Background: The 2010 guidelines by ASCO-CAP have mandated that breast cancer specimens with ≥1% positively staining cells by immunohistochemistry should be considered Estrogen Receptor (ER) positive. This has led to a subclass of low-ER positive (1-10%) breast cancers. We have examined the biology and clinical behavior of these low ER staining tumors.Methods: We have developed a probabilistic score of the “ER-positivity” by quantitative estimation of ER related gene transcripts from FFPE specimens. Immunohistochemistry for ER was done on 240 surgically excised tumors of primary breast cancer. Relative transcript abundance of 3 house-keeping genes and 6 ER related genes were determined by q-RT PCR. A logistic regression model using 3 ER associated genes provided the best probability function, and a cut-off value was derived by ROC analysis. 144 high ER (>10%), 75 ER negative and 21 low-ER (1-10%) tumors were evaluated using the probability score and the disease specific survival was compared.Results: Half of the low-ER positive tumors were assigned to the ER negative group based on the probability score; in contrast 95% of ER negative and 92% of the high ER positive tumors were assigned to the appropriate ER group (p<0.0001). The survival of the low-ER group was intermediate between that of the high ER positive and ER negative groups (p<0.05).Conclusion: Our results suggest that the newly lowered ASCO-CAP criteria for ER positivity, leads to the false categorization of biologically ER negative tumors as ER positive ones. This may have particular relevance to India, where we have a much higher proportion of ER negative tumors in general.

Integrin αvβ6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin β6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvβ6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin β6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvβ6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho–Rac pathway including downstream genes (ACTR2,ACTR3) and effector proteinases (MMP9,MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2‐ and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho–Rac pathway (P‐values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease‐free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9–8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node‐positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho–Rac pathway through increased expression of MMP9 and MMP15.

. Demographic data was collected from the case files. The specimens were received in 10% formalin and fixed for 12-24 hours. Ethical clearance was obtained from the institute.Routine bits were given along with a separate bit for IHC and TMA studies. Sections stained with H&E were studied for histopathological type, grade of tumour and lymph node involvement.Immunohistochemistry: Almost 5 mm sections were cut on polylysine coated slides. Antigen retrieval for ER and PR was done using pressure cooker method in EDTA buffer (pH 9). Heat method was used to retrieve Her2/neu antigen using citrate buffer (pH 6). Sections were then soaked in peroxide block (1%) for 10 minutes. Monoclonal antibodies of ER, PR and Her2/neu {ER alpha, Clone EP1 (Monoclonal, DAKO), PR, Clone PgR 636 (Monoclonal, DAKO) and Her2/neu antibody (Polyclonal, DAKO)} were applied for 30 minutes followed by secondary antibody (HRPO IHC detection system-DAKO) for 30 minutes in a humidified chamber. Sections were then treated with 3,3'-Diaminobenzidine (DAB) chromogen for 10 minute. The sections were washed in distilled water and counterstained with Harri's haematoxylin, dehydrated using alcohol, cleared and mounted. ER and PR scoring was done using quick scoring and Her2/neu scoring was done as per guidelines of CAP/ASCO [5,6].Construction of TMA: H&E sections were assessed and an appropriate area of tumour was marked on the corresponding paraffin block. Care was taken not to include areas of fibrosis, adipose tissue or necrosis . A grid indicating the location of each case on the microarray block was prepared. Using a tissue microarrayer (quick Ray manual tissue microarrayer-Ultima Co. Korea) with a 2 mm diameter, a core was punched from the marked area on the block and transferred to premade recipient Aim: To compare ER, PR and Her2/neu on TMA sections with whole sections and to determine the concordance of results between the two methods. Materials and Methods:A TMA block was constructed by punching out 2 mm cores from appropriately marked paraffin blocks of 53 breast carcinoma cases and embedding them in the recipient block. Immunostaining of TMA sections and whole sections were performed for ER, PR and Her2/neu and the results were compared.Statistical analysis was done using chi square test/FisherExact test. Kappa co-efficient, Jaccard Index and G-Index were computed.Results: Infiltrating Ductal Carcinoma-No Special Type (IDC-NST) was the predominant type of carcinoma and most of the tumours were of Grade II and III. Majority, 38/53 (71.7%) were ER/PR positive and Her2 negative and 9/53 (17%) cases were triple negative. Good concordance between whole sections and TMA sections were noted with kappa value for ER, PR and Her2/neu being 0.671, 0.754, 1.000 respectively which was statistically significant. Conclusion:Immunostaining for ER, PR and Her2/neu done on TMA section using single 2 mm core were comparable with conventional whole section scores. Thus, TMA is a reliable method for evaluating these biomarkers with the advantage of being time an...

