Addition of chromosomal microarray and next generation sequencing to FISH and classical cytogenetics enhances genomic profiling of myeloid malignancies.

Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.

ESR1 and ESR2 polymorphisms in the BIG 1-98 trial comparing adjuvant letrozole versus tamoxifen or their sequence for early breast cancer.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

Endometrial carcinoma with ectopic human chorionic gonadotropin expression.

•Aggressive course and treatment resistance characterize ectopic human chorionic gonadotropin.•Recurrence of endometrial cancer with ectopic hCG was treated with brachytherapy and EMACO.•The serum hCG level can serve as a marker in tumors with ectopic hCG expression.

CYP19A1 polymorphisms and clinical outcomes in postmenopausal women with hormone receptor-positive breast cancer in the BIG 1-98 trial.

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.

Wnt signaling in triple negative breast cancer is associated with metastasis.

BACKGROUND: Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies. METHODS: We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease. RESULTS: The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/ß-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/ß-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or ß-catenin (functional read out of Wnt/ß-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from ß-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/ß-catenin classifier had a greater risk of lung and brain, but not bone metastases. CONCLUSION: These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.

Differential activation of Wnt-ß-catenin pathway in triple negative breast cancer increases MMP7 in a PTEN dependent manner.

Mutations of genes in tumor cells of Triple Negative subset of Breast Cancer (TNBC) deregulate pathways of signal transduction. The loss of tumor suppressor gene PTEN is the most common first event associated with basal-like subtype (Martins, De, Almendro, Gonen, and Park, 2012). Here we report for the first time that the functional upregulation of secreted-MMP7, a transcriptional target of Wnt-ß-catenin signature pathway in TNBC is associated to the loss of PTEN. We identified differential expression of mRNAs in several key-components genes, and transcriptional target genes of the Wnt-ß-catenin pathway (WP), including beta-catenin, FZD7, DVL1, MMP7, c-MYC, BIRC5, CD44, PPARD, c-MET, and NOTCH1 in FFPE tumors samples from TNBC patients of two independent cohorts. A similar differential upregulation of mRNA/protein for beta-catenin, the functional readout of WP, and for MMP7, a transcriptional target gene of beta-catenin was observed in TNBC cell line models. Genetic or pharmacological attenuation of beta-catenin by SiRNA or WP modulators (XAV939 and sulindac sulfide) and pharmacological mimicking of PTEN following LY294002 treatment downregulated MMP7 levels as well as enzymatic function of the secreted MMP7 in MMP7 positive PTEN-null TNBC cells. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA. In cell lines, a high expression of casein-zymograph-positive MMP7 was distinguished by an absence of functional PTEN. A similar inverse relationship between MMP7 and PTEN mRNA levels was observed in the PAM50 data set (a correlation coefficient of -0.54). The PAM50 subtype and outcome data revealed that the high MMP7 group had low pCR (25%) and High Rd (74%) in clinical stage T3 pathologic response in contrast to the high pCR (40%) and low residual disease (RD) (60%) of the low MMP7 group.

Dissecting GRB7-mediated signals for proliferation and migration in HER2 overexpressing breast tumor cells: GTP-ase rules.

Amplification of human Her2 and its aberrant signaling in 20-30% of early breast cancer patients is responsible for highly aggressive tumors with poor outcome. Grb7 is reported to be co-amplified with Her2. We report a concurrent high expression of mRNA (from FFPE tumor samples; mRNA correlation, Pearson r(2)= 0.806), and high levels of GRB7 protein (immunoblot) in HER2+ breast cancer cell lines. We demonstrated the signaling mechanism of HER2 and downstream effectors that contributes to proliferation and migration. Using HER2+ and trastuzumab-resistant breast cancer cell lines, we identified the interaction between GRB7 and HER2 in the control of HER2+ cell proliferation. Our co-IP data show that GRB7 recruits SHC into the HER2-GRB7 signaling complex. This complex formation leads to activation of RAS-GTP. We also observed that following integrin engagement, GRB7 is phosphorylated at tyrosine in a p-FAK (Y397) dependent manner. This FAK-GRB7 complex leads to downstream activation of RAC1-GTP (responsible for migration) probably through the recruitment of VAV2. Our CO-IP data demonstrate that GRB7 directly binds with VAV2 following fibronectin engagement in HER2+ cells. To address whether GRB7 could serve as a pathway specific therapeutic target, we used siRNA to suppress GRB7 expression. Knockdown of GRB7 expression in the HER2+ breast cancer cell lines decreases RAS activation, cell proliferation, 2D and 3D colony formation and also blocked integrin-mediated RAC1 activation along with integrin-directed cell migration. These findings dissected the HER2-mediated signaling cascade into (1) HER2+ cell proliferation (HER2-GRB7-SHC-RAS) and (2) HER2+ cell migration (alpha5 beta1/alpha4 beta1-FAK-GRB7-VAV2-RAC1). Our data clearly demonstrate that a coupling of GRB7 with HER2 is required for the proliferative and migratory signals in HER2+ breast tumor cells.

Company Profile: AKESOgen, Inc.

Rapid advancement of genomics, genetic and bioinformatic technologies have paved the way for an explosion of opportunities in pharmacogenomics, which is reflected by the growing number of biomarkers in the 'personalized medicine cabinet'. AKESOgen, Inc. (GA, USA) has been established to meet and champion these needs. AKESOgen, Inc. is a biomarker, genomics and pharmacogenomics contract research organization that services the academic, pharmaceutical, biotechnology and agricultural sectors. AKESOgen, Inc. performs biomarker profiling and genomics services utilizing different types of markers (e.g., DNA, RNA and methylation) for the research and development market. AKESOgen, Inc. establishes and validates biomarkers in the clinical trials arena and provides expertise in biobanking.

CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer: the breast international group 1-98 trial.

BACKGROUND: Adjuvant tamoxifen therapy is effective for postmenopausal women with endocrine-responsive breast cancer. Cytochrome P450 2D6 (CYP2D6) enzyme metabolizes tamoxifen to clinically active metabolites, and CYP2D6 polymorphisms may adversely affect tamoxifen efficacy. In this study, we investigated the clinical relevance of CYP2D6 polymorphisms. METHODS: We obtained tumor tissues and isolated DNA from 4861 of 8010 postmenopausal women with hormone receptor-positive breast cancer who enrolled in the randomized, phase III double-blind Breast International Group (BIG) 1-98 trial between March 1998 and May 2003 and received tamoxifen and/or letrozole treatment. Extracted DNA was used for genotyping nine CYP2D6 single-nucleotide polymorphisms using polymerase chain reaction-based methods. Genotype combinations were used to categorize CYP2D6 metabolism phenotypes as poor, intermediate, and extensive metabolizers (PM, IM, and EM, respectively; n = 4393 patients). Associations of CYP2D6 metabolism phenotypes with breast cancer-free interval (referred to as recurrence) and treatment-induced hot flushes according to randomized endocrine treatment and previous chemotherapy were assessed. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. RESULTS: No association between CYP2D6 metabolism phenotypes and breast cancer-free interval was observed among patients who received tamoxifen monotherapy without previous chemotherapy (P = .35). PM or IM phenotype had a non-statistically significantly reduced risk of breast cancer recurrence compared with EM phenotype (PM or IM vs EM, HR of recurrence = 0.86, 95% CI = 0.60 to 1.24). CYP2D6 metabolism phenotype was associated with tamoxifen-induced hot flushes (P = .020). Both PM and IM phenotypes had an increased risk of tamoxifen-induced hot flushes compared with EM phenotype (PM vs EM, HR of hot flushes = 1.24, 95% CI = 0.96 to 1.59; IM vs EM, HR of hot flushes = 1.23, 95% CI = 1.05 to 1.43). CONCLUSIONS: CYP2D6 phenotypes of reduced enzyme activity were not associated with worse disease control but were associated with increased hot flushes, contrary to the hypothesis. The results of this study do not support using the presence or absence of hot flushes or the pharmacogenetic testing of CYP2D6 to determine whether to treat postmenopausal breast cancer patients with tamoxifen.

Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.

An important challenge in prostate cancer research is to develop effective predictors of tumor recurrence following surgery to determine whether immediate adjuvant therapy is warranted. To identify biomarkers predictive of biochemical recurrence, we isolated the RNA from 70 formalin-fixed, paraffin-embedded radical prostatectomy specimens with known long-term outcomes to perform DASL expression profiling with a custom panel that we designed of 522 prostate cancer-relevant genes. We identified a panel of 10 protein-coding genes and two miRNA genes (RAD23B, FBP1, TNFRSF1A, CCNG2, NOTCH3, ETV1, BID, SIM2, LETMD1, ANXA1, miR-519d, and miR-647) that could be used to separate patients with and without biochemical recurrence (P < 0.001), as well as for the subset of 42 Gleason score 7 patients (P < 0.001). We performed an independent validation analysis on 40 samples and found that the biomarker panel was also significant at prediction of biochemical recurrence for all cases (P = 0.013) and for a subset of 19 Gleason score 7 cases (P = 0.010), both of which were adjusted for relevant clinical information including T-stage, prostate-specific antigen, and Gleason score. Importantly, these biomarkers could significantly predict clinical recurrence for Gleason score 7 patients. These biomarkers may increase the accuracy of prognostication following radical prostatectomy using formalin-fixed specimens.

alcohol, smoking, and caffeine in relation to fecundability, with effect modification by NAT2.

PURPOSE: Common polymorphisms in the N-acetyltransferase-2 (NAT2) metabolic enzyme determine slow or rapid acetylator phenotypes. We investigated the effects of alcohol, smoking, and caffeine on fecundability, and determined whether the effects were modified by NAT2. METHODS: Three NAT2 polymorphisms were genotyped in 319 women office workers participating in a prospective pregnancy study (1990-1994). Women were ages 20-41 and at risk for pregnancy. Discrete-time survival analysis was used to determine the effects of alcohol, smoking, and caffeine on fecundability and evaluate effect modification by NAT2. RESULTS: We followed 319 women (161 slow acetylators, 158 rapid) for an average of 8 menstrual cycles, resulting in 124 pregnancies. There was no effect of caffeine on fecundability. Drinking ≥1 alcoholic drink per day and current smoking were significantly associated with reduced fecundability, but only among slow acetylators (adjusted fecundability odds ratio [FOR] for smoking = 0.34; 95% confidence interval, 0.22-0.90; adjusted FOR for ≥1 drink per day = 0.20; 0.05-0.92). There was no effect among rapid acetylators. CONCLUSIONS: NAT2 status significantly modified the effects of alcohol and smoking on fecundability, emphasizing the importance of incorporating genetic and metabolic information in studies of reproductive health. Replication of this study is warranted.

Identification of candidate genes for histiocytoid cardiomyopathy (HC) using whole genome expression analysis: analyzing material from the HC registry.

Histiocytoid cardiomyopathy (HC) is a rare but distinctive arrhythmogenic disorder characterized by incessant ventricular tachycardia, cardiomegaly, and often sudden death by age 2 years. The underlying genetic mechanism of HC has eluded researchers for decades. To further identify the potential molecular-genetic bases of HC, molecular analyses of HC hearts and hearts of age-matched controls were performed. Total RNA and genomic DNA were prepared from formalin-fixed, paraffin-embedded cardiac tissue from 12 cases of HC and 12 age-matched controls. To identify genes differentially expressed in HC, whole genome cDNA-mediated annealing, selection, extension, and ligation profiling was performed. TaqMan quantitative polymerase chain reaction confirmed changes in RNA expression. DNA copy number changes were measured by TaqMan copy number variant analysis. Analysis of differential gene expression in HC cases identified 2 significantly downregulated gene sets aligned sequentially along the genome. The 1st gene cluster consisted of genes S100A8 , S100A9 , and S100A12 at 1q21.3c, and the 2nd cluster consisted of genes IL1RL1 ( ST2 ), IL18R1 , and IL18RAP at 2q12.1a. Strong decreases in interleukin 33 expression were also observed. Decreases in copy number of the S100A genes were confirmed by TaqMan copy number variant assays. S100A genes are downstream of the p38-MAPK pathway that can be activated by interleukin 33 signaling. These data suggest a model in which the interleukin 33-IL1RL1/p38-MAPK/ S100A8-S100A9 axis is downregulated in HC cardiac tissue and provide several candidate genes on 1q21.3c and 2q12.1a for inherited mutations that may predispose individuals to HC.

Serotonin transporter gene, depressive symptoms, and interleukin-6.

BACKGROUND: We explored the relationship of genetic variants of the serotonin transporter gene SLC6A4, a key regulator of the serotonergic neurotransmission, with both depressive symptoms and plasma interleukin-6 (IL-6) levels. METHODS AND RESULTS: We genotyped 20 polymorphisms in 360 male twins (mean age, 54 years) from the Vietnam Era Twin Registry. Current depressive symptoms were measured with the Beck Depression Inventory II. IL-6 was assessed using a commercially available ELISA kit. Genotype associations were analyzed using generalized estimating equations. To study how SLC6A4 genetic vulnerability influences the relationship between depressive symptoms and IL-6, bivariate models were constructed using structural equation modeling. Of the 20 polymorphisms examined, the effective number of independent tests was 6, and the threshold of significance after Bonferroni correction was 0.008. There were 6 single-nucleotide polymorphisms significantly associated with Beck Depression Inventory (P< or =0.008), including rs8071667, rs2020936, rs25528, rs6354, rs11080122, and rs8076005, and 1 single-nucleotide polymorphism was borderline associated (rs12150214, P=0.017). Of these 7 single-nucleotide polymorphisms, 3 were also significantly associated with IL-6 (P<0.008), including rs25528, rs6354, and rs8076005, and the other 4 were borderline associated (P=0.009 to 0.025). The subjects with 1 copy of the minor allele of these 7 single-nucleotide polymorphisms had higher Beck Depression Inventory scores and IL-6 levels. Further bivariate modeling revealed that approximately 10% of the correlation between Beck Depression Inventory and IL-6 could be explained by the SLC6A4 gene. CONCLUSIONS: Genetic vulnerability involving the SLC6A4 gene is significantly associated with both increased depressive symptoms and elevated IL-6 plasma levels. Common pathophysiological processes may link depression and inflammation, and implicate the serotonin pathway in neural-immune interactions.

No association between polymorphisms in LEP, LEPR, ADIPOQ, ADIPOR1, or ADIPOR2 and postmenopausal breast cancer risk.

There is evidence that adipokines such as leptin and adiponectin may influence breast tumor development. We conducted a nested case-control study using women in the American Cancer Society Cancer Prevention Study II to examine the association between postmenopausal breast cancer and variability in the genes encoding leptin, the leptin receptor, adiponectin, adiponectin receptor 1, and adiponectin receptor 2. Using 648 cases and 659 controls, we found no statistically significant (P < 0.05) associations between breast cancer risk and any of the single nucleotide polymorphisms. Individual odds ratios ranged from 0.93 to 1.06. We found no evidence of effect modification by body mass index, adult weight gain, location of weight gain, or physical activity. Although we cannot rule out that these genes are involved in gene-gene or gene-environment interactions, our results suggest that individual single nucleotide polymorphisms in these genes do not substantially affect postmenopausal breast cancer risk.

Decreased NK cell frequency and function is associated with increased risk of KIR3DL allele polymorphism in simian immunodeficiency virus-infected rhesus macaques with high viral loads.

NK cells have been established as an important effector of innate immunity in a variety of viral infections. In HIV-1 infection in humans, alterations of NK cell function, frequency, and expression of various NK receptors have been reported to be associated with differential dynamics of disease progression. Expression of certain alleles of KIR3DL and KIR3DS receptors on NK cells was shown to correlate with levels of virus replication. In the SIV-infected rhesus macaque (RM) model of AIDS, several families of killer inhibitory Ig-related receptors (KIR receptors) corresponding to their human counterparts have been characterized, but only at the level of individual sequence variants. Here we define 14 different alleles of KIR3DL expressed among 38 SIV-infected RM, characterized by either high or low levels of SIV replication, by analyzing multiple sequences from individual animals and show an unequal distribution of certain alleles in these cohorts. High levels of SIV replication were associated with significant increases in KIR3DL mRNA levels in addition to decreases in both the frequency and function of NK cells in these animals. The higher frequency of inheritance of two KIR3DL alleles characterized by a single nucleotide polymorphism 159 H/Q was associated with RM that exhibited high plasma viral load. This data for the first time defines multiple alleles of KIR3DL in RM and shows an association between virus control, NK cell function and genetic polymorphisms of KIR receptors.

The endothelin-1 G5665T polymorphism impacts transplant-free survival for single ventricle patients.

BACKGROUND: Early survival for children with single-ventricle congenital heart disease has improved, but late attrition remains a serious problem. The endothelin-1 G5665T single nucleotide polymorphism has been linked to increased vascular reactivity and hypertension. The goal of this study was to determine whether this single nucleotide polymorphism alters transplant-free survival for children with single-ventricle congenital heart disease. METHODS: DNA was isolated from 165 children with single-ventricle congenital heart disease born between January 1980 and December 2006. The endothelin-1 G5665T single nucleotide polymorphism genotype was determined by using real-time polymerase chain reaction. Kaplan-Meier survival curves were generated with a combined end point of death or transplantation. The Cox proportional hazard method was used to evaluate potential covariates. RESULTS: The endothelin-1 G5665T genotype was significantly associated with transplant-free survival for the group as a whole (P = .002), with the greatest effect in children with hypoplastic left heart syndrome (n = 64, P = .0002) as opposed to patients with other types of single-ventricle anatomy (n = 101, P = .1). Cox proportional hazard modeling revealed 3 independent predictors of poor outcome: endothelin G5665T genotype (T/T genotype, P = .001), prematurity (gestational age <37 weeks, P = .02), and era of surgical intervention (1990s vs other decades, P = .02). CONCLUSIONS: Long-term survival of single-ventricle patients is dependent on many factors. These data suggest that genetic variability involving vascular resistance modifiers, such as endothelin-1, might play an important role, particularly in patients with hypoplastic left heart syndrome. Future studies should include evaluation of the relationship between endothelin genotype and plasma endothelin levels and possibly therapeutic trials of endothelin blockers in high-risk patients.

Genetic variation in candidate obesity genes ADRB2, ADRB3, GHRL, HSD11B1, IRS1, IRS2, and SHC1 and risk for breast cancer in the Cancer Prevention Study II.

INTRODUCTION: Obesity has consistently been associated with postmenopausal breast cancer risk. Proteins that are secreted by adipose tissue or are involved in regulating body mass may play a role in breast tumor development. METHODS: We conducted a nested case-control study among postmenopausal women from the American Cancer Society Cancer Prevention Study II Nutrition Cohort to determine whether genes associated with obesity increase risk for breast cancer. Tagging single nucleotide polymorphisms (SNPs) were selected to capture common variation across seven candidate genes that encode adipose-related proteins: ADRB2, ADRB3, GHRL, HSD11B1, IRS1, IRS2, and SHC1. Thirty-nine SNPs were genotyped in 648 cases and 659 controls. Logistic regression models were used to examine the association between each tagging SNP and risk for breast cancer while adjusting for matching factors and potential confounders. We also examined whether these SNPs were associated with measures of adult adiposity. RESULTS: Two out of five tagging SNPs in HSD11B1 were associated with breast cancer (rs11807619, P = 0.006; rs932335, P = 0.0001). rs11807619 and rs932335 were highly correlated (r2 = 0.74) and, when modeled as a haplotype, only haplotypes containing the rs932335 C allele were associated with breast cancer. The rs932335 C allele was associated with a nearly twofold increased risk for breast cancer (odds ratio = 1.83, 95% confidence interval = 1.01-3.33 for C/C versus G/G). Three of the 11 SNPs for IRS2 were associated with breast cancer (rs4773082, P = 0.007; rs2289046, P = 0.016; rs754204, P = 0.03). When these three SNPs were examined as a haplotype, only the haplotype that included the G allele of rs2289046 was associated with breast cancer (odds ratio = 0.76, 95% confidence interval = 0.63-0.92 for TGC versus CAT). IRS2 rs2289046, rs754204, and rs12584136 were also associated with adult weight gain but only among cases. None of the other SNPs in any gene investigated were associated with breast cancer or adiposity. CONCLUSION: Our findings suggest that these tagging SNPs in HSD11B1 and IRS2 mark regions of the genome that may harbor risk alleles for breast cancer, and these associations are probably independent of adiposity.

Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay.

Formalin-fixed paraffin-embedded (FFPE) breast tumor tissues are readily available and represent a largely untapped, vast resource for molecular profiling of clinical samples with long-term follow-up data. We have optimized the conditions and parameters that result in the preparation of total RNA that is of the necessary quality for use in the DASL (cDNA-mediated annealing, selection, extension, and ligation) assay in which expression of 502 genes are analyzed simultaneously using as little as 100 ng of input RNA.