Assessment Of HER2 Gene Amplification Research Articles

The frequency and clinical significance of centromere enumeration probe 17 alterations in human epidermal growth factor receptor 2 immunohistochemistry-equivocal invasive breast cancer.

Background and aimsChromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry‐equivocal score.Methods and resultsA large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in‐situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2‐amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5–< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2‐non‐amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2‐amplified and HER2‐non‐amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2‐amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37–102.00; P = 0.02], and histological grade 3 in HER2 non‐amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61–19.00; P = 0.007).ConclusionThe impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry‐equivocal score who would benefit from NAT.

Quantitative assessment of HER2 gene amplification of breast cancer using droplet digital PCR.

We previously reported the usefulness of droplet digital polymerase chain reaction (ddPCR) for the assessment of Human epithelial growth factor receptor 2 (HER2) gene amplification in breast cancer using formalin-fixed and paraffin-embedded sections. In our previous study, we combined HER2/CEP17 ratio (HER2 gene signals to chromosome 17 signals) with ddPCR and tumor content ratio (TCR) of each sample and determined the HER2 status by adopting a two-dimensional chart. This "ddPCR-TCR method" showed a high concordance with conventional HER2 status. In this study, we updated our method to assess the HER2 status of breast cancer in a more quantitative manner. We combined obtained data of the ddPCR ratio [Rx ] and TCR [x]; we calculated "(Rx - 1)/x + 1" for 41 samples with primary breast cancer and named the value led by this formula as "eHER2 (estimated HER2/CEP17 ratio of a tumor cell)". eHER2 was equivalent to conventional in situ hybridization (ISH) HER2/CEP17 ratio in most cases. eHER2 and ISH ratio showed a strong correlation (Spearman rank correlation ρ = 0.70, p < 0.0001). The obtained results indicated that eHER2 is a potential tool for HER2 status diagnosis in breast cancer.

Advances in HER2 testing.

HER2-positive breast cancer is a particularly aggressive type of breast cancer. Indication of HER2 positivity is essential for its treatment. In addition to a few FDA-approved methods such as immunohistochemical (IHC) detection of HER2 protein expression and in situ hybridization (ISH) assessment of HER2 gene amplification, several novel methods have been developed for HER2 testing in recent years. This chapter provides an overview of HER2 testing with emphasis on those new methods.

Inter-laboratory evaluation of a novel DEPArray-HER2 FISH assay.

e12506 Background: Fluorescent in Situ Hybridization (FISH) is a method currently used for detection and assessment of HER2 gene amplification. Although clinical guidelines set forth by CAP/ASCO exist to ensure accuracy, limitations in HER2test results due to sample preparation, assay-conditions and tumor heterogeneity remain unresolved. We have successfully demonstrated analytical confidence in performing HER2 FISH on DEPArray™ sorted and recovered tumor cells. In this study, we aimed to evaluate inter-laboratory concordance of the DEPArray™ HER2-FISH assay. Methods: Three laboratories equipped with the DEPArray™ were designated as testing sites for this study. Positive control SKBr3 cells embedded in paraffin as well as 20 invasive breast carcinoma FFPE samples were blinded and evaluated by each of the three labs. Control and patient samples were processed through the DEPArray™ beginning with dissociation of the FFPE curls followed by single-cell image-based cell sorting to separate and recover pure distinct tumor cell populations prior to HER2 FISH analysis. Data was only obtained when ∼200 intact cytokeratin+/vimentin-/DAPI+ tumor cells from each sample were recovered and used for subsequent FISH using a standard dual-color HER2/CEP17 FISH procedure. Results: Overall, 80% concordance between DEPArray™-HER2 and conventional HER2 (6 HER2 negative and 10 HER2 positive) was observed between lab(s) and the conventional HER2 method. In each of 4 cases, a discordant HER2 result was reported by one of three sites. In three of these discordant cases, the DEPArray™ HER2 ratio was reported as amplified while the conventional result was negative. In the remaining discordant case, the converse was observed by one site; however, this case was initially evaluated 15 years ago. All three sites correctly scored the SKBr3 positive control cells. Conclusions: The results showed a high concordance rate of correct HER2 status classification. This data further supports the understanding that tissue heterogeneity can indeed give rise to discordant results that may consequently affect treatment options for patients. We demonstrate that sample preparation by DEPArray™ may aid in a more precise classification for tumor biomarker status.

Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong.

To compare the PathVysion fluorescence in-situ hybridisation assay with the INFORM HER2 Dual in-situ hybridisation assay on 104 invasive breast cancers with a broad spectrum of immunohistochemistry scores. This case series involved consecutive patients diagnosed with invasive breast carcinoma with equivocal immunohistochemistry score and referred for further HER2 assessment from the departments of Surgery and/or Clinical Oncology of the two hospitals between January 2013 and February 2014. An additional 10 cases with negative HER2 immunohistochemistry and 11 cases with positive HER2 immunohistochemistry were further included. The results of both fluorescence in-situ hybridisation and dual in-situ hybridisation were available in 99 of 104 cases, respectively. Student’st test showed no statistically significant difference in the mean number of HER2 count, CEP17 copies, or HER2/CEP17 ratio between that obtained by fluorescence in-situ hybridisation and that obtained by dual in-situ hybridisation. Pearson’s correlation of results for the two assays was strong for HER2/CEP17 signal ratio (R=0.963, P<0.001) and mean HER2 copies per nucleus (R=0.897, P<0.001). Overall agreement was 96.0% (95 out of 99 cases, ĸ0.882). Three of the four discordant cases were equivocal for either fluorescence in-situ hybridisation or dual in-situ hybridisation. The results of immunohistochemistry 0/1+ and 3+ cases showed 100% concordance between the two assays. The failure rate was 0.96% for fluorescence in-situ hybridisation and 3.85% for dual in-situ hybridisation. Cases that failed for fluorescence in-situ hybridisation were successful for dual in-situ hybridisation and vice versa. Our study showed that dual in-situ hybridisation is a reliable and useful option for HER2 testing in breast cancer.

Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong

Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong

Assessment of HER2 gene amplification in adenocarcinomas of the stomach or gastroesophageal junction in the INT-0116/SWOG9008 clinical trial

BackgroundTrastuzumab has been approved for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic gastric carcinoma; however, relatively little is known about the role of HER2 in the natural history of this disease. Patients and methodsPatients enrolled in the INT-0116/SWOG9008 phase III gastric cancer clinical trial with available tissue specimens were retrospectively evaluated for HER2 gene amplification by FISH and overexpression by immunohistochemistry (IHC). The original trial was designed to evaluate the benefit of postoperative chemoradiation compared with surgery alone. ResultsHER2 gene amplification rate by FISH was 10.9% among 258 patients evaluated. HER2 overexpression rate by IHC was 12.2% among 148 patients evaluated, with 90% agreement between FISH and IHC. There was a significant interaction between HER2 amplification and treatment with respect to both disease-free survival (DFS) (P = 0.020) and overall survival (OS) (P = 0.034). Among patients with HER2-non-amplified cancers, treated patients had a median OS of 44 months compared with 24 months in the surgery-only arm (P = 0.003). Among patients with HER2-amplified cancers, there was no significant difference in survival based on treatment arm. HER2 status was not a prognostic marker among patients who received no postoperative chemoradiation. ConclusionPatients lacking HER2 amplification benefited from treatment as indicated by both DFS and OS. Clinical trialINT-0116/SWOG9008 phase III.

Prospective multi-centre study to validate chromogenic in situ hybridisation for the assessment of HER2 gene amplification in specimens from adjuvant and metastatic breast cancer patients

As HER2 status is a strong predictor of the response to trastuzumab, clinical guidelines recommend that all breast tumours are first evaluated for HER2 protein expression by immunohistochemistry (IHC) followed by confirmatory testing for HER2 gene amplification using fluorescence in situ hybridisation (FISH) for 2+ cases. Alternatively, chromogenic in situ hybridisation (CISH) offers a simpler, less expensive approach to detect HER2 amplification. In this prospective, multi-centre study, based on the largest dataset for HER2 testing in Germany to date, we evaluated the concordance between FISH and CISH in 399 samples from adjuvant and metastatic breast cancer patients. Tumour specimens from routine diagnostic practice were analysed by IHC, FISH and CISH in four reference centres. FISH and CISH results were strongly concordant (κ=0.83), with 95% of cases showing agreement. Despite variable IHC scoring across testing centres, complete consensus among the three methods was observed for 246 cases, representing 91% of all IHC positive (3+) or negative (0/1+) cases. Confirmatory testing of 132 IHC equivocal (2+) cases also yielded highly concordant results between FISH and CISH. These data validate CISH for the assessment of HER2 gene amplification in breast tumours and, confirm CISH as a valid alternative to FISH in HER2 testing.

