various tumor characteristics with HER2/neu gene amplification and copy numbers of HER2/neu and chr 17 as possible markers for aggressive tumors. Methods: HER2/neu gene amplification (defined as a ratio of HER-2/neu to chr 17 centromere of >2), HER2/neu copy number (cutoff >4.37) and chr 17 copy number (cutoff >2.03) were assessed by FISH in 272 surgically resected colon cancers from 02/00 to 04/03. FISH results were correlated with these tumor characteristics: size, grade (1= well, 2=moderately and 3=poorly differentiated) and TNM stage. Statistical analysis was performed using Fisher’s exact test and Student’s t-test. Results: The mean patient age was 67 and 53% were female. Tumor characteristics: mean size, 4.8 cm; median histologic grade, 2 of 3; and most common stage, III (32%), followed by II (30%), I (23%) and IV (15%). HER2/neu gene amplification was detected in 55 of 272 (20%) tumors. The average HER2/neu and chr 17 copy numbers were 2.78 and 1.98, respectively. There was no statistically significant association between HER2/neu gene amplification, HER2/neu copy number or chr 17 copy number when compared to tumor size, grade or stage. Conclusions: Fifty-five out of 272 (20%) CRCs demonstrated HER2/neu gene amplification but no association was found with tumor size, grade or stage. In view of the 20% amplification rate, which approximates that seen in breast cancer, clinical trials with trastuzumab (Herceptin) appear worth pursuing. Correlation of HER2/neu expression with overall survival in these 272 patients is merited. To the best of our knowledge, ours is the largest reported series examining HER2/neu overexpression, HER2/neu and chr 17 copy numbers in CRC by FISH. No significant financial relationships to disclose.

Feasibility of Using Tissue Microarrays for the Assessment of HER-2 Gene Amplification by Fluorescence In Situ Hybridization in Breast Carcinoma

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.

A comparison of two HER2 FISH methods to measure HER2 amplification and predict clinical outcome

566 Background: Controversy exists regarding the optimal assay to measure HER2 status. We previously reported that breast cancer patients with HER2 amplified tumors benefit from dose intense doxorubicin-based therapy (Muss, NEJM, 1994). In the current study, we directly compare HER2 status, using the FDA-approved PathVysion (PV) FISH kit, and the new Tricolor (TC) FISH system (both from Vysis, Inc), which, in addition to the HER2 and chromosome 17 (CEP17) probe, contains a third probe against the TOPO2 gene, a target of doxorubicin therapy. Because the TC system involves different assay conditions, it was important to determine if this affected HER2 status and predicting outcome. Methods: The PV and TC methods were performed on two sections from the same tumor block from 408 stage II breast cancer patients registered to CALGB 8541 (dose-intensive CAF therapy; cyclophosphamide, doxorubicin and 5FU, Cancer and Leukemia Group B). Three different measures were performed: HER2 amplification status, using the ratio of HER2:CEP17 signals, where a ratio >=2.00 defined a case as amplified, per kit instructions; HER2 amplification status based only on the HER2 signal, where >=4 signals defined a case as amplified; and polyploidy of chromosome 17, where >=3 signals defined polyploidy. Results: There was no significant difference between the two kits in assigning a case as HER2 amplified or not amplified using the ratio definition (McNemar's exact test, p=.18; kappa=0.862). Of the 14 discordant cases, most had ratios near the cutpoint (e.g. 1.71 by PV vs 2.03 by TC). Using the HER2 signal alone (not normalized to CEP 17), we found significant differences between the two kits, with TC giving a higher frequency of amplification (McNemar's test, p=.02). The TC system also identified a higher frequency of chromosome 17 polyploidy. Using the ratio definition, both kits predicted outcome similarly (K-M plots nearly superimposable). Conclusion: Using the chromosome 17 probe to normalize HER2 amplification status yields the most consistent results for HER2 measurement and predicting clinical outcome in a comparison between the PathVysion kit and the new Tricolor probe FISH system. No significant financial relationships to disclose.

A comparison of two HER2 FISH methods to measure HER2 amplification and predict clinical outcome

566 Background: Controversy exists regarding the optimal assay to measure HER2 status. We previously reported that breast cancer patients with HER2 amplified tumors benefit from dose intense doxorubicin-based therapy (Muss, NEJM, 1994). In the current study, we directly compare HER2 status, using the FDA-approved PathVysion (PV) FISH kit, and the new Tricolor (TC) FISH system (both from Vysis, Inc), which, in addition to the HER2 and chromosome 17 (CEP17) probe, contains a third probe against the TOPO2 gene, a target of doxorubicin therapy. Because the TC system involves different assay conditions, it was important to determine if this affected HER2 status and predicting outcome. Methods: The PV and TC methods were performed on two sections from the same tumor block from 408 stage II breast cancer patients registered to CALGB 8541 (dose-intensive CAF therapy; cyclophosphamide, doxorubicin and 5FU, Cancer and Leukemia Group B). Three different measures were performed: HER2 amplification status, using the ratio of HER2:CEP17 signals, where a ratio >=2.00 defined a case as amplified, per kit instructions; HER2 amplification status based only on the HER2 signal, where >=4 signals defined a case as amplified; and polyploidy of chromosome 17, where >=3 signals defined polyploidy. Results: There was no significant difference between the two kits in assigning a case as HER2 amplified or not amplified using the ratio definition (McNemar's exact test, p=.18; kappa=0.862). Of the 14 discordant cases, most had ratios near the cutpoint (e.g. 1.71 by PV vs 2.03 by TC). Using the HER2 signal alone (not normalized to CEP 17), we found significant differences between the two kits, with TC giving a higher frequency of amplification (McNemar's test, p=.02). The TC system also identified a higher frequency of chromosome 17 polyploidy. Using the ratio definition, both kits predicted outcome similarly (K-M plots nearly superimposable). Conclusion: Using the chromosome 17 probe to normalize HER2 amplification status yields the most consistent results for HER2 measurement and predicting clinical outcome in a comparison between the PathVysion kit and the new Tricolor probe FISH system. No significant financial relationships to disclose.

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.

416 UK patients are willing to donate biological material for sub-studies in clinical trials

To evaluate amplification of the HER-2/neu gene by fluorescence in situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining.A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHCstained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0.Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17.We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend antiHER2 therapy.

Impact of Polysomy 17 on HER-2/neu Immunohistochemistry in Breast Carcinomas without HER-2/neu Gene Amplification

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.

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

HER2 Testing in Patients With Breast Cancer: Poor Correlation Between Weak Positivity by Immunohistochemistry and Gene Amplification by Fluorescence In Situ Hybridization

Objective To evaluate amplification of the HER-2/neu gene by fluorescence in situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining. Methods A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHCstained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0. Results Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17. Conclusion We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend antiHER2 therapy. To evaluate amplification of the HER-2/neu gene by fluorescence in situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining. A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHCstained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0. Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17. We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend antiHER2 therapy.

HER2 Testing in Patients With Breast Cancer: Poor Correlation Between Weak Positivity by Immunohistochemistry and Gene Amplification by Fluorescence In Situ Hybridization

To evaluate amplification of the HER-2/neu gene by fluorescence ir situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining. A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHC-stained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0. Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17. We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend anti-HER2 therapy.

HER-2/neu in Breast Cancer: Interobserver Variability and Performance of Immunohistochemistry with 4 Antibodies Compared with Fluorescent In Situ Hybridization

The immunohistochemistry (IHC) performance of 4 anti-HER-2/neu antibodies was compared with fluorescent in situ hybridization (FISH) analysis of HER-2/neu gene expression in breast cancer patients considered for Herceptin (Trastuzumab) therapy. Interobserver variability in IHC interpretation was measured. Formalin-fixed tissue was received from 24 provincial hospital laboratories. The following anti-Her-2 antibodies were used: DAKO A0485 (polyclonal), Novacastra CB11 (monoclonal), Zymed TAB250 (monoclonal), and DAKO HercepTest (polyclonal). Additional sections were analyzed by FISH (Vysis). Three pathologists blinded to FISH results independently interpreted invasive tumor cell membranous staining on a scale of 0 to +3. The HER-2/neu gene was considered amplified when the FISH signal ratio of HER-2/CEP-17 was ≥2.0. Blocks from all hospitals and of all ages were suitable for IHC and FISH analysis. No interlaboratory analysis variability was noted. The interobserver agreement (kappa) for stain intensity for each antibody was good for 0 and +3 but poor for +1 and +2. Reasonable concordance between IHC and FISH was found with three of the four antibodies. TAB250 was the most sensitive antibody. For the three pathologists, the IHC sensitivities and specificities compared with FISH using 0/+1 as negative and +2/+3 as positive were as follows: A0485, 63–84/95–98; CB11, 63–66/97–98; TAB-250, 82–100/94–95; HercepTest, 59–77/91–93. The positive and negative predictive values varied by stain intensity. Stain scores of 0 and +3 were highly predictive of gene status. Stain scores of +1 and +2 were not sufficiently predictive to classify cases as amplified versus nonamplified. IHC is a reasonable first test to assess HER-2/neu status in patients with breast cancer. For most cases, DAKO A0485, TAB250, and HercepTest adequately predicted gene status. In cases with stain intensity of +1 or +2, the interobserver agreement is poor, and the predictive value is unsatisfactory for clinical use. Additional testing, preferably with FISH, is recommended.

HER-2/neu oncogene amplification in prostate cancer

Oncogene amplification is a manifestation of genetic instability, which has been implicated in the pathogenesis of many cancers. Work performed in this laboratory has focused on HER-2/neu amplification in breast cancer, ovarian cancer, cervical cancer and rhabdomyosarcoma, all of which revealed HER-2/neu amplification in at least a proportion of tumors. In the present project, we studied HER-2/neu oncogene amplification in prostate carcinoma, 244,000 new cases of which are diagnosed yearly, and which affects a significant number of men. Fluorescent in situ hybridization (FISH) using the Vysis HER-2/neu probe with a chromosome 17 centromere control probe was performed on formalin-fixed paraffin-embedded archival prostate cancer tissues. Out of a total of 85 cases studied, 7 cases (8.2%) were found to be amplified. Of those 7 amplified cases, 2 were also trisomic for chromosome 17. Amplification is defined as a signal ratio of HER-2/neu to chromosome 17 of >1.5, after Mark et al. (1998). Clinicopathologic characteristics of the amplified cases versus the nonamplified cases will be compared. Although the results of the present study still need to be confirmed and extended by additional cases in other centers, the data thus far do indicate that a small subset of prostate cancer is characterized by HER-2/neu gene amplification, as in other cancers. (This study was partially funded by Vysis, Inc., Downers Grove, IL).