Abstract PS6-55: The prognostic utility of AR/ER ratio in young women with breast cancer.

Background: World over, less than a quarter of breast cancer diagnoses are in premenopausal women. However, in India premenopausal women constitute half of all women with breast cancer in most hospital case series. Most of these women present at advanced stages with aggressive subtypes of disease and hence the high mortality.The role and utility of detecting androgen receptor (AR) expression in the different sub-types of breast cancer, especially the ones without hormone receptor expression is yet to be firmly established. Evidence from previous studies is suggestive of its beneficial role in hormone receptor positive (HR+) breast cancer. The biological function of AR on the mammary epithelium is determined by the Estrogen receptor (ER) context, in that, it is found to be anti-proliferative in ER positive tumors while it is thought to promote growth in the absence of ER activity. An interesting approach to representing this interplay is as a ratio between AR/ER expressions. As expected, the ratio has been shown to be positively correlated with better outcomes in hormone receptor cancers, mostly in postmenopausal women. The effect of a high ratio in ER negative tumors seems more complicated. In this study, we have evaluated the AR/ER ratio specifically in patients younger than 50 years in whom the estrogenic influence is dominant due to their premenopausal status. Materials and Methods: Tumor samples from patients 50 years or younger were chosen from a larger cohort of 275 patients with median follow up of 72 months. Expression of ER and AR proteins were detected by immunohistochemistry (IHC), and the transcript levels of ESR1 and AR were determined by quantitative PCR. Relative normalized units of their gene expression were used to calculate the AR/ER ratio. A cut-off at the 3rd quartile was used to divide tumors into categories of high and low ratios. Clinical characteristics were compared between the low and high ratio groups along with IHC subtype distribution (HR+, HER2+ and Triple negative (TNBC)). Kaplan Meier curves was used for survival analysis and Cox proportional hazard analysis model was used to calculate the hazard ratio (HR). The results were validated in METABRIC dataset. Results: Eighty-eight (32%) patients were <50 years with a mean age of 43 years. AR/ER ratio ranged between 0.6 to 3.5 with a mean of 1.5. Sixty-six tumors were categorized as low and 22 were high based on the 3Q cut off (1.7). Clinical characters such as age, tumor size, grade, stage of disease was not different between the high and low ratio categories. Distribution of IHC subtypes among each group showed high ratio category had 64% TNBC tumors (p<0.0001). Tumors with high ratio had poor disease-free survival, (HR-2.6(95% CI-1-6.9) p-0.03). Trends in the METABRIC dataset was similar with 411(21%) patients <50 years. Ninety-seven patients with high ratio had significantly poor disease-free survival (HR-1.95 (95% CI-1.3-2.7) p-0.000). Conclusion: Interaction between AR and ER is known to influence the AR activity and our results reiterate prognostic ability of AR/ER ratio even in young patients of breast cancer. Our results suggest androgenic influences on clinical progression of breast cancer in this age group mediated through AR, has to be examined by its level in relation to the activity of ER, particularly in hormone receptor negative breast cancers. Even more importantly, examining these influences in the context of the menopausal status might help identify subgroups of patients most likely to benefit from interventions targeted at AR.

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

Purpose: Women 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. Methods: Tumor 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). Results: Eighty-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. Conclusion: Our 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.

miRNAs: Critical mediators of breast cancer metastatic programming.

MicroRNA mediated aberrant gene regulation has been implicated in several diseases including cancer. Recent research has highlighted the role of epigenetic modulation of the complex process of breast cancer metastasis by miRNAs. miRNAs play a crucial role in the process of metastatic evolution by facilitating alterations in the phenotype of tumor cells and the tumor microenvironment that promote this process. They act as critical determinants of the multi-step progression starting from carcinogenesis all the way to organotropism. In this review, we focus on the current understanding of the compelling role of miRNAs in breast cancer metastasis.

NGS-based profiling of key cancer genes in Indian triple-negative breast cancer patients reinforces molecular heterogeneity of the disease.

Triple-negative breast cancers (TNBC) are one of the most aggressive forms of breast cancers. With poor patient outcomes, it presents a great burden on the healthcare systems. There have been some efforts to explore the genomic changes that occur in TNBCs. However, there is not enough data on Indian TNBCs. We sought to understand the mutational landscape of key cancer-associated genes in Indian TNBC patients using TruSeq Cancer Amplicon Panel. We sequenced 51 TNBC patient samples and found great heterogeneity amongst samples with respect to the genomic variants. Several previously reported including alterations in PI3K-AKT pathway genes were also identified. Likewise, we identified several novel high-frequency variants, for example, GNAQ F341S (17%), the functional role of which remains unclear. Our study lays the foundation of larger efforts needed to understand the genomic landscape of Indian TNBCs which can aid in classification and better therapeutic management of patients.

Prognostic role of microRNA 182 and microRNA 18a in locally advanced triple negative breast cancer.

BACKGROUND: The study assessed the epigenetic regulation and the role of microRNA (miR) expression in locally advanced triple negative breast cancers (TNBC) and comparison with the clinico-pathological variables and survival. METHODS: Fifty patients of locally advanced TNBC during the period 2011-2013 were included. Expression level of test microRNA (miR-182 and miR-18a) was determined using Taqman quantitative Real time polymerase chain reaction (qRT-PCR) from formalin fixed paraffin embedded biopsy blocks. Clinical and demographic information and survival data was retrieved from the Hospital medical records. RESULTS: An improved clinical complete response (cCR) was observed in patients with age ≥ 45 years (80%), premenopausal status (70%), tumor size < 6 cms (80%), nodal status N0-N1 (95%) and grade II-III tumor (80%). A statistically significant correlation was observed on comparison of cCR with menopausal status (p-value 0.020), T category (p-value 0.018) and the clinical nodal status (p-value 0.003). pCR also correlated with clinical nodal status (p-value 0.008). Epigenetically, miR-18a under expression (< 8.84) was most commonly associated with tumor size < 6 cms (76.7%), clinical nodal status N0-N1 (90%), cCR (60%) and pCR (53.3%). A similar trend was observed with miR-182. Statistical significance was observed with T category (p-values 0.003 and 0.004), clinical nodal status (p-values 0.001 and 0.001), clinical response (p-values 0.002 and 0.002) and pathological response (p-values 0.007 and 0.006) with respect to miR-18a and miR-182, respectively. Also, the menopausal status significantly correlated with the miR-182 expression (p-value 0.009). miR-182 overexpression (≥ 6.32) was not observed in any of the postmenopausal patients. A univariate cox proportional hazard regression model also showed statistical interactions (p-values <0.004). CONCLUSION: miR-182 and miR-18a overexpression correlates with worse clinical and pathological tumor characteristics in locally advanced TNBC and hence could be used to predict the outcomes and prognosis in these patients.

Genome-Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

The Impact of Breast Cancer on the Patient and the Family in Indian Perspective.

PURPOSE: To understand the role played by the immediate family in treatment decision and support in patients diagnosed with breast cancer, the influence of demographic factors on psychosocial roles of women within the family. METHODS: A mixed method design used for data collection on family support, financial arrangement and psychosocial impact of cancer from 378 women with breast cancer recruited at first diagnosis between 2008 and 2012, during multiple counseling sessions. The median follow-up is 7 years with only 2% lost to follow-up. RESULTS: Most patients (99%) had support from family members. 57% of patients met the costs of treatment through personal savings and health insurance. The rest (43%) had difficulty and had to resort to desperate measures such as selling their property or taking on high-interest personal loans. Patients with higher education and urban settings had better financial management. A male member of the family (husband or son) was the main decision maker in half of the cases. Concerns over women's responsibilities within the family varied by the age of the patient. The vast majority of women (90%) experienced social embarrassment in dealing with the disease and its aftermath. CONCLUSION: In India, it is the family that provides crucial support to a woman with breast cancer during her ordeal with the disease and its treatment. This study has implications on the psychosocial support beyond the cancer patients alone, to include the immediate family and consider aspects of finance and social adjustments as critical in addition to the routine medical aspects of the disease.

Eccentric placentae have reduced surface area and are associated with lower birth weight in babies small for gestational age.

Placental structure and function determine birth outcomes. Placental mass does not always correlate with fetal birth weight (BW) in uncomplicated pregnancies which raises the possibility of other variables such as placental shape and cord insertion being the determinants of placental efficiency. In total, 160 women with singleton pregnancy, recruited into a pregnancy cohort were studied. Placental weight (PW) was measured and other data were obtained from clinical records. Birth outcomes were classified as small for gestational age (SGA) and appropriate for gestational age (AGA) based on fetal gender, gestational age (GA) and BW. High-resolution images of the chorionic plate were recorded. The shape of the placenta and the insertion of the cord were measured using eccentricity index (EI) and cord centrality index (CCI). Only placentae with eccentrically inserted cords (n=136) were included. The mean BW and PW were 2942 (±435) g and 414 (±82) g with average GA of 38.6 weeks. The mean CCI and EI was 0.483 (±0.17) and 0.482 (±0.16). Neither of these correlated with placental efficiency. However, EI showed negative correlation with placental surface area and breadth. Upon sub-grouping the cohort into SGA (n=32) and AGA (n=104), the SGA babies with the highest EI (third tertile) had significantly lower BW than those with the least eccentric placentae (first tertile). Although eccentric-shaped placentae were present in both SGA and AGA groups, the effect on BW was observed only in the SGA group.

NLScore: a novel quantitative algorithm based on 3 dimensional structural determinants to predict the probability of nuclear localization in proteins containing classical nuclear localization signals.

The presence of a nuclear localization signal (NLS) in proteins can be inferred by the presence of a stretch of basic amino acids (KRKK). These NLSs are termed classical NLS (cNLS). However, only a fraction of proteins containing the cNLS pattern are transported into the nucleus by binding to importin α. Hence, there must exist, additional structural determinants that guide the appropriate interaction between putative NLSs containing cargo and importin α. Using 52 protein structures containing cNLS obtained from RCSB PDB, we assembled a training set and a validation set such that both sets were comprised of a combination of proteins with proven nuclear localization and ones that were non-nuclear. We modeled the interface between cargoes containing cNLS and importin α. We conducted rigid body docking and produced induced-fit modes by allowing both side chain and the backbone to be flexible. The output of these studies and additional determinants such as energy of interaction, atomic contacts, hydrophilic interaction, cationic interaction, and penetration of the cargo protein were used to derive a 26 parameter quantitative structure activity relationship based regression equation. This was further optimized by a step-wise backward elimination approach to derive a 15 parameter score. This NLScore was not only able to correctly classify confirmed nuclear and non-nuclear localized proteins but it was able to perform better than currently implemented algorithms like NucPred, Euk-mPLoc 2.0, cNls Mapper, and NLStradamus. Leave-one-out cross validation (LOOCV) showed that NLScore correctly predicted 78.6% and 81.6% of non-nuclear and nuclear proteins respectively. Graphical abstract NLScore: a novel quantitative algorithm based on 3 dimensional structural determinants to predict the probability of nuclear localization in proteins.

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.

Placental expression of DNA methyltransferase 1 (DNMT1): Gender-specific relation with human placental growth.

AIMS: Placental physiology and morphology is critically regulated by DNA methylation. As such, placental global DNA methylation and transcript abundance of placental DNA methyltransferases (DNMT1 and DNMT3A) may relate to placental and fetal growth in human pregnancies. We aimed to test correlations of human fetoplacental parameters and birth weight with the placental expression of DNA methyltransferases (DNMT1 and DNMT3A) and placental global methylation. SUBJECTS AND METHODS: Placentae (n = 109) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 56 SGA and 53 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of DNMT1, DNMT3A and DNTMT3B normalized to a panel of reference genes. LINE-1 methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 68). Associations of placental transcript abundances of DNMT1, DNMT3A and DNMT3B and of LINE-1 methylation levels with maternal, placental and neonatal parameters were tested. RESULTS: Placental DNMT1 transcript abundance associated positively with placental weight (ß = 10.21, P = 0.013). This association was specific to the AGA births (ß = 12.77, P = 0.022) and was absent in the SGA births. Association of DNMT1 expression with placental weight and birth weight within the AGA births was specific to the female gender (Birth weight: ß = 83.61, P = 0.043; Placental weight: ß = 23.92, P = 0.025). Placental DNMT1 transcript levels were not different according to SGA status or gender. Placental DNMT3A transcript levels and LINE-1 methylation levels did not show any associations with maternal, placental and neonatal parameters. CONCLUSIONS: Placental DNMT1 expression was found to be associated positively with placental weight and birth weight, specifically in the female AGA births. Thus, we hypothesize that placental DNMT1 participates in fetoplacental growth in a fetal gender-specific manner.

Data on alteration of hormone and growth factor receptor profiles over progressive passages of breast cancer cell lines representing different clinical subtypes.

Human breast cancers are a highly heterogeneous group of tumours consisting of several molecular subtypes with a variable profile of hormone, growth factor receptors and cytokeratins [1]. Here, the data shows immunofluorescence profiling of four different cell lines belonging to distinct clinical subtypes of breast cancer. Post revival, the cell lines were passaged in culture and immunophenotyping was done for ER, HER-2, AR and EGFR. Data for the markers from early passage (5th) through passages as late as 25 for the different cell lines is presented.

The ethics of research on stored biological samples: outcomes of a Workshop.

Research is often conducted using laboratory samples and data. The ethical issues that arise in a study involving residual samples are considerably different from those arising in a prospective study. Some of these ethical issues concern the risks to confidentiality, individual autonomy, trust in and credibility of the researcher or the research, commercialisation and even the nomenclature involved.

ß3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD.

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin ß3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin ß3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin ß3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin ß3 brought about a reversal of this effect and chemosensitized the cells. These results identify ß3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

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.

Placental expression of the insulin receptor binding protein GRB10: Relation to human fetoplacental growth and fetal gender.

INTRODUCTION: Imprinted genes play an important role in mammalian fetoplacental growth and development. We have evaluated whether the placental expression of two imprinted genes, growth factor receptor-binding protein 10 (GRB10) and pleckstrin homology-like domain, family A, member 2 (PHLDA2) correlate with human fetoplacental growth parameters. METHODS: Placentae (n = 77) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 36 SGA and 41 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of GRB10 and PHLDA2 normalized to a panel of reference genes. RESULTS: Placental GRB10 transcript abundance associated positively with placental weight (r = 0.307, P = 0.007), birth weight (r = 0.267, P = 0.019) and neonatal head circumference (r = 0.280, P = 0.014). Placental GRB10 transcript levels were significantly lower in male SGA placentae compared to the male AGA placentae. Placental PHLDA2 transcript abundance did not show any associations with maternal, placental or neonatal parameters. DISCUSSION: Placental GRB10 expression was found to be associated positively with placental weight, birth weight, and neonatal head circumference, especially in males. Hence, we speculate that placental GRB10 plays a role in regulating fetoplacental growth and thereby in the pathophysiology of fetal growth restriction in the context of fetal gender.

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.

Separate quality-control measures are necessary for estimation of RNA and methylated DNA from formalin-fixed, paraffin-embedded specimens by quantitative PCR.

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.