Follicular lymphoma subgroups with and without t(14;18) differ in their N-glycosylation pattern and IGHV usage

Genetic testing to detect somatic alterations is usually performed on formalin-fixed paraffin-embedded tumor samples. However, tumor molecular profiling through ctDNA analysis may be particularly interesting with the emergence of targeted therapies for ovarian cancer (OC), mainly when tumor is not available and biopsy is not viable, also allowing representation of multiple neoplastic subclones. Using a custom panel of 27 genes, next-generation sequencing (NGS) was performed on tumor and matched plasma samples from 96 OC patients, which were combined in two groups (treatment naive and post-treatment). Overall, at least one somatic variant present in the tumor sample was also detected in the matched plasma sample in 35.6% of the patients, a percentage that increased to 69.6% of the treatment naive patients and 83.3% of those with stage IV disease, showing the potential of ctDNA analysis as an alternative to identify somatic variants in these patients, namely those that have predictive value for targeted therapy. In fact, of the two treatment-naive patients with somatic BRCA1 variants identified in tumor samples, in one of them we detected in ctDNA a BRCA1 somatic variant that was present in the tumor with a VAF of 53%, but not in the one that had a VAF of 5.4%. We also showed that ctDNA analysis has a complementary role to molecular unraveling of inter- and intra-tumor heterogeneity, as exemplified by one patient diagnosed with bilateral OC in which different somatic variants from both tumors were detected in ctDNA. Interestingly, as these bilateral tumors shared a rare combination of two of the three variants identified in ctDNA, we could conclude that these morphologically different tumors were clonally related and not synchronous independent neoplasias. Moreover, in the post-treatment group of patients with plasma samples collected after surgery, those with detectable somatic variants had poor prognosis when compared with patients with no detectable somatic variants, highlighting the potential of ctDNA analysis to identify patients at higher risk of recurrence. Concluding, this study demonstrated that somatic variants can be detected in plasma samples of a significant proportion of OC patients, supporting the use of NGS-based ctDNA testing for noninvasive tumor molecular profiling and to stratify patients according to prognosis.

Abstract Background: Detection and characterization of clonal IG/TR rearrangements and translocations in lymphoproliferative neoplasms provides critical information in the diagnostic pathway in several clinical scenarios and is a valuable tool to address research questions around B and T cells. This includes ascertaining the clonal nature of lymphoid proliferations, characterization of translocations in lymphomas and leukemias, characterization of CDR3 regions for MRD target identification and stereotyping analysis, amongst others. Until now, collecting this information required a combination of different methodologies, such as Gene-scanning/heteroduplex analysis, FISH and Sanger sequencing. Material and methods: As part of the EuroClonality-NGS consortium, we have designed a capture-based protocol covering the coding V, D and J genes of the IG/TR regions, as well as switch regions in the IGH locus. The assay uses Nimblegen (Roche Molecular Systems) capture baits spanning a total of 180kb and the products are analyzed on a MiSeq Illumina sequencer and MiSeq v3 sequencing chemistry with 2 x 120bp pair-end reads. This design allows the identification of D-J and V(D)J rearrangements as well as chromosomal translocations involving IG/TR genes by Next-generation sequencing (NGS). We piloted this approach for clonality and translocation detection in a cohort of 21 peripheral blood, bone marrow and fresh-frozen samples (3 precursor B-cell acute lymphoblastic leukemias [B-ALL], 4 Burkitt lymphoma [BL], 5 chronic lymphocytic leukemias [CLL], 2 splenic marginal zone lymphomas [SMZL], 2 diffuse large B cell lymphomas [DLBCL], 2 follicular lymphomas [FL], 2 precursor T-cell lymphoblastic leukemias [T-ALL] and 1 T-cell non-Hodgkin lymphoma [T-HNL]) with well-characterized translocations by FISH/Karyotype and/or clonal rearrangements by PCR and Sanger sequencing. Results: We were able to detect the described IG/TR translocation in 18/21 samples, including translocations into the J, D and switch regions of the IG/TR genes. Three samples failed to produce results, two concerned fresh-frozen lymphomas with low quality DNA, and one concerned a technical error in a B-ALL case. The translocation partner and the breakpoint was identified in 15/18 evaluable cases, including CRLF2, MYC, BCL11A, BCL2, CCND3, IRF4, BCL11B, and TLX1. In three cases with only karyotyping data available, a translocation involving the IGH locus was identified with no clear leukemia/lymphoma-related genes in the neighboring regions of the reciprocal chromosome, suggesting the potential for new translocation partners and/or mechanism of disease. In all 14 samples with well-characterized D-J/V(D)J rearrangements by PCR and sequencing, NGS was able to detect the same rearrangements. These included IGH and IGK/IGL rearrangements in B-cell proliferations and TRB, TRG and TRD in T-cell proliferations. Additionally, aberrant clonal rearrangements were seen that were not detected with conventional PCR-based approaches (e.g. IGKV to IGK intron). Conclusion: The EuroClonality NGS IG/TR capture-based approach is a promising tool for the simultaneous detection and characterization of IG/TR translocation and rearrangements in a clinical setting. A formal pan-European validation study is underway within the EuroClonality-NGS consortium. Disclosures No relevant conflicts of interest to declare.

