Dissecting the Biological Heterogeneity within Hormone Receptor Positive HER2 Negative Breast Cancer by Gene Expression Markers Identifies Indolent Tumors within Late Stage Disease.

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.

HR+HER2- breast cancers with growth factor receptor-mediated EMT have a poor prognosis and lapatinib downregulates EMT in MCF-7 cells.

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 epithelial-to-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 ß6) versus those with low levels. Within the epithelial-to-mesenchymal transition+ tumors, there was a positive correlation between the transcripts of integrin ß6 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 ß6 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 αvß6, and matrix metalloproteinase 9 protein. The results suggest that synergistic interactions between growth factor receptors and integrin ß6 could mediate epithelial-to-mesenchymal transition and migration in a subset of luminal breast cancers and lapatinib might be effective in disrupting this interaction.

High expression of integrin ß6 in association with the Rho-Rac pathway identifies a poor prognostic subgroup within HER2 amplified breast cancers.

Integrin αvß6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin ß6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvß6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin ß6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvß6 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.

Identification of BRCA1 Deficiency Using Multi-Analyte Estimation of BRCA1 and Its Repressors in FFPE Tumor Samples from Patients with Triple Negative Breast Cancer.

PURPOSE: Apart 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: We 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.

A Majority of Low (1-10%) ER Positive Breast Cancers Behave Like Hormone Receptor Negative Tumors.

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.

The epigenetic silencing of the estrogen receptor (ER) by hypermethylation of the ESR1 promoter is seen predominantly in triple-negative breast cancers in Indian women.

The proportion of estrogen receptor (ER)-negative and triple-negative (TN) breast cancer in Indian women is higher than that reported in the West, and this difference persists even after their migration to the West. The causes for this significant difference are not entirely clear. Hypermethylation of the ER promoter, an epigenetic alteration, is known to be one of the mechanisms by which the expression of ER is suppressed. Two thirds of breast cancer specimens from an Indian center tested, using the highly sensitive, methylation-specific polymerase chain reaction (MSP) technique, were reported positive. We have used a quantitative assay, the MethyLight, to better assess the extent of methylation in the ESR1 promoter region in 98 breast cancer tumor specimens from Indian women. In addition, the amount of ER transcripts was determined by quantitative reverse transcriptase polymerase chain reaction. Using the stringent cutoff of at least 4% of the target sequence being methylated, 27% of TN tumors were methylated. In addition they demonstrated the highest levels of methylation. In contrast less than 2% ER-positive tumors were hypermethylated. While the proportion of hypermethylated tumors are lower in this study than that estimated using MSP, our results support the notion of increased epigenetic deregulations in ER-negative tumors in general and TN tumors in particular. The development of this assay also permits a rational approach to the selection of patients for clinical trials examining the efficacy of demethylating agents in the treatment of ER-negative breast cancer.