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

Supplementary Tables 1-5 from microRNA miR-196a-2 and Breast Cancer: A Genetic and Epigenetic Association Study and Functional Analysis

e12541 Abstract Background: The KRAS-variant is a functional, microRNA binding site variant that predicts increased cancer risk as well as response to cancer therapies. Here we evaluate the impact of estrogen exposure on breast cancer (BC) cell transformation, as well as BC risk and tumor biology in women with the KRAS-variant. We further study the association of this variant with multiple primary breast cancer (MPBC). Methods: The impact of estrogen withdrawal on transformation in isogenic breast epithelial cell lines with or without the KRAS-variant was studied. Women with BC (n= 1712) and KRAS-variant unaffected controls (n= 80) were recruited, and hormonal exposures, KRAS-variant status, and pathology were compared. Presentation characteristics of MPBC were also studied. Results: Isogenic lines with the KRAS-variant had unique biology, and acute estrogen withdrawal led to oncogenic transformation. Consistent with our biologic findings, KRAS-variant BC patients were significantly more likely to have oophorectomy pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients appeared to have a lower estrogen state than KRAS-variant controls, with a lower BMI (p < 0.001). HRT discontinuation in KRAS-variant BC patients was significantly associated with a diagnosis of triple negative BC. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42-37.87], p < 0.001). Conclusions: Estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant. These women are also significantly more likely to present with a second, independent breast cancer.

18th Meeting of the Irish Society

The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer.

A 30-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma C. H. Chung1,2*, J. W. Lee3, R. J. Slebos4, J. D. Howard1, J. Perez1, H. Kang1, E. J. Fertig1, M. Considine1, J. Gilbert5, B. A. Murphy5, S. Nallur6, T. Paranjape6, R. C. Jordan9, J. Garcia10, B. Burtness7, A. A. Forastiere1 & J. B. Weidhaas6,8 Departments of Oncology; Otolaryngology-Head and Neck Surgery, Sidney Kimmel Cancer Center, Johns Hopkins University, Baltimore; Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston; Department of Cancer Biology; Division of Hematology/Oncology, Department of Medicine, Vanderbilt University, Nashville; Department of Therapeutic Radiology; Section of Medical Oncology, Department of Internal Medicine, Yale University School of Medicine, New Haven; Department of Radiation Oncology, University of California, Los Angeles; Department of Pathology, University of California, San Francisco; Mayo Clinic, Rochester, USA

Ovarian cancer is a major cause of cancer deaths, yet there have been few known genetic risk factors identified, the best known of which are disruptions in protein coding sequences (BRCA1 and 2). Recent findings indicate that there are powerful genetic markers of cancer risk outside of these regions, in the noncoding mRNA control regions. To identify additional cancer-associated, functional non-protein-coding sequence germline variants associated with ovarian cancer risk, we captured DNA regions corresponding to all validated human microRNAs and the 3′ untranslated regions (UTRs) of ~6000 cancer-associated genes from 31 ovarian cancer patients. Multiple single-nucleotide polymorphisms in the 3′UTR of the vascular endothelial growth factor receptor/FLT1, E2F2 and PCM1 oncogenes were highly enriched in ovarian cancer patients compared with the 1000 Genome Project. Sequenom validation in a case–control study (267 cases and 89 controls) confirmed a novel variant in the PCM1 3’UTR is significantly associated with ovarian cancer (P=0.0086). This work identifies a potential new ovarian cancer locus and further confirms that cancer resequencing efforts should not ignore the study of noncoding regions of cancer patients.

While cancer is a serious health issue, there are very few genetic biomarkers that predict predisposition, prognosis, diagnosis, and treatment response. Recently, sequence variations that disrupt microRNA (miRNA)-mediated regulation of genes have been shown to be associated with many human diseases, including cancer. In an early example, a variant at one particular single nucleotide polymorphism (SNP) in a let-7 miRNA complementary site in the 3' untranslated region (3' UTR) of the KRAS gene was associated with risk and outcome of various cancers. The KRAS oncogene is an important regulator of cellular proliferation, and is frequently mutated in cancers. To discover additional sequence variants in the 3' UTR of KRAS with the potential as genetic biomarkers, we resequenced the complete region of the 3' UTR of KRAS in multiple non-small cell lung cancer and epithelial ovarian cancer cases either by Sanger sequencing or capture enrichment followed by high-throughput sequencing. Here we report a comprehensive list of sequence variations identified in cases, with some potentially dysregulating expression of KRAS by altering putative miRNA complementary sites. Notably, rs712, rs9266, and one novel variant may have a functional role in regulation of KRAS by disrupting complementary sites of various miRNAs, including let-7 and miR-181.

A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) which disrupts microRNA regulation has previously been associated with altered patient outcome and drug sensitivity in various cancers. Our study suggests this KRAS-variant is a potential predictive biomarker for poor platinum response in recurrent/metastatic head and neck squamous cell carcinoma patients.A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings.KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients.NCT00503997, NCT00425750, NCT00003809.

BACKGROUND:A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).PATIENTS AND METHODS:We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings.RESULTS:KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).CONCLUSIONS:The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients.CLINICAL TRIAL REGISTRATION NUMBERS:NCT00503997, NCT00425750, NCT00003809.

The KRAS-variant is a germline single nucleotide polymorphism (SNP) within the 3′ UTR of the KRAS gene predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA (miRNA). The KRAS-variant is associated with increased risk of various cancers, including lung cancer, ovarian cancer and triple-negative breast cancer, and is associated with altered tumor biology in head and neck cancer, colon cancer and melanoma. To better understand the molecular pathways that may be regulated or affected by the presence of the KRAS-variant allele in cancer cells, we examined its prevalence in the NCI-60 panel of cell lines and sought to identify common features of the cell lines that carry the variant allele. This study provides a step forward towards understanding the molecular and pathological significance of the KRAS-variant.

Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk.