Supplementary Table 1 from A KRAS-Variant in Ovarian Cancer Acts as a Genetic Marker of Cancer Risk

Supplementary Figure 2 from A KRAS-Variant in Ovarian Cancer Acts as a Genetic Marker of Cancer Risk

Background We previously identified a functional variant in a let-7 microRNA (miRNA) complementary site in the 3'-untranslated region of the KRAS oncogene (rs61764370) which is associated with cancer. We aimed to investigate the association of this KRAS variant with breast cancer and tumour biology.Methods We assessed frequency distributions of the KRAS variant in 415 patients with histologically confirmed breast cancer and 457 controls from Connecticut, USA (study group 1) and association of this variant with breast-cancer subtypes in 690 Irish women with known oestrogen receptor (ER), progesterone receptor (PR), and HER2 statuses, and 360 controls (study group 2). We pooled data for study groups 1 and 2 with a cohort of 140 women with triple-negative breast cancer and 113 controls to assess the association of the KRAS variant with triple-negative breast cancer risk, and genome-wide mRNA and specific miRNA expression in patients with triple-negative breast cancer.Findings Although frequency distributions of the KRAS variant in study group 1 did not differ between all genotyped individuals, eight (33%) of 24 premenopausal women with ER/PR-negative cancer had the KRAS variant, compared with 27 (13%) of 201 premenopausal controls (p=0.015). In study group 2, the KRAS variant was significantly enriched in women with triple-negative breast cancer (19 [21%] of 90 cases) compared with 64 (13%) of 478 for luminal A, 13 (15%) of 87 for luminal B, and two (6%) of 35 for HER2-positive subgroups (p=0.044). Multivariate analysis in the pooled study groups showed that the KRAS variant was associated with triple-negative breast cancer in premenopausal women (odds ratio 2.307, 95% CI 1.261-4.219, p=0.0067). Gene-expression analysis of triple-negative breast-cancer tumours suggested that KRAS-variant positive tumours have significantly altered gene expression, and are enriched for the luminal progenitor and BRCA1 deficiency signatures. miRNA analysis suggested reduced levels of let-7 miRNA species in KRAS-variant tumours.Interpretation The KRAS variant might be a genetic marker for development of triple-negative breast cancer in premenopausal women, and altered gene and miRNA expression signatures should enable molecular and biological stratification of patients with this subgroup of breast cancer.

Recently, a variant allele in the 3′UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as ovarian cancer, and was most enriched in ovarian cancer patients from hereditary breast and ovarian cancer families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, we have genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, we also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Importantly, results from the current study suggest that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers.

Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3' untranslated region (3'UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3'UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3'UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3'UTR variants in a large population of controls and breast cancer patients (n = 221) with known breast cancer subtypes and ethnicities. We identified three 3'UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p = 0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3'UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients = 0.78%). These rare BRCA1 haplotypes and 3'UTR SNPs may represent new genetic markers of breast cancer risk.

The KRAS variant is a functional microRNA-binding disrupting variant, discovered through a targeted gene approach, which is a hypothesis-driven investigation of a planned region of the genome to find functional variants. This technique differs from the approach used in genome-wide association studies, in which a large number of non-functional tagging single nucleotide polymorphisms are tracked throughout the genome on the basis of coverage. We regard the application of genome-wide association approaches and datasets to functional variants, as proposed by Pharoah and colleagues, as invalid. Association between KRAS rs61764370 and triple-negative breast cancer—a false positive?In their study,1 Paranjape and colleagues suggest an association between a single nucleotide polymorphism, rs61764370, in the KRAS 3′-untranslated region and risk of triple-negative breast cancer in women younger than 52 years. However, this association should not be regarded as confirmed. Full-Text PDF

MicroRNAs (miRNAs) are well established as global gene regulators and thus, slight alterations in miRNA levels as well as their ability to regulate their targets may cause important cellular changes leading to cancer risk. 3´ untranslated region (UTR) miRNA binding site single nucleotide polymorphisms (SNPs) have added another layer of possible genetic variation involved in the complex process of oncogenesis. Identifying these key genetically inherited effectors of miRNA functioning has improved our understanding of the complexity of disease. Interest in the field has grown rapidly in only the last 5 years, with several studies reporting on the role of 3´UTR binding site SNPs as genetic markers of increased cancer susceptibility, as well as biomarkers of cancer type, outcome and response to therapy. Currently, there are numerous known miRNA binding site SNPs associated with multiple cancer subtypes.

Downlo rian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advanced e at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogene tered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solid s. In this study, we investigated a hypothesized association between an increased risk of OC and a varlele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site in ncogene. Specimens obtained were tested for the presence of the KRAS-variant from nonselected OC ts in three independent cohorts, two independent ovarian case-control studies, and OC patients with tary breast and ovarian cancer syndrome (HBOC) as well as their family members. Our results indicate e KRAS-variant is associated with more than 25% of nonselected OC cases. Further, we found that it is a r for a significant increased risk of developing OC, as confirmed by two independent case-control analastly, we determined that the KRAS-variant was present in 61% of HBOC patients without BRCA1 or mutations, previously considered uninformative, as well as in their family members with cancer. Our gs strongly support the hypothesis that the KRAS-variant is a genetic marker for increased risk of defindin veloping OC, and they suggest that the KRAS-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities. Cancer Res; 70(16); 6509–15. ©2010 AACR.

Abstract The purpose of our study is to determine the role of a previously identified novel germline polymorphism in the let-7 miRNA binding site in the 3’UTR of the KRAS oncogene, referred to as the KRAS-variant, as a biomarker of breast cancer risk. We have shown that the KRAS-variant is a biomarker of increased risk of ovarian cancer for patients from families with Hereditary Breast and Ovarian Cancer. Thus, in this study, we tested the association of this biomarker with breast cancer using: i) A BC case-control recruited at Yale, ii) A cohort of Irish Caucasian women diagnosed with known BC subtypes and iii) A cohort of triple negative (TN) BC patients at Yale, all with known demographics and clinical variables. We assayed for the prevalence of the KRAS-variant by SNP genotyping to determine its association with BC. We compared the prevalence of the KRAS-variant in different BC subtypes and evaluated the association with age and ethnicity. MiRNA profiling was performed on the subjects in the TN BC cohort using Taqman low density miRNA microarrays. We analyzed the miRNA expression patterns within the TN BC cohort and also compared the miRNA expression patterns in the KRAS-variant positive and negative patients also evaluating the association with the age and ethnicity using linear modeling methods. We are currently performing gene expression analysis on the same TN BC patients that we have miRNA expression analysis and KRAS-variant testing completed on. We will compare gene expression patterns with the unique miRNA signatures in the KRAS-variant positive versus negative cases. We plan to use network programs to evaluate similarities in gene expression by miRNA, and also evaluate known targets of misexpressed miRNAs and determine their levels. In the BC case-control the KRAS-variant was found to be significantly associated with the ER/PR- BC patients below 51 yrs (33% vs. 10.5% for controls and ER/PR+). In the Irish Caucasian BC cohort the KRAS-variant was most prevalent in the TN BC patients (21.7% vs. 12-14% for Luminal A and Bs) below 51yrs. Consistently in the TN BC cohort the KRAS-variant was most prevalent in non-African American (AA) women below 51 yrs (33% vs. 21.5% in women of all races below 51 yrs). Through miRNA profiling we found that miRNA signatures vary across the TN BC patients, suggesting the existence of different groups within the TN cohort itself. The young non-AA TN BC patients appeared to have lower miRNA expression overall and clustered together. We also found that the KRAS-variant is associated with a unique miRNA expression pattern in TN BC patients with the variant compared to those without. A miRNA binding site polymorphism in the 3’UTR of the KRAS gene, the KRAS-variant, is a new biomarker of risk for developing triple negative BC in young non-AA women. In addition, KRAS-variant patient tumors have unique miRNA signatures and preliminary data supports they also have common gene expression differences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3029.

Abstract Ovarian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advanced disease at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogene and altered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solid tumors. In this study, we investigated a hypothesized association between an increased risk of OC and a variant allele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site in this oncogene. Specimens obtained were tested for the presence of the KRAS-variant from nonselected OC patients in three independent cohorts, two independent ovarian case-control studies, and OC patients with hereditary breast and ovarian cancer syndrome (HBOC) as well as their family members. Our results indicate that the KRAS-variant is associated with more than 25% of nonselected OC cases. Further, we found that it is a marker for a significant increased risk of developing OC, as confirmed by two independent case-control analyses. Lastly, we determined that the KRAS-variant was present in 61% of HBOC patients without BRCA1 or BRCA2 mutations, previously considered uninformative, as well as in their family members with cancer. Our findings strongly support the hypothesis that the KRAS-variant is a genetic marker for increased risk of developing OC, and they suggest that the KRAS-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities. Cancer Res; 70(16); 6509–15. ©2010 AACR.

Objectives: The multilaminar structure of the zona forms during oogenesis, therefore, zona properties might reflect follicular development, oocyte cytoplasmic maturation and egg quality. In addition, appropriate thinning of the zona during embryo preimplantation development might be required for successful embryo hatching. We sought to quantitatively characterize the morphology of sub-zonal layers, particularly their thickness, retardance, and degree of irregularity in living human eggs and embryos using the non-invasive Polscope. Design: The Polscope was employed to examine and quantify the properties of the layers of the zona pellucida of eggs prior to ICSI and cleavage stage embryos prior to transfer. Materials and Methods: Eggs and embryos from 29 consenting patients who were undergoing IVF/ICSI were imaged at an equatorial plane using and Axiovert S100TC microscope (0.55 condenser, 40X 0.6 NA objective) (Zeiss, Germany) fitted with the Polscope (CRI Inc, Woburn, MA, USA). Eggs and embryos were placed in 5μl drops of modified HEPES-buffered HTF covered with warm paraffin oil in a WilCo glass bottom petri dish (WPI, Sarasote (Sarasota?), FL) and maintained at 37 ± 0.5°C during examination with a TakaiHit Thermo Plate (Zander, Varo Beach, FL) and objective heater (CRI). Zona parameters were digitally measured at eight octants and compared using the f-test, student?s t-test and ANOVA. Results: Cleavage stage embryos have thinner zonae than both immature and mature eggs primarily due to thinning of the outermost layer. The thicker the zona layer the greater its retardance. Considerable variation exists in the thickness and retardance of zona from eggs/embryos of a cohort. Some individual eggs exhibit highly irregular zona layers. Conclusions: The thickness and organization of the zona of human eggs and embryos varies considerably, can be quantified using the PolScope and may someday provide a non-invasive marker for egg and embryo quality.