HER2 testing on core needle biopsy specimens from primary breast cancers: interobserver reproducibility and concordance with surgically resected specimens

<p>Abstract</p> <p>Background</p> <p>Accurate evaluation of human epidermal growth factor receptor type-2 (HER2) status based on core needle biopsy (CNB) specimens is mandatory for identification of patients with primary breast cancer who will benefit from primary systemic therapy with trastuzumab. The aim of the present study was to validate the application of HER2 testing with CNB specimens from primary breast cancers in terms of interobserver reproducibility and comparison with surgically resected specimens.</p> <p>Methods</p> <p>A total of 100 pairs of archival formalin-fixed paraffin-embedded CNB and surgically resected specimens of invasive breast carcinomas were cut into sections. All 100 paired sections were subjected to HER2 testing by immunohistochemistry (IHC) and 27 paired sections were subjected to that by fluorescence in situ hybridization (FISH), the results being evaluated by three and two observers, respectively. Interobserver agreement levels in terms of judgment and the concordance of consensus scores between CNB samples and the corresponding surgically resected specimens were estimated as the percentage agreement and κ statistic.</p> <p>Results</p> <p>In CNB specimens, the percentage interobserver agreement of HER2 scoring by IHC was 76% (κ = 0.71) for 3 × 3 categories (0-1+ <it>versus </it>2+ <it>versus </it>3+) and 90% (κ = 0.80) for 2 × 2 categories (0-2+ <it>versus </it>3+). These levels were close to the corresponding ones for the surgically resected specimens: 80% (κ = 0.77) for 3 × 3 categories and 92% (κ = 0.88) for 2 × 2 categories. Concordance of consensus for HER2 scores determined by IHC between CNB and the corresponding surgical specimens was 87% (κ = 0.77) for 3 × 3 categories, and 94% (κ = 0.83) for 2 × 2 categories. Among the 13 tumors showing discordance in the mean IHC scores between the CNB and surgical specimens, the results of consensus for FISH results were concordant in 11. The rate of successful FISH analysis and the FISH positivity rate in cases with a HER2 IHC score of 2+ differed among specimens processed at different institutions.</p> <p>Conclusion</p> <p>It is mandatory to study HER2 on breast cancers, and either CNB or surgical specimen can be used.</p

Abstract PD02-04: Automated Quantitative RNA In Situ Hybridization for Resolution of Equivocal and Heterogeneous ERBB2 (HER2) Status in Invasive Breast Carcinoma

Abstract Background: Breast carcinomas that demonstrate a heterogeneous ERBB2 (HER2) status or equivocal results by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) present diagnostic challenges for which there is neither a standard methodology to achieve resolution in the clinical laboratory nor a uniform approach to management. We assessed the feasibility of using a novel automated and quantitative HER2 mRNA bright field in situ hybridization (ISH) assay capable of single molecule detection to determine HER2 status in invasive breast carcinomas that demonstrated significant tumor heterogeneity or failed to be resolved by standard IHC and FISH algorithmic testing. Design: Formalin-fixed, paraffin-embedded (FFPE) breast carcinomas from a non-consecutive series of 163 patients were analyzed for HER2 mRNA using a fully automated bright field RNA ISH assay (RNAscope, Advanced Cell Diagnostics, Hayward, CA). Cases were assigned into either a training set (n = 34) or a validation set (n = 129) and analyzed by both Q-RT-PCR and RNAscope. automated image analysis was used to numerate the punctate signal dots per cell in RNAscope-stained slides. A HER2 mRNA score based on single-cell quantification by RNAscope was developed and correlated to HER2 FISH and HER2 mRNA Q-RT-PCR results. A simple cutoff value was derived using the training set and applied to the validation set. Results: Evaluable HER2 results were obtained for 154 cases (94.5%) by RNAscope and 163 cases (100%) by Q-RT-PCR. In the training set, both FISH/IHC positive and negative cases were definitively separated by both Q-RT-PCR and RNAscope. HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.77) and Q-RT-PCR (r = 0.81). Application of both methods to the validation set resulted in correct identification of 31/31 positive cases and 41/43 negative (overall concordance=97.3%) for both RNAscope and Q-RT-PCR. RNAscope showed a significant advantage over Q-RT-PCR in correctly identifying cases equivocal by FISH that were resolved by reflex IHC testing. RNAscope classified 7 of 26 (26.9%) FISH/IHC double equivocal cases as positives. In cases with HER2 protein heterogeneity, RNAscope showed a 100% concordance with FISH results, whereas Q-RT-PCR showed a 42.9% concordance. Conclusion: RNAscope analysis of HER2 mRNA is an effective means to resolve HER2 status in double equivocal cases and cases that demonstrate heterogeneity. Automation and image analysis-based quantification minimize analytical and post-analytical variability. Quantification of single RNA transcripts in situ at single-cell level demonstrates superiority over qRTPCR and great potential in predictive biomarker analysis. Further studies of larger cohorts correlating clinical response with HER2 mRNA expression in situ are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-04.</jats:p

MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35-4.30 and P = 0.003)-though not overall survival (HR = 1.64; 95% CI, 0.92-2.94 and P = 0.095)-after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET