Child, J. Anthony

Abstract There is a high background risk of venous thrombotic events (VTE) in patients with Multiple Myeloma (MM) and this is significantly increased following treatment with Thalidomide. The risk may be mediated by the production of a prothrombotic state, via an angiopathic mechanism, or alternatively it may arise from the release of prothrombotic factors from dying myeloma cells targeted by the treatment. No unifying evidence of a prothrombotic state has been demonstrated to date and it remains important to understand the mechanism underlying VTEs in order to prevent them effectively. Inherited genetic variants in the form of SNPs can affect the behaviour of clotting factors. These SNPs are functional and well characterised in the coagulation system and we have used them to analyse the mechanism underlying thalidomide induced VTE. As part of the Bank On A Cure TM project we have carried out an analysis of SNPs in 11 genes: GPIIA, ITGA, MTHFR, MTR, PPARG2, TFP1, VCAM, factor V, HABP2, prothrombin and THDB in a case series of 90 patients treated with thalidomide, with and without DVTs. This study showed a significant association with MTHFR and factor V polymorphisms. In order to validate and extend these initial observations we carried out an analysis of a second nested case control study using 131 Myeloma patients from within the MRC Myeloma IX trial, comprising 47 cases with a VTE and 94 age and sex matched controls exposed to the same treatment. We used a SNPlex panel containing 16 functional venous thrombosis associated SNPs, all of these functional polymorphisms have reported mechanisms and biology. We looked at polymorphisms in the genes: fibrinogen A (Ala331Thr), B (-455G>A, Lys478Arg) and G polypeptides (His140Tyr); Coagulation factors V(Arg534Gln), VIII (Glu1260Asp), IX(Ala194Thr), XI (Phe339Cys) and XII(-4G>T); PROCR(Gly219Ser); prothrombin(19911A>G ,20210G>A); MTHFR(Glu429Ala, Ala222Val); Serpine1(-844G>A) and TFPI(Met292Val). We have typed our selected SNPs using the SNPlex™ Genotyping System, which is based on the oligonucleotide ligation/PCR assay with a universal fluorescently labeled ZipChute™ probe detection. The probes are hybridized to complementary ZipCode™ sequences that are part of genotype-specific amplicons. Probes are eluted and detected on a Applied Biosystems 3130x DNA Analyser. This approach is an attractive alternative to existing genotyping methodologies, as it requires only three unlabeled probes per SNP, and consumes very little gDNA. The data analysis on the polymorphisms frequencies is ongoing to examine associations of thalidomide treatment with VTE events and will be presented. These datasets will allow a better definition of the pathogenesis of thalidomide related VTE, so that we can intervene therapeutically in a rational fashion.

AbstractA venous thromboembolism (VTE) with the subsequent risk of pulmonary embolism is a major concern in the treatment of patients with multiple myeloma with thalidomide. The susceptibility to developing a VTE in response to thalidomide therapy is likely to be influenced by both genetic and environmental factors. To test genetic variation associated with treatment related VTE in patient peripheral blood DNA, we used a custom-built molecular inversion probe (MIP)–based single nucleotide polymorphism (SNP) chip containing 3404 SNPs. SNPs on the chip were selected in “functional regions” within 964 genes spanning 67 molecular pathways thought to be involved in the pathogenesis, treatment response, and side effects associated with myeloma therapy. Patients and controls were taken from 3 large clinical trials: Medical Research Council (MRC) Myeloma IX, Hovon-50, and Eastern Cooperative Oncology Group (ECOG) EA100, which compared conventional treatments with thalidomide in patients with myeloma. Our analysis showed that the set of SNPs associated with thalidomide-related VTE were enriched in genes and pathways important in drug transport/metabolism, DNA repair, and cytokine balance. The effects of the SNPs associated with thalidomide-related VTE may be functional at the level of the tumor cell, the tumor-related microenvironment, and the endothelium. The clinical trials described in this paper have been registered as follows: MRC Myeloma IX: ISRCTN68454111; Hovon-50: NCT00028886; and ECOG EA100: NCT00033332.