Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach

INTRODUCTION: Overexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization. METHODS: This study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections. RESULTS: For this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors. CONCLUSIONS: Retesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories

Tumor immune microenvironmental characteristics in Human Epidermal Growth Factor-2 (HER2) positive esophageal adenocarcinoma: A comparative analysis and biomarker study

Background: HER2 targeting in esophageal adenocarcinoma (EAC) has shown potential, but often fails to show durable response. Given the contributions of the tumor immune microenvironment (TIME) to therapeutic responses, we aimed to chart the TIME characteristics of HER2 positive tumors. Methods: 84 biopsies were taken from the TRAP cohort (neoadjuvant chemoradiotherapy (nCRT) according to CROSS with trastuzumab and pertuzumab; n = 40; HER2+ n = 40) and a control cohort with nCRT only (n = 44; HER2- n = 40, HER2+ n = 4) before treatment. Biopsies were analysed using targeted gene expression analysis (Nanostring immune-oncology panel, 750 genes). Differential gene expression was assessed between HER2 positive (n = 44) vs. negative biopsies (n = 40), and non-responders (n = 17) vs. responders (n = 23) to anti-HER2 treatment. Statistical significance was determined as p-value <0.05, adjusted for multiple testing correction. Results: 83 biopsies were eligible for analyses following quality control (TRAP cohort n = 40; control cohort n = 43); there were no significant differences in clinical characteristics between the TRAP vs. control the cohort or HER2 positive vs. HER2 negative biopsies. HER2 expression was found to associate with epithelial markers (EPCAM p < 0.001; E-cadherin p < 0.001). Moreover, HER2 expression was associated with a lower expression of immune cell infiltration, such as NK-cells (p < 0.001) and CD8 T-cells (p < 0.001), but also lower expression of immune exhaustion markers (PDCD1LG2, CTLA4; p < 0.001). In non-responders to anti-HER2 treatment, baseline biopsies showed increased expression of immune exhaustion markers, as well as hypoxia and VEGF signalling. Discussion: HER2 expression was associated with epithelial tumor characteristics. The HER2 positive TIME showed reduced immune cell infiltration but also lower expression of inhibitory signals associated with immune exhaustion, questioning the mechanism behind potential clinical benefit of co-administration of anti-HER2 agents and checkpoint inhibitors. As limited response was associated with increased VEGF signalling, studies could investigate potential synergism of targeting VEGF and HER2

Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Inadequate detection of the FSHR complicates future research on extragonadal FSHR localization

Introduction: Recently, follicle stimulating hormone (FSH) through interaction with its receptor (FSHR) has been proposed to play a role in postmenopausal osteoporosis and cardiovascular disease, rather than the loss of estrogen. To explore this hypothesis, unravelling which cells express extragonadal FSHR on protein level is key. Methods: We used two commercial anti-FSHR antibodies and validated them by performing immunohistochemistry on positive (ovary, testis) and negative controls (skin). Results: The monoclonal anti-FSHR antibody could not identify the FSHR in ovary or testis. The polyclonal anti-FSHR antibody stained the granulosa cells (ovary) and Sertoli cells (testis), yet there was equally intense staining of other cells/extracellular matrix. Furthermore, the polyclonal anti-FSHR antibody also stained skin tissue extensively, suggesting that the antibody stains more than just FSHR. Discussion: The findings in this study may add accuracy to literature on extragonadal FSHR localization and warrants attention to the use of inadequate anti-FSHR antibodies to value the potential role of FSH/FSHR in postmenopausal disease

Inadequate detection of the FSHR complicates future research on extragonadal FSHR localization

Introduction: Recently, follicle stimulating hormone (FSH) through interaction with its receptor (FSHR) has been proposed to play a role in postmenopausal osteoporosis and cardiovascular disease, rather than the loss of estrogen. To explore this hypothesis, unravelling which cells express extragonadal FSHR on protein level is key. Methods: We used two commercial anti-FSHR antibodies and validated them by performing immunohistochemistry on positive (ovary, testis) and negative controls (skin). Results: The monoclonal anti-FSHR antibody could not identify the FSHR in ovary or testis. The polyclonal anti-FSHR antibody stained the granulosa cells (ovary) and Sertoli cells (testis), yet there was equally intense staining of other cells/extracellular matrix. Furthermore, the polyclonal anti-FSHR antibody also stained skin tissue extensively, suggesting that the antibody stains more than just FSHR. Discussion: The findings in this study may add accuracy to literature on extragonadal FSHR localization and warrants attention to the use of inadequate anti-FSHR antibodies to value the potential role of FSH/FSHR in postmenopausal disease

Retrospective analysis of metastatic behaviour of breast cancer subtypes

Among breast cancer patients who develop distant metastases, there is marked variability in the clinical course, including metastasis pattern. Here, we present a retrospective study of breast cancer patients who all developed distant metastases focusing on the association between breast cancer subtype and clinical course, including organ-specific metastasis. Tissue microarrays (TMAs) were assembled and stained for ER, PR, HER2, EGFR, CK5/6, CK14, E-Cadherin, TP53 and Ki67 for 263 breast cancer patients with metastatic disease. Tumours were classified into ER+/HER2-/Ki67high, ER+/HER2-/Ki67low, ER+/HER2+, ER-/HER2+ and ER-/HER2- groups. Relevant data related to metastasis pattern, metastasis timeline, systemic treatment and survival were retrieved. Associations between site-specific relapse and patient/tumour characteristics were assessed with multivariate models using logistic regression. Median time for development of distant metastasis was 30 months (range 0-15.3 years); 75.8 % of the distance metastases developed in the first 5 years after treatment of the primary tumour. Patients with ER-/HER2- tumours had a median overall survival of 27 months; those with HER2+ tumours of 52 months; those with ER+/HER2-/Ki67high of 76 months and those with ER+/HER2-/Ki67low of 79 months. Bone was the most common site for distant metastasis (70.6 %) followed by liver (54.5 %) and lung (31.4 %), respectively. Visceral metastasis was found in 76.8 % of the patients. Patients with ER-/HER2- tumours developed visceral metastases in 81 % and bone metastases in 55.2 %; those with HER2+ tumours developed visceral metastases in 77.4 % and bone metastases in 69.8 %; those with ER+/HER2-/Ki67high developed visceral metastases in 75.7 % and bone metastases in 87.8 % and those with ER+/HER2-/Ki67low developed visceral metastases in 76.9 % and bone metastases in 73.1 %. In metastatic breast cancer patients, tumour subtypes are associated with survival and pattern of distant metastases. These associations are of help in choices for surveillance and therapy in individual patient