Betül Yesilyurt

VEGF pathway genetic variants as biomarkers of treatment outcome with bevacizumab: an analysis of data from the AViTA and AVOREN randomised trials

BACKGROUND No biomarkers that could guide patient selection for treatment with the anti-VEGF monoclonal antibody bevacizumab have been identified. We assessed whether genetic variants in the VEGF pathway could act as biomarkers for bevacizumab treatment outcome. METHODS We investigated DNA from white patients from two phase 3 randomised studies. In AViTA, patients with metastatic pancreatic adenocarcinoma were randomly assigned to receive gemcitabine and erlotinib plus either bevacizumab or placebo. In AVOREN, patients with metastatic renal-cell carcinoma were randomly assigned to receive interferon alfa-2a plus either bevacizumab or placebo. We assessed the correlation of 138 SNPs in the VEGF pathway with progression-free survival and overall survival in a subpopulation of patients from AViTA. Significant findings were confirmed in a subpopulation of patients from AVOREN and functionally studied at the molecular level. FINDINGS We investigated DNA of 154 patients from AViTA, of whom 77 received bevacizumab, and 110 patients from AVOREN, of whom 59 received bevacizumab. Only rs9582036, a SNP in VEGF receptor 1 (VEGFR1 or FLT1), was significantly associated with overall survival in the bevacizumab group of AViTA after correction for multiplicity (per-allele hazard ratio [HR] 2·1, 95% CI 1·45-3·06, p=0·00014). This SNP was also associated with progression-free survival (per-allele HR 1·89, 1·31-2·71, p=0·00081) in bevacizumab-treated patients from AViTA. AC and CC carriers of this SNP exhibited HRs for overall survival of 2·0 (1·19-3·36; p=0·0091) and 4·72 (2·08-10·68; p=0·0002) relative to AA carriers. No effects were seen in placebo-treated patients and a significant genotype by treatment interaction (p=0·041) was recorded, indicating that the VEGFR1 locus containing this SNP serves as a predictive marker for bevacizumab treatment outcome in AViTA. Fine-mapping experiments of this locus identified rs7993418, a synonymous SNP affecting tyrosine 1213 in the VEGFR1 tyrosine-kinase domain, as the functional variant underlying the association. This SNP causes a shift in codon usage, leading to increased VEGFR1 expression and downstream VEGFR1 signalling. This VEGFR1 locus correlated significantly with progression-free survival (HR 1·81, 1·08-3·05; p=0·033) but not overall survival (HR 0·91, 0·45-1·82, p=0·78) in the bevacizumab group in AVOREN. INTERPRETATION A locus in VEGFR1 correlates with increased VEGFR1 expression and poor outcome of bevacizumab treatment. Prospective assessment is underway to validate the predictive value of this novel biomarker. FUNDING F Hoffmann-La Roche.

Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks

DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR)-deficient tumors and convey them with resistance to standard therapy. Using whole-genome and whole-exome sequencing, we here confirm an MMR-deficient mutation signature that is distinct from other tumor genomes, but surprisingly similar to germ-line DNA, indicating that a substantial fraction of human genetic variation arises through mutations escaping MMR. Moreover, we identify a large set of recurrent indels that may serve to detect microsatellite instability (MSI). Indeed, using endometrial tumors with immunohistochemically proven MMR deficiency, we optimize a novel marker set capable of detecting MSI and show it to have greater specificity and selectivity than standard MSI tests. Additionally, we show that recurrent indels are enriched for the ‘DNA double-strand break repair by homologous recombination’ pathway. Consequently, DSB repair is reduced in MMR-deficient tumors, triggering a dose-dependent sensitivity of MMR-deficient tumor cultures to DSB inducers. DOI: http://dx.doi.org/10.7554/eLife.02725.001

Investigation of gene‐environment interactions between 47 newly identified breast cancer susceptibility loci and environmental risk factors

A large genotyping project within the Breast Cancer Association Consortium (BCAC) recently identified 41 associations between single nucleotide polymorphisms (SNPs) and overall breast cancer (BC) risk. We investigated whether the effects of these 41 SNPs, as well as six SNPs associated with estrogen receptor (ER) negative BC risk are modified by 13 environmental risk factors for BC. Data from 22 studies participating in BCAC were pooled, comprising up to 26,633 cases and 30,119 controls. Interactions between SNPs and environmental factors were evaluated using an empirical Bayes‐type shrinkage estimator. Six SNPs showed interactions with associated p‐values (pint) <1.1 × 10−3. None of the observed interactions was significant after accounting for multiple testing. The Bayesian False Discovery Probability was used to rank the findings, which indicated three interactions as being noteworthy at 1% prior probability of interaction. SNP rs6828523 was associated with increased ER‐negative BC risk in women ≥170 cm (OR = 1.22, p = 0.017), but inversely associated with ER‐negative BC risk in women <160 cm (OR = 0.83, p = 0.039, pint = 1.9 × 10−4). The inverse association between rs4808801 and overall BC risk was stronger for women who had had four or more pregnancies (OR = 0.85, p = 2.0 × 10−4), and absent in women who had had just one (OR = 0.96, p = 0.19, pint = 6.1 × 10−4). SNP rs11242675 was inversely associated with overall BC risk in never/former smokers (OR = 0.93, p = 2.8 × 10−5), but no association was observed in current smokers (OR = 1.07, p = 0.14, pint = 3.4 × 10−4). In conclusion, recently identified BC susceptibility loci are not strongly modified by established risk factors and the observed potential interactions require confirmation in independent studies.

Somatic copy number alterations predict response to platinum therapy in epithelial ovarian cancer

OBJECTIVE Platinum resistance remains an obstacle in the treatment of epithelial ovarian cancer (EOC). The goal of this study was to profile EOCs for somatic copy number alterations (SCNAs) as predictive markers of platinum response. METHODS SCNAs were assessed in a discovery (n=86) and validation cohort (n=115) of high risk stage I or stage II-IV EOCs using high-resolution SNP arrays. ASCAT and GISTIC identified all significantly overrepresented amplified or deleted chromosomal regions. Cox regression and univariate analysis assessed which SCNAs correlated with overall survival (OS), progression-free survival (PFS), platinum-free interval (PFI) and platinum response. Relevant SCNAs were also assessed in a pooled analysis involving both cohorts and published SCNA data from The Cancer Genome Atlas (TCGA; n=227). RESULTS We identified 53 regions to be significantly overrepresented in EOC. Of these, 6 were associated with OS, PFS or PFI in the discovery cohort at P<0.05. In the validation cohort, amplifications of chromosomal region 14q32.33, which contains AKT1 as a potential driver gene, also correlated with OS (OR=1.670; P=0.018). In a pooled analysis of 428 tumors, involving the discovery, validation and TCGA cohorts, 14q32.33 amplifications significantly reduced OS, PFS and PFI (HR=2.69, P=1.7×10(-4); HR=1.82, P=1.9×10(-2) and HR=1.80, P=2.2×10(-2) respectively). Moreover, AKT1 mRNA expression correlated with the number of chromosomal copies of the 14q32.33 region (P=2.8×10(-11);R(2)=0.26). CONCLUSIONS We established that amplifications in 14q32.33 were associated with reduced OS, PFS, PFI and platinum resistance in three independent cohorts, suggesting that AKT1 amplifications act as a potentially predictive marker for EOC treated with platinum-based chemotherapy.

Abstract 2006: Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR) deficient tumors and convey them with different prognostic and therapeutic outcome upon chemotherapy. Using whole-genome and -exome sequencing, we here characterize the somatic mutation patterns underlying MMR-deficient tumors. In particular, we show that mutation frequencies are >30-fold increased relative to MMR-proficient tumors: insertion-deletion (indel) mutations are largely confined to homopolymers, whereas substitutions mainly consist of single basepair transitions located near indels. Since exonic indels result in loss-of-function frameshift mutations, indels were less frequent in the exome due to negative selection. However, a small fraction of exonic indels was positively selected and occurred as hotspot mutations, preferentially affecting the ‘DNA double-strand break (DSB) repair by homologous recombination (HR)’ pathway (on average, each MMR-deficient tumor contained 2.6 indels in this pathway). We quantified DSB repair by immunofluorescent staining of RAD51 foci in 8 MMR-deficient and 4 MMR-proficient tumor cultures in the presence of the PARP inhibitor olaparib. We did not observe a difference in RAD51 foci formation between MMR-deficient and MMR-proficient tumor cultures in the absence of olaparib (10±2% of cells versus 13±2% showed RAD51 foci formation, P=0.74). Upon 10μM olaparib exposure, MMR-deficient tumor cultures accumulated less RAD51-positive foci (19±3% in MMR-deficient versus 37±4% in MMR-proficient cells; P=0.02), thereby confirming that these tumors were at least partly deficient in DNA DSB repair by HR. We also investigated the degree of unrepaired DNA damage by H2AX immunofluorescence and olaparib drastically increased the number of H2AX foci in all cells regardless of their MMR status. Furthermore, MMR-deficient cultures exhibited a dose-dependent reduction in proliferation under olaparib, whereas MMR-proficient cells did not respond (P<0.001 by repeated measurement). On average, 1, 3 and 10μM of olaparib decreased proliferation of MMR-deficient cells by respectively 15%, 20% and 42% (P=0.02, P<0.001 and P<0.001, respectively versus untreated cells), whereas no effect was seen in MMR-proficient tumors (P=NS for all concentrations of olaparib). Finally, high-throughput profiling of hotspot mutations also more accurately detected microsatellite instability (MSI) in several cancer types compared to standard diagnostic methods. Sequencing of MMR-deficient tumors thus identified an unexpected synthetically lethal interaction between the major DNA repair pathways, offering novel treatment opportunities for MMR-deficient tumors. Citation Format: Betul T. Yesilyurt, Hui Zhao, Xavier Sagaert, Lieve Coenegrachts, Zeynep Kalender, Diego Garcia, Kim De Keersmaker, Gert Matthijs, Bernard Thienpont, Stein Aerts, Jan Cools, Frederic Amant, Diether Lambrechts. Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2013-2006

Abstract LB-447: Functional validation of a genetic locus in the VEGFR-1 tyrosine kinase (TK) domain as a predictive marker for bevacizumab

Background There are no validated biomarkers to guide patient selection for treatment with the anti-VEGF antibody bevacizumab. We recently identified a locus, consisting of 4 SNPs in the VEGFR-1 TK domain, that correlated with progression-free (PFS) and overall survival (OS) of bevacizumab-treated pancreatic cancer patients in the AViTA phase III trial. The causal SNP underlying this association and its effect on VEGFR-1 expression or function are not yet known. Methods We conducted a fine-mapping study using data from the 1000 Genomes Project to identify the causal variant in this locus. The candidate SNP was functionally validated by studying effects on VEGFR-1 RNA (RT-PCR) or protein (ELISA) expression. Transcription and translation were measured using transient transfection and rabbit reticulocyte assays. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy newborns. Association of causal SNPs was replicated in bevacizumab-treated renal cancer patients from the AVOREN phase III trial via Sequenom genotyping. Results Fine-mapping identified 48 common SNPs linked to one of the 4 SNPs in the VEGFR-1 locus. In silico functional prediction revealed that only one SNP (rs7993418) was located in exon 28. No other SNPs were located in exons or functionally important regions. Remarkably, rs7993418 is a synonymous SNP introducing a shift in codon usage (TAT to TAC codon) at an evolutionary conserved tyrosine (Tyr1213Tyr). In vitro transcription/translation of VEGFR-1 cDNAs carrying either the wildtype TAT or mutant TAC codon revealed that both constructs transcribe equal amounts of mRNA, whereas a 27% increased VEGFR-1 protein expression was found for TAC versus TAT-carrying constructs (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-447. doi:1538-7445.AM2012-LB-447