ALBERT

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.

Description: Background Chronic lymphocytic leukaemia (CLL) is characterised by clinical and biological heterogeneity. Despite significant advances in therapeutic management, CLL remains largely incurable. Current risk stratification is based on cytogenetic features (del(17p), del(11q), del(13q), +12). So far, sequencing studies in CLL have focussed predominantly on the exome. These have identified a number of genes that are recurrently mutated at low frequency such as TP53, SF3B1, ATM, NOTCH1, MYD88, and BIRC3. Apart from TP53 abnormalities, none of these are currently used to guide clinical decisions and it is unclear how they are implicated in disease pathogenesis. Methods In this study, we sought to further refine the molecular landscape of CLL using whole genome sequencing (WGS) of paired tumour and germline DNA samples from a cohort of clinically annotated patients with CLL. We sequenced a heterogeneous cohort of 41 samples (25 males, 16 females, median age 69 (range 49-94)) with a range of clinical features (49% fludarabine refractory, 61% unmutated IgVH). Whole genome sequencing libraries were generated using the Illumina TruSeq PCR-free sample preparation kit, with a median insert size of 400bp, and subjected to 100bp paired-end sequencing on an Illumina HiSeq 2500 platform. Both tumour and germline libraries were sequenced to an average depth of 38x. Sequencing reads were aligned using the Isaac algorithm and the Starling and Strelka algorithms were used for SNV and Indel calling in germline and tumour samples respectively. All variants with a read depth

Description: Key Points Targeted NGS of relapsed/refractory CLL reveals a high incidence of concurrent mutations that mostly affect the TP53, ATM, and SF3B1 genes. Concurrent mutations of the TP53, ATM, and/or SF3B1 genes confer short survival in patients with relapsed/refractory CLL.

Description: Background Treatment decisions in Multiple Myeloma (MM) have been driven by a patient's age or ability to tolerate therapy, but as yet, not by their tumour genetics, even though understanding of the prognostic utility of genetic features continues to develop. The ability to identify genetic prognostic markers and potential therapeutic targets in a timely fashion within a health service is a vital step in delivering precision medicine to patients. The aims of this study were to assess the implementation of clinical grade whole genome sequencing (WGS) data at diagnosis to replicate, and ultimately replace, standard-of-care Fluorescence In Situ Hybridisation (FISH) cytogenetics; to provide markers for prognostication or MRD; and to identify actionable targets for clinical trials in precision therapy of MM. Methods Bone marrow aspirates from patients with newly-diagnosed myeloma were collected between June 2017 and April 2018 in a single tertiary hospital in the United Kingdom. The population comprised seven male and seven female patients with a mean age of 78 years. From the first-draw of bone marrow aspirate, standard-of-care FISH cytogenetic analyses were performed locally according to criteria from the International Myeloma Working Group (IMWG). From the remnant aspirate samples, CD138-positive plasma cells were enriched by magnetic bead-sorting and genomic DNA was extracted locally for WGS with a success rate of approximately 70%. Fourteen samples underwent successful plasma cell purification (78 - 99% morphological purity) with a yield of at least 0.5 μg DNA. To identify germline genomic variants, DNA was extracted from peripheral blood samples obtained simultaneously. WGS was performed at a centralised facility and mean coverage for germline samples was 35.1x and for plasma cell-enriched samples was 100.9x. Conventional cytogenetic FISH data were compared with genomic data for chromosome-level alterations. Identified somatic variants were automatically cross-referenced against publicly available databases that describe somatic mutations in cancer as well as a virtual panel of potentially actionable therapeutic targets including : NRAS, KRAS, BRAF, CDKN2C, FGFR3 and IDH2. Results In paired samples, WGS replicated all 13 translocation and chromosomal loss/gain events identified by FISH (Figure 1). Furthermore, three translocations involving the IGH locus suggested by FISH analysis were characterised by WGS. Using samples derived from surplus material, fast-track turnaround of 14 days was attainable. Five patients had no identifiable marker by FISH cytogenetics. In these patients, WGS found all five to possess somatic variants could be used as prognostic or potential Measurable Residual Disease (MRD) markers. Nine patients exhibited somatic variants in genes that may be subject to targetable therapy as determined by trials available on ClinicalTrials.gov: five NRAS, two KRAS, one BRAF and one FGFR3 mutation as of July 2019. Conclusion WGS assessment of newly diagnosed myeloma provides accurate, timely and actionable information beyond what is available from standard-of-care FISH. The application of WGS to myeloma diagnostics presents a number of advantages. Firstly, WGS can replicate and exceed existing myeloma FISH assessment of translocation and loss/gain events in a prompt turnaround time. Secondly, germline variants are deducted from tumour variants to provide a gold standard description of the somatic mutational landscape in the patient's disease. Thirdly, virtual panels of known somatic variants can be applied to the data as knowledge accumulates about the role of specific mutations on prognosis or therapeutic response. We demonstrate that centrally provided WGS and its analysis can be incorporated into routine local assessment of newly diagnosed myeloma. Therefore, these observations show that such technology has the potential to be rapidly scalable across existing hospital networks. Disclosures Gooding: Celgene Corporation: Research Funding. Ramasamy:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Background:Up to 10% of patients with chronic lymphocytic leukemia (CLL) undergo a high-grade transformation called Richter Syndrome (RS), most commonly of the diffuse large B cell lymphoma type. In ~80% of cases, the lymphoma originates from the CLL clone and is associated with a median survival inferior to 1 year after diagnosis. Prognosis following standard immunochemotherapy remains very poor and new therapeutic targets for RS are urgently needed. Methods:Only samples from patients with newly diagnosed RS recruited into the UK CHOP-OR (CHOP in combination with Ofatumumab, EudraCT number 2009-016459-23) were included (Eyre et al, Br J Haematol. 2016). DNA was extracted from 35 peripheral blood mononuclear cells (leukemic phase) and 21 FFPE-lymph node slides (Richter phase). For 15 patients, paired CLL and RS phase were available. A TruSeq Custom Amplicon panel (TSCA, Illumina) was designed targeting 28 recurrently mutated genes in CLL based on recent publications. Average sequencing depth was 2311x. Single nucleotide variant (SNV) were filtered with a variant allele frequency (VAF) 〉10%, with a depth 〉20, with an altered variant read 〉6 and by excluding synonymous mutation and mutation with a frequency 〉5% in the general population. Mutation frequencies were compared to those of Landau et al based on a CLL population of 538 patients. Results: Pathogenic mutations were identified in 28/30 CLL phase (93%) and 18/18 (100%) of RS phase. In the majority of paired cases (80%), mutations identified in the CLL phase were confirmed in the RS phase. Additional mutations were acquired in the RS phase compared to the CLL phase (277 versus 82) with a median of 5.5 mutations/RS phase versus 3 mutations/CLL phase (P=0.0003). When compared to the frequency in a general CLL population, we observed a significant increase of mutations in ATM (15%, 57%, 78% for general CLL population, CLL phase of RS, RS phase, respectively), TP53 (7%, 23%, 56%), NOTCH1 (6%, 23%, 50%), CDKN2A (23.1 months vs 6.6 months, P=0.0242). Conclusion: We show that CLL patients developing RS carry a high mutation burden and recurrent driver mutations in the leukemic clone and that additional mutations are acquired in the RS phase. We confirm the association of mutations in TP53, NOTCH1 and CDKN2A with RS and show in addition that SAMHD1 mutations are also frequently seen in RS. Finally, we propose that patients with biopsy findings of classical RS but no mutation in any of the 6 selected genes have an improved PFS and OS whereas patients without the classical RS biopsy features but a high-risk mutation profile fare poorly. Disclosures Eyre: GSK: Honoraria; Celgene: Other: Travel, Accomodation; Gilead: Honoraria, Other: Travel, Accomodation, Speakers Bureau; Takeda: Honoraria, Other: Travel, Speakers Bureau. Schuh:Gilead: Consultancy, Honoraria, Research Funding; Roche, Janssen, Novartis, Celgene, Abbvie: Consultancy, Honoraria.

