ALBERT

Description: Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism.

Description: Background:The relationship between metastatic colorectal cancer (CRC) and venous thromboembolism (VTE) is not well defined in the modern treatment era. Previous population-based studies date back to a period marked by inpatient intravenous heparin therapy and inferior survival due to paucity of therapeutic anti-cancer options, and before the advent of newer therapies including oxaliplatin, irinotecan, and anti-angiogenic treatment with bevacizumab. The objectives of this retrospective study were to examine the impact of multiple putative risk factors on VTE incidence in a large representative modern cohort of older patients with metastatic CRC. Methods:We performed a retrospective analysis of SEER-Medicare data on elderly patients with metastatic CRC diagnosed in 2004-2011. VTE and associated risk factors were analyzed using multivariate Cox proportional hazards models adjusted for sex, age at diagnosis, race, ethnicity, tumor anatomy (left/right/unknown), calendar year of diagnosis, Charlson comorbidity score, location of SEER registry and urban residence, with time-varying covariates for use of cancer therapies. Results:Of 339,778 records, 11,086 metastatic colon cancer cases were identified. 1,338 cases had VTE with a cumulative incidence of 13% at 1 year and 19% at 3 years. The mean age was 77.9 years (range 65-106). 49.7% were women and 83.5% white. 60.5% had a Charlson comorbidity score of zero at diagnosis; 6% had scores of 6-18. Significant predictors of VTE included female sex (Hazard Ratio (HR) 1.22; 95% Confidence Interval (CI) 1.10, 1.36; P

Description: Background: The cumulative incidence of symptomatic venous thromboembolism (VTE) among patients with malignant glioma (MG) is high, with estimates ranging from 20-35% during the course of therapy. VTE is the second leading cause of death among ambulatory patients receiving cancer chemotherapy, with a 2-fold or greater increase in mortality compared to those without VTE, even after adjusting for stage. In patients with MG and VTE the chance of two-year survival is significantly reduced. The development of VTE is also associated with increased morbidity, including increased risk of hospitalization, delays in cancer treatment, and an increased risk of complications including pain, bleeding, and bruising from high dose therapeutic anticoagulation. The economic burden of VTE in cancer patients is estimated at a 2-fold increase in cost. Data regarding VTE burden encompasses all cancer types and additional data is needed to characterize VTE burden in specific cancer types, particularly rarer cancers. The aim of this study was to assess the healthcare burden associated with the development of VTE in patients with MG. Methods: A retrospective chart review of patients with MG at an academic medical center was conducted from 2009-2017. Sixty-seven patients, age 18 years and older, with a histologic diagnosis of MG (WHO grade III-IV) were assessed. The number of office visits, emergency room visits, and inpatient hospitalizations with the associated costs of VTE management and its complications were collected. Using SPSS, linear regression models and descriptive statistics were used to determine the relationship between the development of VTE and healthcare consumption. Results: Of the 67 patients in the study, 18 developed VTE (27%). All patients who developed a VTE were placed on therapeutic anticoagulation. Fifty percent of patients developed complications related to anticoagulation, including gastrointestinal, retroperitoneal and intracranial bleeding events. Two patients required placement of an IVC filter after experiencing a bleeding complication. Patients that developed VTE had an increase in inpatient days (16.6 inpatient days) as compared to their non-VTE counterparts (8.8 inpatient days), (p=0.012). There was increased utilization of the emergency room with an average of 3.94 visits in those patients with VTE as compared to those without (1.84), (p=0.003). A full cost analysis found that the average primary total cost for ED visits and inpatient hospitalizations in patients with VTE was $48,863 while those without VTE averaged $35,948. This represented a 26% increase in the average primary total cost in those patients with VTE. Discussion: The development of VTE in patients with MG increases inpatient admissions days and incurs additional pharmaceutical costs related to anticoagulation. This study represents the first assessment of VTE-associated health care burden specific to primary brain cancer. We confirm that VTE affects a large number of patients with MG (27%) and limits the time spent at home due to increased hospitalizations and emergency room visits. VTE may be a preventable complication and further studies are needed to investigate safe prevention strategies for patients with MG. Disclosures No relevant conflicts of interest to declare.

Description: The storage and release by platelets of the pro-angiogenic protein, vascular endothelial growth factor (VEGF) and the anti-angiogenic protein, endostatin may influence not only wound healing but also tumor angiogenesis and metastasis. The intra-platelet mechanisms and platelet activators that control angiogenic protein release are incompletely elucidated. Previous work demonstrated that the proteinase-activated receptors (PARs), PAR-1 and PAR-4 differentially impact platelet release of these angiogenic proteins (Ma et al., PNAS 102:216). We determined the influence of the adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12 on platelet release of VEGF and endostatin. Minimally altered whole blood from healthy volunteers was incubated with ADP alone [12.5 μM] or in combination with a reversible P2Y1 antagonist [MRS2179 (1.25 mM)] or a reversible P2Y12 antagonist [cangrelor (1mM)]. VEGF and endostatin release was measured with the use of an ELISA assay. ADP-induced activation of platelets increased the concentration of VEGF released by an average of 19.7 +/− 14 ng/mL (p= 0.019) but did not change the concentration of endostatin. Inhibition of the P2Y12 receptor prevented the ADP mediated release of VEGF and decreased VEGF release by 27.9 +/− 14 ng/ml (p=0.005) whereas inhibition of the P2Y1 receptor did not significantly reduce the release of VEGF (p=0.3, mean decrease 14.3 +/− 30 ng/ml) relative to ADP stimulated control. In summary, ADP-induced activation of platelets leads to the release of VEGF but not endostatin. Release of VEGF is attenuated by a P2Y12 but not a P2Y1 receptor antagonist. Accordingly, activation of platelets by ADP appears to be pro-angiogenic and inhibition by a P2Y12 antagonist should attenuate this effect.

Description: Abstract 3590 Genome-wide array or sequencing analyses are powerful tools for identifying genetic aberrations in cancers including leukaemias. However, the majority of these aberrations are likely to be random passenger events that do not drive clonal expansion. Currently, it is unknown whether cancers are maintained by a finite set of recurrent mutations similar for each patient or whether and to what extent malignancies are ‘personalised’, and also how molecular disease drivers evolve over time in the context of clinical intervention. The answers to these questions will determine whether future treatment modalities must be tailored according to individual and dynamic cancer characteristics. We hypothesized that differential quantitative high-resolution genome-wide array analysis of sequential samples from the same patients before treatment and at subsequent relapse would have the potential to identify emerging structural abnormalities with relevance to disease progression and/or response to treatment in a given patient. In order to test this hypothesis, we chose B-cell chronic lymphocytic leukaemia (CLL), because of its unique clinical characteristics, as our model to begin to evaluate the potential role of ‘companion diagnostics’ for this condition. We analysed DNA samples of 80 patients with CLL using a 1 million high resolution SNP array. On 34 of them, sequential pre-treatment and relapse samples were available. The raw data was analysed using the OncoSNP analysis tool designed in-house specifically for cancer samples as it enables quantification of copy number alterations (CNA) and copy neutral loss of heterozygosity (cnLOH) based on B-allele frequency plots in complex mixtures. This allows low levels of aberrations to be detected and for mosaic samples to be identified. Results were compared against the data from the Wellcome Trust Case Control Consortium, the DGA and germline DNA in selected cases. Large CNAs (〉1Mb) and cnLOH (〉5Mb) without deletions of 11q22.3 or 17p13.1 were identified in a third of patients. These patients had an intermediate clinical risk score that increased with the number of large CNAs. SNP array demonstrated clonal evolution in 32% of patients in the sequential sample cohort. These consisted of extension of the 13q abnormality (2), loss of the 13q deletion (1), a 10q23.1-q25.1 deletion (1), gain of 2pter-p14 (1), deletion of 2q33.1-q36.3, (1) a heterozygous deletion of 2q37.1 (1), gain of 8q22.2-qter (1), deletion of 8p (1), amplification of 8q (1), deletion of 8q (1), loss of 16p13.3 (2), mosaic deletion of 17q11.2 (1), an expansion of chromosomes carrying a 19p13.2-p13.11 gain and a 19p13.11 loss (1), deletions within 3p (1), conversion of a gain of 12p12.2-q21.31 to a copy neutral loss of heterozygosity (cnLOH) (1), deletion of 17pter-13.1 (1), increased proportion of chromosomes with the 7q33–34 deletion (1), expansion of cnLOH for 20q11.22-qter (1) and an increased number of cells with a deletion of 2q22.2-q24.1 (1) found at relapse. Importantly, most CNAs occurring at relapse were recurrent in the entire cohort implying that these are non-random events that are important in disease progression. Analysis of the minimal deleted region (MDR) of these recurrent and relapse associated CNAs revealed genes important in lymphoid development, such as NFκB2 and TRAP1 found in the alternative NFκB pathway and BLIMP1 involved in B-cell differentiation. There were also recurrent abnormalities in the region coding for SP140 which has been implicated in familial CLL. Known cancer genes were also affected by these recurrent and relapse-associated CNAs such as RND3, RIF1, RFXANK, and RHOT1, which are all members of the RAS pathway family. Using the OncoSNP program it was possible to determine that most of these emerging abnormalities were present in low numbers at diagnosis (fig. 1) suggesting that treatment does not induce the genetic alterations but may select for them. Fig .1 CNA and B-allele frequency plots of pre-treatment and relapse samples of a patient demonstrating clonal evolution. A low level of cnLOH can be seen in the B-allele frequency plot at diagnosis (a) at the end of chromosome 8 (dashed box) which becomes more apparent at relapse (b). Fig .1. CNA and B-allele frequency plots of pre-treatment and relapse samples of a patient demonstrating clonal evolution. A low level of cnLOH can be seen in the B-allele frequency plot at diagnosis (a) at the end of chromosome 8 (dashed box) which becomes more apparent at relapse (b). In conclusion, this is the first attempt to quantify CNAs in sequential leukaemia samples. The results demonstrate that recurrent and relapse associated CNAs affect genes important in B-cell development and cancer progression. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Major progress has been made in understanding disease biology and therapeutic options for patients with chronic lymphocytic leukaemia (CLL). Recurrent mutations have been discovered using next generation sequencing, but with the exception of TP53 disruption their potential impact on response to treatment is unknown. In order to address this question, we characterised the genomic landscape of 250 first-line chemo-immunotherapy treated CLL patients within UK clinical trials using targeted resequencing and whole-genome SNP array. Methods: We studied patients from two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC) receiving FCR-based treatment in a first-line treatment setting. A TruSeq Custom Amplicon panel (TSCA, Illumina) was designed targeting 10 genes recurrently mutated in CLL based on recent publications.Average sequencing depth was 2260X. The cumulated length of targets sequenced was 7.87 kb from 330 amplicons covering 160 exons. Alignment and variant calling included a combination of three pipelines to confidently detect SNVs, indels and low level frequency mutations. SNP array testing was performed using HumanOmni2.5-8 BeadChips, (Illumina) and data analysed using Nexus 6.1 Discovery Edition, Biodiscovery. We performed targeted resequencing and genome-wide SNP arrays using selected samples’ germline material to confirm somatic mutations (n=40). Univariate and multivariate analyses using minimal residual disease (MRD) as the outcome measure were performed for 220 of the 250 patients. Results: Pathogenic mutations were identified in 165 (66%) patients, totalling 268 mutations in 10 genes. ATM was the most frequently mutated gene affecting 67 patients (29%) followed by SF3B1 (n=56, 24%), NOTCH1 (n= 32, 14%), TP53 (n= 21, 9%), BIRC3 (n= 17, 7%) and XPO1 (n=14, 6%). Less frequently recurrent mutations were seen in SAMHD1 (n=8, 3%), MYD88 (n= 4, 2%), MED12 (n=7, 3%) and ZFPM2 (n=5, 2%). Integrating sequencing and array results increased the patients with one or more CLL driver mutation from 66% to 94%. As previously reported del17p and TP53 mutations are co-occurring and associate with MRD positivity in all cases (n=15, p=0.0002). We report on minor TP53 subclones in 11 patients (VAF 1-5%), 8 of whom have MRD data available and were also associated with MRD positivity. Deletions of 11q were present in 44 patients. These lesions always included ATM but not always BIRC3. Bialleleic disruption was present in ATM for 27 patients (significantly associated with MRD positivity) and in BIRC3 for 4 patients. Rather surprisingly, trisomy 12 (n=33) and NOTCH1 mutations (n=28) were associated with MRD negativity (p=0.006 and 0.097, respectively). Analysing clonal and subclonal mutations per gene revealed the majority of mutations in SF3B1 and BIRC3 were subclonal (65% and 87% respectively). In contrast almost all SAMHD1 and MYD88 mutations were clonally distributed. There was an association between NOTCH1 subclonal mutations and MRD negativity, compared to clonal mutations, but this difference was not seen in the remaining mutated genes. From our copy number data, the presence of subclones was associated with MRD positivity (p=0.05). Combining important lesions in a multiple logistic regression analysis to predict MRD positivity, bialleleic ATM disruption, together with TP53 disruption, were the strongest predictors, followed by SAMHD1, whereas BIRC3 monoalleleic mutations were a medium predictor for MRD negativity. Conclusion: This is the first integrated genome-wide analysis of the distribution and associations of CLL drivers, using targeted deep resequencing and whole genome SNP arrays in an FCR-based first-line treatment setting. We have shown subclonal and clonal mutation profiles in all patients. For patients with two or more CLL-associated mutations we have begun to unravel clonal hierarchies. We have developed a comprehensive model using MRD as an outcome measure and have found bialleleic ATM mutations and SAMHD1 disruption to strongly predict for MRD positivity. Using MRD status as a robust proxy for PFS not only enables us to confirm results of previous studies, but is advantageous also in considerably reducing the timeframe for results. Indeed, we suggest that MRD status should be assessed routinely in future studies to complement modern integrated genomics approaches. Disclosures Hillmen: Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding.