Reliability of a computational platform as a surrogate for manually interpreted immunohistochemical markers in breast tumor tissue microarrays.

BACKGROUND: Pathologist and computational assessments have been used to evaluate immunohistochemistry (IHC) in epidemiologic studies. We compared Definiens Tissue Studio® to pathologist scores for 17 markers measured in breast tumor tissue microarrays (TMAs) [AR, CD20, CD4, CD8, CD163, EPRS, ER, FASN, H3K27, IGF1R, IR, Ki67, phospho-mTOR, PR, PTEN, RXR, and VDR]. METHODS: 5 914 Nurses' Health Study participants, diagnosed 1976-2006 (NHS) and 1989-2006 (NHS-II), were included. IHC was conducted by the Dana-Farber/Harvard Cancer Center Specialized Histopathology Laboratory. The percent of cells staining positive was assessed by breast pathologists. Definiens output was used to calculate a weighted average of percent of cells staining positive across TMA cores for each marker. Correlations between pathologist and computational scores were evaluated with Spearman correlation coefficients. Receiver-operator characteristic curves were constructed, using pathologist scores as comparison. RESULTS: Spearman correlations between pathologist and Definiens assessments ranged from weak (RXR, rho=-0.05; CD163, rho = 0.10) to strong (Ki67, rho = 0.79; pmTOR, rho = 0.77). The area under the curve was >0.70 for all markers except RXR. CONCLUSION: Our data indicate that computational assessments exhibit variable correlations with interpretations made by an expert pathologist, depending on the marker evaluated. This study provides evidence supporting the use of computational platforms for IHC evaluation in large-scale epidemiologic studies, with the caveat that pilot studies are necessary to investigate agreement with expert assessments. In sum, computational platforms may provide greater efficiency and facilitate high-throughput epidemiologic analyses.

S-nitrosylated and non-nitrosylated COX2 have differential expression and distinct subcellular localization in normal and breast cancer tissue.

Immunohistochemical (IHC) staining in breast cancer shows both gain and loss of COX2 expression with disease risk and progression. We investigated four common COX2 antibody clones and found high specificity for purified human COX2 for three clones; however, recognition of COX2 in cell lysates was clone dependent. Biochemical characterization revealed two distinct forms of COX2, with SP21 recognizing an S-nitrosylated form, and CX229 and CX294 recognizing non-nitrosylated COX2 antigen. We found S-nitrosylated and non-nitrosylated COX2 occupy different subcellular locations in normal and breast cancer tissue, implicating distinct synthetic/trafficking pathways and function. Dual stains of ~2000 breast cancer cases show early-onset breast cancer had increased expression of both forms of COX2 compared to postmenopausal cases. Our results highlight the strengths of using multiple, highly characterized antibody clones for COX2 IHC studies and raise the prospect that S-nitrosylation of COX2 may play a role in breast cancer biology.

Sex-Stratified Polygenic Risk Score Identifies Individuals at Increased Risk of Basal Cell Carcinoma.

The incidence of basal cell carcinoma (BCC) is higher among men than women. Susceptibility loci for BCC have been identified through genome-wide association studies, and two previous studies have found polygenic risk scores (PRS) to be significantly associated with the risk of BCC. However, to our knowledge, sex-stratified PRS analyses examining the genetic contribution to BCC risk among men and women have not been previously reported. To quantify the contribution of genetic variability on the BCC risk by sex, we derived a polygenic risk score and estimated the genetic relative risk distribution for men and women. Using 29 published single nucleotide polymorphisms, we found that the estimated relative risk of BCC increases with higher percentiles of the polygenic risk score. For men, the estimated risk of BCC is twice the average population risk at the 88th percentile, while for women, this occurs at the 99th percentile. Our findings indicate that there is a significant impact of genetic variation on the risk of developing BCC and that this impact may be greater for men than for women. Polygenic risk scores may be clinically useful tools for risk stratification, particularly in combination with other known risk factors for BCC development.

Research Techniques Made Simple: Interpreting Measures of Association in Clinical Research.

To bring evidence-based improvements in medicine and health care delivery to clinical practice, health care providers must know how to interpret clinical research findings and critically evaluate the strength of evidence. This requires an understanding of differences in clinical study designs and the various statistical methods used to identify associations. We aim to provide a foundation for understanding the common measures of association used in epidemiologic studies to quantify relationships between exposures and outcomes, including relative risks, odds ratios, and hazard ratios. We also provide a framework for critically assessing clinical research findings and highlight specific methodologic concerns.

Genetic variations, reproductive aging, and breast cancer risk in African American and European American women: The Women's Circle of Health Study.

Reproductive aging phenotypes, including age at menarche (AM) and age at natural menopause (ANM), are well-established risk factors for breast cancer. In recent years, many genetic variants have been identified in association with AM and ANM in genome-wide association studies among European populations. Using data from the Women's Circle of Health Study (WCHS) of 1,307 European-American (EA) and 1,365 African-American (AA) breast cancer cases and controls, we aimed to replicate 53 earlier GWAS variants for AM and ANM in AA and EA groups and to perform analyses on total and net reproductive lifespan (TRLS; NRLS). Breast cancer risk was also examined in relation to a polygenic risk score (PRS) for each of the reproductive aging phenotypes. We replicated a number of variants in EA women, including rs7759938 in LIN28B for AM and rs16991615 in MCM8 for ANM; whereas in the AA group, only one SNP (rs2947411 in TMEM18) for AM was directionally consistent and nominally significant. In analysis of TRLS and NRLS, several SNPs were significant, including rs466639 in RXRG that was associated with both phenotypes in both AA and EA groups. None of the PRS was associated with breast cancer risk. Given the paucity of data available among AA populations, our study contributes to the literature of genetics of reproductive aging in AA women and highlights the importance of cross population replication of GWAS variants.

Single nucleotide variants in metastasis-related genes are associated with breast cancer risk, by lymph node involvement and estrogen receptor status, in women with European and African ancestry.

Single nucleotide polymorphisms (SNPs) in pathways influencing lymph node (LN) metastasis and estrogen receptor (ER) status in breast cancer may partially explain inter-patient variability in prognosis. We examined 154 SNPs in 12 metastasis-related genes for associations with breast cancer risk, stratified by LN and ER status, in European-American (EA) and African-American (AA) women. Two-thousand six hundred and seventy-one women enrolled in the Women's Circle of Health Study were genotyped. Pathway analyses were conducted using the adaptive rank truncated product (ARTP) method, with pARTP ≤ 0.10 as significant. Multi-allelic risk scores were created for the ARTP-significant gene(s). Single-SNP and risk score associations were modeled using logistic regression, with false discovery rate (FDR) P-value adjustment. Although single-SNP associations were not significant at pFDR < 0.05, several genes were significant in the ARTP analyses. In AA women, significant ARTP gene-level associations included CDH1 with LN+ (pARTP = 0.10; multi-allelic OR = 1.13, 95%CI 1.07-1.19, pFDR = 0.0003) and SIPA1 with ER- breast cancer (pARTP = 0.10; multi-allelic OR = 1.16, 95%CI 1.02-1.31, pFDR = 0.03). In EA women, MTA2 was associated with overall breast cancer risk (pARTP = 0.004), regardless of ER status, and with LN- disease (pARTP = 0.01). Also significant were SATB1 in ER- (pARTP = 0.03; multi-allelic OR = 1.12, 95%CI 1.05-1.20, pFDR = 0.003) and KISS1 in LN- (pARTP = 0.10; multi-allelic OR = 1.18, 95%CI 1.08-1.29, pFDR = 0.002) analyses. Among LN+ cases, significant ARTP associations were observed for SNAI1, CD82, NME1, and CTNNB1 (multi-allelic OR = 1.09, 95%CI 1.04-1.14, pFDR = 0.001). Our findings suggest that variants in several metastasis genes may affect breast cancer risk by LN or ER status, although verification in larger studies is required. © 2016 Wiley Periodicals, Inc.

Genetic variants in the mTOR pathway and interaction with body size and weight gain on breast cancer risk in African-American and European American women.

PURPOSE: Positive energy imbalance and growth factors linked to obesity promote the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway. As the obesity-breast cancer associations differ between European American (EA) and African-American (AA) women, we investigated genetic variants in the mTOR pathway and breast cancer risk in these two racial groups. METHODS: We examined 400 single-nucleotide polymorphisms (SNPs) in 31 mTOR pathway genes in the Women's Circle of Health Study with 1263 incident breast cancers (645 EA, 618 AA) and 1382 controls (641 EA, 741 AA). Multivariable logistic regression was performed separately within racial groups. Effect modification was assessed for measured body size and weight gain since age 20. RESULTS: In EA women, variants in FRAP1 rs12125777 (intron), PRR5L rs3740958 (synonymous coding), and CDKAL1 rs9368197 (intron) were associated with increased breast cancer risk, while variants in RPTOR rs9900506 (intron) were associated with decreased risk (nominal p-trend for functional and FRAP1 SNPs or p adjusted for correlated test [p ACT] < 0.05). For AA women, variants in RPTOR rs3817293 (intron), PIK3R1 rs7713645 (intron), and CDKAL1 rs9368197 were associated with decreased breast cancer risk. The significance for FRAP1 rs12125777 and RPTOR rs9900506 in EA women did not hold after correction for multiple comparisons. The risk associated with FRAP1 rs12125777 was higher among EAs who had body mass index ≥30 kg/m(2) (odds ratio = 7.69, 95 % CI 2.11-28.0; p-interaction = 0.007) and gained weight ≥35 lb since age 20 (odds ratio = 3.34, 95 % CI 1.42-7.85; p-interaction = 0.021), compared to their counterparts. CONCLUSIONS: The mTOR pathway may be involved in breast cancer carcinogenesis differently for EA and AA women.

Variants of estrogen-related genes and breast cancer risk in European and African American women.

It has been observed previously that compared with women of European ancestry (EA), those of African ancestry (AA) are more likely to develop estrogen receptor (ER)-negative breast cancer, although the mechanisms have not been elucidated. We tested the associations between breast cancer risk and a targeted set of 20 genes known to be involved in estrogen synthesis, metabolism, and response and potential gene-environment interactions using data and samples from 1307 EA (658 cases) and 1365 AA (621 cases) participants from the Women's Circle of Health Study (WCHS). Multivariable logistic regression found evidence of associations with single-nucleotide polymorphisms (SNPs) in the ESR1 gene in EA women (rs1801132, odds ratio (OR)=1.47, 95% CI=1.20-1.80, P=0.0002; rs2046210, OR=1.24, 95% CI=1.04-1.47, P=0.02; and rs3020314, OR=1.43, 95% CI=1.19-1.70, P=0.00009), but not in AA women. The only other gene associated with breast cancer risk was CYP1A2 in AA women (rs2470893, OR=1.42, 95% CI=1.00-2.02, P=0.05), but not in EA women. When stratified by ER status, ESR1 rs1801132, rs2046210, and rs3020314 showed stronger associations in ER-positive than in ER-negative breast cancer in only EA women. Associations with the ESR1 SNPs in EA women also appeared to be stronger with longer endogenous estrogen exposure or hormonal replacement therapy use. Our results indicate that there may be differential genetic influences on breast cancer risk in EA compared with AA women and that these differences may be modified by tumor subtype and estrogen exposures. Future studies with a larger sample size may determine the full contribution of estrogen-related genes to racial/ethnic differences in breast cancer.

Case-only analyses of the associations between polymorphisms in the metastasis-modifying genes BRMS1 and SIPA1 and breast tumor characteristics, lymph node metastasis, and survival.

Lymph node metastases and tumor characteristics predict breast cancer prognosis but correlate imperfectly with likelihood of metastatic relapse. Discovery of genetic polymorphisms affecting metastasis may improve identification of patients requiring aggressive adjuvant therapy to prevent recurrence. We investigated associations between several variants in the BRMS1 and SIPA1 metastasis-modifying genes and lymph node metastases, tumor subtype and grade, recurrence, disease-free survival, and overall survival. This cross-sectional and prospective prognostic analysis included 859 patients who received surgery for incident breast cancer at Roswell Park Cancer Institute, participated in the DataBank and BioRepository shared resource, and had DNA, clinical, and pathology data available for analysis. Genotyping for BRMS1 (rs11537993, rs3116068, and rs1052566) and SIPA1 (rs75894763, rs746429, rs3741378, and rs2306364) polymorphisms was performed using Sequenom(®) iPLEX Gold and Taqman(®) real-time PCR assays. Logistic and Cox proportional hazards regressions were used to estimate odds ratios (OR) and hazard ratios (HR), respectively. BRMS1 rs1052566 heterozygous individuals were more likely to have node-positive tumors (OR = 1.58, 95 % CI 1.13-2.23), although there was no dose-response relationship, and those with at least one variant allele were less likely to have the luminal B subtype (AG + AA: OR = 0.59, 95 % CI 0.36-0.98). BRMS1 rs3116068 was associated with increased likelihood of having the luminal B and the HER2-enriched tumor subtype (P trend = 0.03). Two SIPA1 SNPs, rs746429 and rs2306364, were associated with decreased risk of triple-negative tumors (P trend = 0.04 and 0.07, respectively). Presence of 8 or more risk alleles was associated with an increased likelihood of having a node-positive tumor (OR = 2.14, 95 % CI 1.18-3.36, P trend = 0.002). There were no significant associations with survival. Polymorphisms in metastasis-associated genes may be related to tumor characteristics and lymph node metastasis, but not survival. Future evaluation of metastasis-modifying gene variants is necessary to better understand the biology of metastasis.

Single-nucleotide polymorphisms in DNA repair genes and association with breast cancer risk in the web study.

Base excision repair (BER) and nucleotide excision repair (NER) pathways repair damaged DNA, and polymorphisms in these genes might affect breast cancer susceptibility. We evaluated associations between seven single-nucleotide polymorphisms in four DNA repair genes (ERCC4 rs1799801, XPC rs2227998, rs2228001, rs2228000, OGG1 rs1052133 and XRCC1 rs25487 and rs25486) and breast cancer risk, examining modification by smoking and alcohol consumption, using data from the Western New York Exposures and Breast Cancer Study. Women aged 35-79 years with incident breast cancer (n = 1170) and age- and race-matched controls (n = 2115) were enrolled. Genotyping was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). No significant associations were observed in premenopausal women. Among postmenopausal women, rs25487 and rs25486 (OR = 1.24; 95% CI 1.01-1.51 and OR = 1.23; 95% CI 1.01-1.49, respectively, for combined heterozygous and homozygous variant compared with reference) were associated with increased risk of breast cancer. Postmenopausal women carrying the variant allele of the synonymous XPC polymorphism (rs2227998) were also at borderline significantly increased risk (OR = 1.24; 95% CI 1.01-1.52, heterozygous variant compared with reference; OR = 1.22; 95% CI 1.01-1.48, for combined heterozygous and homozygous variant compared with reference). There was no evidence of genotype-smoking and genotype-alcohol consumption interactions for pre- and postmenopausal women. These results indicate that some of the variants in BER and NER genes may influence risk of postmenopausal breast cancer.