Evaluation of automated silver‐enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens

To validate the use of the silver-enhanced in situ hybridization (SISH) technique in assessing HER2 status of breast carcinoma in excision biopsy specimens, and to assess its reliability in determining HER2 status in core biopsy specimens. Routinely processed paraffin sections of 65 excised breast carcinomas and 56 available preoperative core biopsy specimens from the same patients were selected from the archives for testing with the SISH technique using the automated Ventana Benchmark XT machine. For each case, two sections were used, one for the assessment of HER2 gene amplification and the other for assessment of chromosome 17. Of the 65 excision specimens tested, sections of 53 cases were also available for fluorescence in situ hybridization (FISH) examination. HER2 gene amplification was detected by SISH in 14 (21%) out of 65 excision specimens and in eight (14%) out of 56 core biopsy specimens. The results of SISH and FISH were identical in 50 (94%) out of the 53 excision cases examined by the two techniques. Two cases were SISH-, FISH+, and one case was the other way round. SISH results of core biopsy specimens and corresponding excision biopsy specimens were identical in 50 (89%) out of 56 cases. Four cases (7%) were SISH- in cores but positive in excision specimens, whereas two cases were the other way round. The results validate the use of the SISH technique for assessing HER2 status of excised breast carcinoma tissue sections. The results are comparable to those obtained with FISH, but SISH has the advantage of having a permanent end result that can be visualized by an ordinary light microscope. There is a reasonable 89% concordance between SISH results obtained in core and excision biopsy specimens. However, it may be prudent to postpone doing SISH, if possible, until sections of the resected specimen are available, as these seem to be more reliable.

HER2 gene amplification and EGFR expression in a large cohort of surgically staged patients with nonendometrioid (type II) endometrial cancer

Type II endometrial cancers (uterine serous papillary and clear cell histologies) represent rare but highly aggressive variants of endometrial cancer (EC). HER2 and EGFR may be differentially expressed in type II EC. Here, we evaluate the clinical role of HER2 and EGFR in a large cohort of surgically staged patients with type II (nonendometrioid) EC and compare the findings with those seen in a representative cohort of type I (endometrioid) EC. In this study HER2 gene amplification was studied by fluorescence in situ hybridisation (FISH) and EGFR expression by immunohistochemistry. Tissue microarrays were constructed from 279 patients with EC (145 patients with type I and 134 patients with type II EC). All patients were completely surgically staged and long-term clinical follow up was available for 258 patients. The rate of HER2 gene amplification was significantly higher in type II EC compared with type I EC (17 vs 1%, P<0.001). HER2 gene amplification was detected in 17 and 16% of the cases with uterine serous papillary and clear cell type histology, respectively. In contrast, EGFR expression was significantly lower in type II compared with type I EC (34 vs 46%, P=0.041). EGFR expression but not HER2 gene amplification was significantly associated with poor overall survival in patients with type II EC, (EGFR, median survival 20 vs 33 months, P=0.028; HER2, median survival 18 vs 29 months, P=0.113) and EGFR expression retained prognostic independence when adjusting for histology, stage, grade, and age (EGFR, P=0.0197; HER2, P=0.7855). We conclude that assessment of HER2 gene amplification and/or EGFR expression may help to select type II EC patients who could benefit from therapeutic strategies targeting both HER2 and EGFR.

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.

Correlation of chromogenic in-situ hybridisation (CISH) with FISH and IHC for assessment of HER2 gene amplification: an international validation ring study

Correlation of chromogenic in-situ hybridisation (CISH) with FISH and IHC for assessment of HER2 gene amplification: an international validation ring study