Pre-Menopausal Women With Breast Cancers Having High AR/ER Ratios in the Context of Higher Circulating Testosterone Tend to Have Poorer Outcomes

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

Differential role of glucocorticoid receptor based on its cell type specific expression on tumor cells and infiltrating lymphocytes

Background: The glucocorticoid receptor (GR) is frequently expressed in breast cancer (BC), and its prognostic implications are contingent on estrogen receptor (ER) status. To address conflicting reports and explore therapeutic potential, a GR signature (GRsig) independent of ER status was developed. We also investigated cell type-specific GR protein expression in BC tumor epithelial cells and infiltrating lymphocytes. Methods: GRsig was derived from Dexamethasone treated cell lines through a bioinformatic pipeline. Immunohistochemistry assessed GR protein expression. Associations between GRsig and tumor phenotypes (proliferation, cytolytic activity (CYT), immune cell distribution, and epithelial-to-mesenchymal transition (EMT) were explored in public datasets. Single-cell RNA sequencing data evaluated context-dependent GR roles, and a cell type-specific prognostic role was assessed in an independent BC cohort. Results: High GRsig levels were associated with a favorable prognosis across BC subtypes. Tumor-specific high GRsig correlated with lower proliferation, increased CYT, and anti-tumorigenic immune cells. Single-cell data analysis revealed higher GRsig expression in immune cells, negatively correlating with EMT while a positive correlation was observed with EMT primarily in tumor and stromal cells. Univariate and multivariate analyses demonstrated the robust and independent predictive capability of GRsig for favorable prognosis. GR protein expression on immune cells in triple-negative tumors indicated a favorable prognosis. Conclusion: This study underscores the cell type-specific role of GR, where its expression on tumor cells is associated with aggressive features like EMT, while in infiltrating lymphocytes, it predicts a better prognosis, particularly within TNBC tumors. The GRsig emerges as a promising independent prognostic indicator across diverse BC subtypes

An androgen receptor regulated gene score is associated with epithelial to mesenchymal transition features in triple negative breast cancers

Background: Androgen receptor (AR) is considered a marker of better prognosis in hormone receptor positive breast cancers (BC), however, its role in triple negative breast cancer (TNBC) is controversial. This may be attributed to intrinsic molecular differences or scoring methods for AR positivity. We derived AR regulated gene score and examined its utility in BC subtypes. Methods: AR regulated genes were derived by applying a bioinformatic pipeline on publicly available microarray data sets of AR+ BC cell lines and gene score was calculated as average expression of six AR regulated genes. Tumors were divided into AR high and low based on gene score and associations with clinical parameters, circulating androgens, survival and epithelial to mesenchymal transition (EMT) markers were examined, further evaluated in invitro models and public datasets. Results: 53% (133/249) tumors were classified as AR gene score high and were associated with significantly better clinical parameters, disease-free survival (86.13 vs 72.69 months, log rank p = 0.032) when compared to AR low tumors. 36% of TNBC (N = 66) were AR gene score high with higher expression of EMT markers (p = 0.024) and had high intratumoral levels of 5α-reductase, enzyme involved in intracrine androgen metabolism. In MDA-MB-453 treated with dihydrotestosterone, SLUG expression increased, E-cadherin decreased with increase in migration and these changes were reversed with bicalutamide. Similar results were obtained in public datasets. Conclusion: Deciphering the role of AR in BC is difficult based on AR protein levels alone. Our results support the context dependent function of AR in driving better prognosis in ER positive tumors and EMT features in TNBC tumors

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

<div><p>Purpose</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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 <b><i>deficient (0)</i></b> and <b><i>adequate (1)</i></b>. 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.</p><p>Conclusion</p><p>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.</p></div

BRCA1 Deficiency Score (BDS).

<p>BRCA1 Deficiency Score (BDS): Amalgamated scores range from a low of 0 to a high of 3. Scores of 0 and 1 are almost exclusively seen in TNBCs. Scores of 2 and 3 were seen in both HR+HER2- as well as TNBCs. 23/27 HR+HER2- tumors scored adequate on all measures and hence had a score of 3. This method of selection classified 40% of TNBCs being BRCA1 deficient and only 3% of HR+HER2-ve to be BRCA1 deficient.</p

Transnscript abundance of BRCA1 and ID4 in TNBC Vs HR+HER2-ve tumors.

<p>Transcript abundance of BRCA1 and ID4 in HR+HER2-veand TNBCs. Median expression of BRCA1 and ID4 transcripts is significantly different in the two groups; *p = 0.008 for BRCA1, and **p = 0.001 for ID4, (Mann Whitney U Test). The high ranges of BRCA1 are exclusively HR+, and the high-ranges of ID4 are all TNBCs.</p

Relationship between BRCA1 and ID4.

<p>Relationship between BRCA1 and ID4.A four quadrant plot with “cut-off” lines for both transcripts that are at 1 SD above the mean value of all specimens. The upper right quadrant representing high ID4 and high BRCA1 is empty. The upper left quadrant of high ID4 and low BRCA1 is almost exclusively TNBCs while the lower right quadrant of high BRCA1 and low ID4 is exclusively luminal tumors.</p

Distribution of TNBCs and HR+HER2- tumors adequate and deficient in the three measurements.

<p>Distribution of TNBCs and HR+HER2- tumors adequate and deficient in the three measurements.</p

Distribution of BRCA1/ID4 ratio values in luminal tumors compared to TNBCs.

<p>Distribution of BRCA1/ID4 ratio values in luminal tumors compared to TNBCs. An overlay of the histograms in panel A clearly shows the marked “Left-Skew” of TNBCs and a relatively “Right-shifted” distribution of luminal (HR+) tumors. The median value is significantly different in the two classes (*p<0.01- Mann Whitney U test).</p

Assay part numbers (Gene expression and MicroRNA analysis).

<p>Assay part numbers (Gene expression and MicroRNA analysis).</p