The vitamin D receptor gene as a determinant of survival in pancreatic cancer patients: Genomic analysis and experimental validation

Purpose: Advanced pancreatic cancer is a highly refractory disease almost always associated with survival of little more than a year. New interventions based on novel targets are needed. We aim to identify new genetic determinants of overall survival (OS) in patients after treatment with gemcitabine using genome-wide screens of germline DNA. We aim also to support these findings with in vitro functional analysis. Patients and methods: Genome-wide screens of germline DNA in two independent cohorts of pancreatic cancer patients (from the Cancer and Leukemia Group B (CALGB) 80303 and the Mayo Clinic) were used to select new genes associated with OS. The vitamin D receptor gene (VDR) was selected, and the interactions of genetic variation in VDR with circulating vitamin D levels and gemcitabine treatment were evaluated. Functional effects of common VDR variants were also evaluated in experimental assays in human cell lines. Results: The rs2853564 variant in VDR was associated with OS in patients from both the Mayo Clinic (HR 0.81, 95% CI 0.70–0.94, p = 0.0059) and CALGB 80303 (HR 0.74, 0.63–0.87, p = 0.0002). rs2853564 interacted with high pre-treatment levels of 25-hydroxyvitamin D (25(OH)D, a measure of endogenous vitamin D) (p = 0.0079 for interaction) and with gemcitabine treatment (p = 0.024 for interaction) to confer increased OS. rs2853564 increased transcriptional activity in luciferase assays and reduced the binding of the IRF4 transcription factor. Conclusion: Our findings propose VDR as a novel determinant of survival in advanced pancreatic cancer patients. Common functional variation in this gene might interact with endogenous vitamin D and gemcitabine treatment to determine improved patient survival. These results support evidence for a modulatory role of the vitamin D pathway for the survival of advanced pancreatic cancer patients.</p

Refining tumor-associated aneuploidy through ‘genomic recoding’ of recurrent DNA copy number aberrations in 150 canine non-Hodgkin lymphomas

Identification of the genomic regions most intimately associated with non-Hodgkin's lymphoma (NHL) pathogenesis is confounded by the genetic heterogeneity of human populations. We hypothesize that the restricted genetic variation of purebred dogs, combined with the contrasting architecture of the human and canine karyotypes, will increase the penetrance of fundamental NHL-associated chromosomal aberrations in both species. We surveyed non-random aneuploidy in 150 canine NHL cases, revealing limited genomic instability compared to their human counterparts and no evidence for CDKN2A/B deletion in canine B-cell NHL. ‘Genomic recoding’ of canine NHL data into a ‘virtual human’ chromosome format showed remarkably few regions of copy number aberration (CNA) shared between both species; restricted to regions of dog chromosomes 13 and 31, and human chromosomes 8 and 21. Our data suggest that gene discovery in NHL may be enhanced through comparative studies exploiting the less complex association between CNAs and tumor pathogenesis in canine patients

FLT3 mutations in canine acute lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit <it>FLT3 </it>ITD mutations.</p> <p>Methods</p> <p>We molecularly characterized <it>FLT3 </it>mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via <it>in vitro </it>proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.</p> <p>Results</p> <p>The canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have <it>FLT3 </it>ITD mutations and <it>FLT3 </it>mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the <it>FLT3 </it>mutation. Finally, western blots were used to confirm the conserved downstream mediators of <it>FLT3 </it>activating mutations.</p> <p>Conclusions</p> <p>These results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.</p

Variation in the Glucose Transporter gene <i>SLC2A2 </i>is associated with glycaemic response to metformin

Metformin is the first-line antidiabetic drug with over 100 million users worldwide, yet its mechanism of action remains unclear1. Here the Metformin Genetics (MetGen) Consortium reports a three-stage genome-wide association study (GWAS), consisting of 13,123 participants of different ancestries. The C allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (P = 6.6 × 10−14) greater metformin-induced reduction in hemoglobin A1c (HbA1c) in 10,577 participants of European ancestry. rs8192675 was the top cis expression quantitative trait locus (cis-eQTL) for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Among obese individuals, C-allele homozygotes at rs8192675 had a 0.33% (3.6 mmol/mol) greater absolute HbA1c reduction than T-allele homozygotes. This was about half the effect seen with the addition of a DPP-4 inhibitor, and equated to a dose difference of 550 mg of metformin, suggesting rs8192675 as a potential biomarker for stratified medicine

Cis eQTL analysis of SLC2A2 in human liver tissue

A three-stage genome wide association study (GWAS) reports the finding of the C-allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (p=6.6x10^-14) greater metformin induced HbA1c reduction in 10,577 participants of European ancestry (doi:10.1038/ng.3632).  The SNP, rs8192675 is the top cis-eQTL for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Here, we deposit the SLC2A2 transcript levels (see Supplemental Table A) in 583 human liver samples and their genotypes within the SLC2A2 region (see Supplemental Table B). The data here are referred to as data set 4 in the published GWAS (doi:10.1038/ng.3632)

The vitamin D receptor gene as a determinant of survival in pancreatic cancer patients: Genomic analysis and experimental validation.

PURPOSE:Advanced pancreatic cancer is a highly refractory disease almost always associated with survival of little more than a year. New interventions based on novel targets are needed. We aim to identify new genetic determinants of overall survival (OS) in patients after treatment with gemcitabine using genome-wide screens of germline DNA. We aim also to support these findings with in vitro functional analysis. PATIENTS AND METHODS:Genome-wide screens of germline DNA in two independent cohorts of pancreatic cancer patients (from the Cancer and Leukemia Group B (CALGB) 80303 and the Mayo Clinic) were used to select new genes associated with OS. The vitamin D receptor gene (VDR) was selected, and the interactions of genetic variation in VDR with circulating vitamin D levels and gemcitabine treatment were evaluated. Functional effects of common VDR variants were also evaluated in experimental assays in human cell lines. RESULTS:The rs2853564 variant in VDR was associated with OS in patients from both the Mayo Clinic (HR 0.81, 95% CI 0.70-0.94, p = 0.0059) and CALGB 80303 (HR 0.74, 0.63-0.87, p = 0.0002). rs2853564 interacted with high pre-treatment levels of 25-hydroxyvitamin D (25(OH)D, a measure of endogenous vitamin D) (p = 0.0079 for interaction) and with gemcitabine treatment (p = 0.024 for interaction) to confer increased OS. rs2853564 increased transcriptional activity in luciferase assays and reduced the binding of the IRF4 transcription factor. CONCLUSION:Our findings propose VDR as a novel determinant of survival in advanced pancreatic cancer patients. Common functional variation in this gene might interact with endogenous vitamin D and gemcitabine treatment to determine improved patient survival. These results support evidence for a modulatory role of the vitamin D pathway for the survival of advanced pancreatic cancer patients