Description: Background: Previous studies using next-generation sequencing (NGS) have led to the identification of a number of genes mutated frequently in CLL. Recent publications focus on the most recurrently mutated genes (TP53, SF3B1 and NOTCH1) which tend to be mutually exclusive. Large series of untreated patients have shown that these mutations have a prognostic impact. Relapse may be associated with more frequent mutational events. Further investigation of relapsed CLL genomes within a clinical trial setting using a comprehensive NGS gene panel is required. Methods: Using targeted NGS we determined the mutational spectrum of 118 refractory/relapsing CLL patients enrolled in one French and two UK prospective trials (ICLL01 from the French intergroup GCFLLC/MW-GOELAMS, NCRNCLL201, NCRNCLL202 respectively). Eighty percent of patients had an unmutated IGHV status and 21 (18%) patients carried a 17p deletion. Sequencing libraries were composed of a panel of nine recurrently mutated genes in CLL (i.e.TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3 and MYD88) and run on the Illumina MiSeq instrument (Illumina Inc). On average 14.1 M reads were obtained per run of which 96.8% were identified reflecting an acceptable signal to noise ratio. Yield was 4.1 Gb and 95.9% of reads were above Q30 across 6 MiSeq runs. Data was analysed using our in-house bioinformatics pipeline consisting of a combination of two different aligners (Custom Amplicon Alignment, Illumina Inc and Stampy, Wellcome Trust centre for Human Genetics), two variant callers (GATK, Broad Institute and Platypus, Wellcome Trust centre for Human Genetics) and a stringent filtering process in order to detect SNVs and indels with a variant allele frequency down to 7%. Results: We identified a total of 196 mutations (mean=1.7/sample) in 95 (80%) patients: 138 missense mutations, 41 substitutions/indels, 12 nonsense and 5 splicing mutations. TP53, SF3B1 and ATM mutations occurred frequently in 29 (24.6%), 33 (28%) and 29 (24.6%) patients, respectively. Eighteen (15.3%) patients harbored a NOTCH1 mutation matching the range of reported frequency. Mutations in the other genes sequenced were distributed as follows: XPO1 mutations in 17 (14.4%), SAMHD1 mutations in 12 (10.2%), MED12 mutations in 10 (8.5%), BIRC3 mutations in 6 (5.1%) and MYD88 mutations in 3 (2.5%) patients. Twenty-three (20%) patients did not have any mutations present (Figure 1, cluster #1). A total of 51 (43%) patients had one gene mutated (Figure 1, cluster #2) and the remaining 44 (37%) patients had two or more genes mutated (Figure 1, clusters #3 & #4). Recurrent combinations of mutations (affecting more than 5% of patients) were found in a group of 23 (20%) patients. These combinations of mutations comprised of at least two of the following genes: TP53, SF3B1 and ATM (Figure 1, cluster #3, so called multiple-hit (MH) profile). Remarkably, mutations in these 3 genes were found significantly more frequently associated than in isolation. We then investigated the potential clinical relevance of the MH profile. This profile was associated with poorer ORR than the remaining cohort (43% vs 80%, P

Description: The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter's Syndrome (RS) and it is a rare event with dismal prognosis. In this study, we conducted whole genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 clinical trial (CHOP-O) patients. We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumour necrosis factor receptor associated factor three (TRAF3). In the non-coding genome, we discovered AID-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2 andTRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal de-convolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study (NCT03899337).