Ayse Latif

CYP2D6 Genotype and Adjuvant Tamoxifen: Meta‐Analysis of Heterogeneous Study Populations

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta‐analysis on data from 4,973 tamoxifen‐treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor–positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease–free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow‐up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Breast cancer susceptibility variants alter risks in familial disease

Background Recent candidate and genome-wide association studies have identified variants altering susceptibility to breast cancer. Objective To establish the relevance of these variants to breast cancer risk in familial breast cancer cases both with and without BRCA1 or BRCA2 (BRCA1/2) mutations. Methods A cohort of unrelated individuals with breast cancer due to the presence of either BRCA1 (121) or BRCA2 mutations (109) and individuals with familial breast cancer not due to BRCA1/2 mutations (722) were genotyped using Taqman SNP Genotyping Assays. Allele frequencies were compared with an ethnically and gender-matched group (436). Results A synonymous variant (Ser51) in TOX3 (previously TNRC9) was associated with an increased risk of breast cancer (OR=1.82, p<0.001) in BRCA2 mutation carriers. The associations for FGFR2 (OR=1.20, p=0.046), TOX3 (OR=1.5, p<0.001), MAP3K1 (OR=1.26 p=0.03), CASP8 (OR=0.73 p=0.02) and the chromosome 8-associated SNP (OR=1.31, p=0.004) were replicated in individuals without BRCA1/2 mutations. In addition, homozygote carriers of MAP3K1 variants were shown to have a significantly lower Manchester Score (mean 13.8–17.6, p=0.003), whereas individuals carrying one or two copies of the FGFR2 variant had a higher Manchester Score (mean 17.5–17.9, p=0.01). Conclusions This study confirms that susceptibility variants in FGFR2, TOX3 and MAP3K1 and on chromosome 8q are all associated with increased risk of cancer in individuals with a family history of breast cancer, whereas CASP8 is protective in this context. The level of risk is dependent on the strength of the family history and the presence of a BRCA1/2 mutation and contributes to the understanding of the use of these variants in clinical risk prediction.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Background: Recently, we demonstrated that the glycoprotein opticin is anti-angiogenic. Here, the underpining mechanism is explored. Results: By binding to collagen, opticin competitively inhibits integrin-mediated endothelial cell adhesion. Conclusion: Opticin inhibits angiogenesis by weakening endothelial cell adhesion to the surrounding extracellular matrix. Significance: Elucidating the regulatory mechanisms of angiogenesis is important for understanding pathology and drug discovery. Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and MatrigelTM. We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α1β1 and α2β1 integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ∼2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ∼5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750–1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ∼650 years ago, and into the Iraqi–Jewish community ∼450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.

Fine-Mapping CASP8 Risk Variants in Breast Cancer

Background: Multiple genome-wide and candidate gene association studies have been conducted in search of common risk variants for breast cancer. Recent large meta analyses, consolidating evidence from these studies, have been consistent in highlighting the caspase-8 (CASP8) gene as important in this regard. To define a risk haplotype and map the CASP8 gene region with respect to underlying susceptibility variant/s, we screened four genes in the CASP8 region on 2q33-q34 for breast cancer risk. Methods: Two independent data sets from the United Kingdom and the United States, including 3,888 breast cancer cases and controls, were genotyped for 45 tagging single nucleotide polymorphisms (tSNP) in the expanded CASP8 region. SNP and haplotype association tests were carried out using Monte Carlo-based methods. Results: We identified a three-SNP haplotype across rs3834129, rs6723097, and rs3817578 that was significantly associated with breast cancer (P < 5 × 10−6), with a dominant risk ratio and 95% CI of 1.28 (1.21–1.35) and frequency of 0.29 in controls. Evidence for this risk haplotype was extremely consistent across the two study sites and also consistent with previous data. Conclusion: This three-SNP risk haplotype represents the best characterization so far of the chromosome upon which the susceptibility variant resides. Impact: Characterization of the risk haplotype provides a strong foundation for resequencing efforts to identify the underlying risk variant, which may prove useful for individual-level risk prediction, and provide novel insights into breast carcinogenesis. Cancer Epidemiol Biomarkers Prev; 21(1); 176–81. ©2011 AACR.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP.

Background: Pancreatic cancer cells exhibit up-regulated glycolysis (the “Warburg effect”). Results: Reversing the Warburg phenotype protects pancreatic cancer cells from glycolytic inhibitor-induced ATP depletion, plasma membrane calcium pump (PMCA) inhibition, and [Ca2+]i overload. Conclusion: Glycolytic ATP is critical for PMCA function in pancreatic cancer. Significance: The glycolytic dependence of the PMCA may represent a novel therapeutic target in pancreatic cancer. Evidence suggests that the plasma membrane Ca2+-ATPase (PMCA), which is critical for maintaining a low intracellular Ca2+ concentration ([Ca2+]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca2+]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca2+]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca2+]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

Lack of caveolin-1 (P132L) somatic mutations in breast cancer

We were interested to read Patani et al.’s article [1] where they reported that they did not detect the p.P132L somatic mutation in CAV1 in a series of 240 breast tumors. We have also undertaken a study to determine the prevalence of the CAV1 p.P132L mutation in a series of 537 frozen tissue samples collected at the time of primary surgery from women with stages I, II, or III breast cancer from Manchester, UK between 1989 and 1998 (Table 1). Clinical data was collected by a comprehensive retrospective case note review to supplement prospectively collected data. The study was approved by local research ethics committee (08/H1006/77). Genomic DNA was extracted from fresh-frozen tissues using the EZ1 DNA tissue kit on EZ1 biorobot (Qiagen, Valencia, CA). CAV1 p.P132L mutation status was not identified by Pyrosequencing (Qiagen) in genomic DNA extracted from 537 tumor samples (call rate: 99.3%) (for full method see Supplementary material). To confirm the absence of p.P132L, samples with the highest allele quantification were analyzed by both direct DNA sequencing and restriction fragment length polymorphism analysis with the Nla-III restriction endonuclease. No mutations were detected. Furthermore, to ensure that mutations were not present at a level below the threshold of test sensitivity, we used laser capture microscopy (LCM) in a subset of 15 specimens from patients theoretically enriched for p.P132L mutation (ER-positive and well-differentiated tumors) [2]. DNA extracted from isolated tumor cells was sequenced in both orientations and no mutation was detected. The existence of CAV1 mutations in human breast cancers is controversial. In 2001 Hayashi et al. [3] reported that up to 16% of breast cancer tumor samples (mainly in ‘‘scirrhous’’ carcinomas) harbor a somatic mutation, p.P132L, in CAV1. Subsequent studies demonstrated that the p.P132L mutation was associated with ER-positive, but not ER-negative, breast tumors [4, 5]. However, other groups have not confirmed the existence of this somatic mutation in human breast cancers [6, 7]. Discordant results may reflect differences in patient populations, source of tumor materials and methods used [8]. DNA sequencing of formalin-fixed paraffin-embedded samples is fraught with difficulties, owing to the high prevalence of artefacts, with up to one artefactual mutation per 500 bases has been reported [9]. Importantly, in our study fresh-frozen samples were used whereas formalin-fixed paraffin-embedded samples were screened by groups where mutations were Electronic supplementary material The online version of this article (doi:10.1007/s10549-012-1981-0) contains supplementary material, which is available to authorized users.

Expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is increased in the endometrium of women with endometrial cancer and women with Polycystic Ovary Syndrome

Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC).