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.

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.

PurposeWomen with breast tumors with higher expression of AR are in general known to have better survival outcomes while a high AR/ER ratio is associated with poor outcomes in hormone receptor positive breast cancers mostly in post menopausal women. We have evaluated the AR/ER ratio in the context of circulating androgens specifically in patients younger than 50 years most of whom are pre-menopausal and hence have a high estrogenic hormonal milieu.MethodsTumor samples from patients 50 years or younger at first diagnosis were chosen from a larger cohort of 270 patients with median follow-up of 72 months. Expression levels of ER and AR proteins were detected by immunohistochemistry (IHC) and the transcript levels by quantitative PCR. Ciculating levels of total testosterone were estimated from serum samples. A ratio of AR/ER was derived using the transcript levels, and tumors were dichotomized into high and low ratio groups based on the third quartile value. Survival and the prognostic significance of the ratio was compared between the low and high ratio groups in all tumors and also within ER positive tumors. Results were further validated in external datasets (TCGA and METABRIC).ResultsEighty-eight (32%) patients were ≤50 years, with 22 having high AR/ER ratio calculated using the transcript levels. Circulating levels of total testosterone were higher in women whose tumors had a high AR/ER ratio (p = 0.02). Tumors with high AR/ER ratio had significantly poorer disease-free survival than those with low AR/ER ratio [HR-2.6 (95% CI-1.02–6.59) p = 0.04]. Evaluation of tumors with high AR/ER ratio within ER positive tumors alone reconfirmed the prognostic relevance of the high AR/ER ratio with a significant hazard ratio of 4.6 (95% CI-1.35–15.37, p = 0.01). Similar trends were observed in the TCGA and METABRIC dataset.ConclusionOur data in pre-menopausal women with breast cancer suggest that it is not merely the presence or absence of AR expression but the relative activity of ER, as well as the hormonal milieu of the patient that determine clinical outcomes, indicating that both context and interactions ultimately influence tumor behavior.

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.