PurposeApart from germ-line BRCA1-mutated breast cancers, a significant proportion of women with sporadic triple negative breast cancer (TNBC) sub-type are known to harbour varying levels of BRCA1-dysfuction. There is currently no established diagnostic method to identify these patients.MethodsThe analysis was performed on 183 primary breast cancer tumor specimens from our longitudinal case-series archived as formalin-fixed-paraffin-embedded (FFPE) blocks comprising 71 TNBCs and 112 Hormone receptor positive HER2 negative (HR+HER2-) tumors. Transcript levels of BRCA1 and two of its repressors ID4 and microRNA182 were determined by TaqMan quantitative PCR. BRCA1 protein was detected immunohistochemically with the MS110 antibody.ResultsThe representation of BRCA1 and its repressor ID4 as a ratio led to improved separation of TNBCs from HR+HER2- compared to either measure by itself. We then dichotomised the continuous distribution of each of the three measurements (Protein, MIRNA and transcript:repressor ratio) into categories of deficient (0) and adequate (1). A composite BRCA1 Deficiency Score (BDS) was computed by the addition of the score for all three measures. Samples deficient on 2 or more measures were deemed to be BRCA1 deficient; and 40% of all TNBCs met this criterion.ConclusionWe propose here a simple multi-level assay of BRCA1 deficiency using the BRCA1:ID4 ratio as a critical parameter that can be performed on FFPE samples in clinical laboratories by the estimation of only 3 bio-markers. The ease of testing will hopefully encourage adoption and clinical validation.

Hormone receptor positive (HR+) breast cancers are a heterogeneous class with differential prognosis. Although more than half of Indian women present with advanced disease, many such patients do well. We have attempted identification of biologically indolent tumors within HR+HER2- tumors based on gene expression using histological grade as a guide to tumor aggression. 144 HR+HER2- tumors were divided into subclasses based on scores derived by using transcript levels of multiple genes representing survival, proliferation, and apoptotic pathways and compared to classification by Ki-67 labeling index (LI). Clinical characters and disease free survival were compared between the subclasses. The findings were independently validated in the METABRIC data set. Using the previously established estrogen receptor (ER) down stream activity equation, 20% of the tumors with greater than 10% HR positivity by immunohistochemistry (IHC) were still found to have inadequate ER function. A tumor aggression probability score was used to segregate the remainder of tumors into indolent (22%) and aggressive (58%) classes. Significant difference in disease specific survival was seen between the groups (P = .02). Aggression probability based subclassification had a higher hazard ratio and also independent prognostic value (P < .05). Independent validation of the gene panel in the METABRIC data set showed all 3 classes; indolent (24%), aggressive (68%), and insufficient ER signaling (7%) with differential survival (P = .01). In agreement with other recent reports, biologically indolent tumors can be identified with small sets of gene panels and these tumors exist in a population with predominantly late stage disease.

Despite an overall good prognosis, a significant proportion of patients with hormone receptor positive human epidermal growth factor receptor 2 negative breast cancers develop distant metastases. The metastatic potential of epithelial cells is known to be regulated by tumor-stromal interaction and mediated by epithelial-to-mesenchymal transition. Hormone receptor positive human epidermal growth factor receptor 2 negative tumors were used to estimate markers of epithelialto-mesenchymal transition, and the luminal breast cancer cell line MCF-7 was used to examine the interactions between integrins and growth factor receptors in causation of epithelial-to-mesenchymal transition. A total of 140 primary tumors were sub-divided into groups enriched for the markers of epithelial-to-mesenchymal transition (snail family transcriptional repressor 2 and integrin β6) versus those with low levels. Within the epithelial-to-mesenchymal transition+ tumors, there was a positive correlation between the transcripts of integrin β6 and growth factor receptors-human epidermal growth factor receptor 2 and epidermal growth factor receptor. In tumors enriched for epithelial-to-mesenchymal transition markers, patients with tumors with the highest quartile of growth factor receptor transcripts had a shorter disease-free survival compared to patients with low growth factor receptor expression by Kaplan-Meier analysis (log rank, p = 0.03). Epithelial-to-mesenchymal transition was induced in MCF-7 cells by treatment with transforming growth factor beta 1 and confirmed by upregulation of SNAI1 and SNAI2 transcripts, increase of vimentin and integrin β6 protein, and repression of E-cadherin. Treatment of these cells with the dual-specificity tyrosine-kinase inhibitor lapatinib led to downregulation of epithelial-to-mesenchymal transition as indicated by lower levels of SNAI1 and SNAI2 transcripts, integrin αvβ6, and matrix metalloproteinase 9 protein. The results suggest that synergistic interactions between growth factor receptors and integrin β6 could mediate epithelial-to-mesenchymal transition and migration in a subset of luminal breast cancers and lapatinib might be effective in disrupting this interaction.

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Introduction: The initial identification of HER2 as a driver in a subset of breast cancers was at the level of DNA (amplification), and subsequently noted at the level of transcripts and protein as well. However, the clinical selection of patients for treatment with Trastuzumab, has been through either IHC (protein) or FISH (DNA amplification) and not through transcript abundance. Interestingly, in most studies that have estimated transcript abundance in primary tumors, the proportion of patients that demonstrate increased transcript levels (termed HER2 Enriched) have tended to be slightly larger than the clinical HER2+ category. A more clinically useful measure might be proof of HER2 downstream activity that might help separate tumors being driven significantly by HER2 from ones where its role is supportive. One of the many consequences of HER2 over-expression is activation of the oncomiR, miR-21 via the MAPK pathway. miR-21 in turn is known to epigenetically regulate multiple targets including the tumor suppressors PTEN and PDCD4. While these molecular mechanisms have been demonstrated convincingly in breast cancer cell lines, clinical studies of these alterations in large numbers are yet to be reported. In this study we have examined the relationship between clinical HER2 positivity and miR-21 levels in 124 surgically excised breast cancer specimens. Methods: We selected 124 surgically excised specimens of primary breast cancers from our cohort that by HER2 immunohistochemistry (IHC) comprised 42 positive, 62 negative and 20 equivocal. Relative abundance of miR-21 was assessed using a TaqMan qRT-PCR, with normalization by RNU48. Relative transcript abundance of a set of 6 genes (HER2, GRB7, MLN64 and 3 reference genes) were evaluated by SYBR Green real time qPCR. Results: The majority of tumors that were clinically HER2+ over expressed miR-21. A concordance with an AUC of 96% at 100% sensitivity and 85% specificity was noted. There is a highly significant differential expression of miR-21 between HER2 positive, negative and equivocal samples (P &lt; 0.0001). HER2 enriched score determined by using the expression levels of 3 genes (HER2, GRB7, MLN64) identified 35% (44/124) of the samples to be HER2 enriched. 72% of these (32/44) were also clinical HER2 positive by IHC. As expected, miR-21 was significantly over expressed in these tumors as well (P&lt;0.0001). To identify all samples which might show HER2 downstream activity, a logistic regression model was built using expression of miR-21, HER2, MLN64 and GRB7 as the determinants of HER2 status. The best fitting model classified 91% (38/42) of HER2 +, 95% (59/62) of the HER2 negative accurately with 94% specificity and an AUC of 0.96. The model helped identify 10% of clinical HER2 negative samples (6/20 equivocal & 3/62 HER-2 negative) to have a high probability of being HER2+. Conclusion: Identification of HER2+ tumors with evidence of downstream activity may help identify patients with tumors being driven significantly by HER2 from ones where its role is supportive. The possibility of targeting miR-21 raises the tantalizing prospect of effecting change by altering the epigenetic regulation of multiple targets including tumor-suppressors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-09.