298 Background: The type and frequency of recurrent, targetable alterations in muscle-invasive, treatment-naïve urothelial cancer (UC) have been well-catalogued through the bladder TCGA. We sought to validate these findings within a prospective cohort of patients (pts) with progressive UC treated at our institution. This cohort typifies the UC patient population managed by medical oncologists. Methods: Pts with a diagnosis of UC were enrolled onto an IRB-approved protocol between 1/14/14 and 7/29/14, which allowed for sequencing of all exons in 341 oncogenes and tumor suppressor genes using an exon capture next generation sequencing assay (NGS) platform (MSK-IMPACT) in a CLIA lab from tumor and matched germline DNA. Somatic point mutations, truncations, copy number alterations, and insertions/deletions were detected. Results: 49 UC pts were sequenced, 5 (10%) having upper tract disease. 33 (67%) tumors were pure UC histology with the remainder containing divergent differentiation. 17 samples (24%) were metastatic in origin, and 40 samples were analyzed from pts who had received prior chemotherapy. The most common alteration identified was point mutations within the TERT promoter (71%), a region not sequenced by TCGA. Additional alteration frequencies were similar between both sets. FGFR3 mutations overlapped with both PIK3CA and TSC1 alterations and co-alterations were observed between FGFR3 and CDKN2A as well as PIK3CA and CCND1. Conclusions: TERT promoter mutations were found at high frequency in the MSK-IMPACT tumor cohort. The frequency and type of alterations identified in the bladder TCGA are similar to the MSK-IMPACT cohort, suggesting that TCGA data can be used to guide clinical trials in the metastatic, pre-treated population. Overlap of alterations within and across core signaling pathways underscores the need for functional validation of alterations and for rational combinations of targeted therapies to effectively treat advanced UC. [Table: see text]

Background: Rapid progress in next-generation sequencing (NGS) technologies make it possible to spell out the mutational status, the genetic and epigenetic variability of chronic lymphocytic leukemia (CLL). The identification of driver mutations allows us to expand understanding of the pathogenesis of CLL, to identify prognostic groups and to select potential targets for therapy, contributing to the development and implementation of new targeted drugs and its combinations. Nevertheless, the course of CLL does not always correspond to the existing prognostic risk groups, assessed by "standard" cytogenetic and molecular genetic methods. The NGS technology admits establishing markers of an unfavorable course of the disease. Aim: To assess the mutational status of patients (pts) with CLL using the developed Lymphoid Targeted NGS Panel and to study the possible correlation of the mutational status with the clinical characteristics of the disease. Method: In this prospective study were included 24 pts with CLL: treatment-naïve (n=8) and relapsed/refractory (n=16). The diagnosis of CLL was established according to iwCLL criteria (iwCLL, Hallek et al., 2018) and show only typical immunophenotype. The pts were divided into three prognostic groups according to cytogenetic assay: favorable (n=14), neutral (n=2), and unfavorable (n=8). Although, 20/24 pts were divided into two prognostic groups taking into account the data on the mutational status of the immunoglobulin heavy-chain variable (IGHV) region: favorable (n=8) and unfavorable (n=12). Four patients have no available data on IGHV mutational status. All patients had indications for starting treatment: FCR (n=7), RB (n=6), ibrutinib (n=3), venetoclax (n=1), acalabrutinib (n=5), combination of venetoclax and acalabrutinib (n=2). DNA samples were extracted from peripheral B-cell lymphocytes via the standard phenol-chloroform method. Average reading depth of 1000x is produced on a MiSeq platform (Illumina, USA). The 2% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using the following databases: COSMIC, ClinVar, gnomAD with application in silico analysis (Cscape, Cancer Genome Interpreter, SNPs&Go). The Lymphoid Panel includes 117 genes, part of which is involved in the main 8 cellular signaling pathways underlying the pathogenesis of CLL. We have completed a pilot NGS study using the developed Lymphoid Targeted Gene Panel on DNA samples of six treatment-naïve pts. Results: Genetic aberrations were identified in all DNA samples using NGS. Somatic mutations were detected in 82.9% of cases, in an amount from 15 to 37. In four pts (4/6) with an unfavorable prognosis (cytogenetics and unmutated IGHV), known pathogenic variants of mutations were identified: JAK3 V722L, NOTCH1 P2514fs*4, IDH2 T352P, TP53 Lys120Glu, BRAF D594G. The existing approach to the interpretation of the results does not allow making an unambiguous conclusion about the clinical significance of variants in the IDH2 and JAK3 genes, despite the known pathogenic effect of the variants. The detected variant of the mutation (Lys120Glu) in the TP53 gene is often associated with the presence of a 17p13 deletion, which was confirmed by the FISH assay and correlated with the unfavorable clinical course of the disease in patient CLL-024. Twenty-two mutations were identified, the pathogenicity of which has not yet been determined, in the amount of 2 to 5 (median=3.5) mutations per patient. It should be noted that two patients (CLL-023, CLL-024) with unfavorable prognosis had mutations both in BCR gene and in NOTCH2 gene of unknown significance. Conclusion: The data obtained from a pilot study demonstrate the possibility of using NGS technology in clinical practice. The assessment of the mutational status of pts with CLL using NGS correlates with the clinical parameters of pts. Considering that there is currently no information about prognostic significances of identified mutations, additional research is required. Disclosures Martynkevich: Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Shuvaev:Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Voloshin:Novartis: Honoraria, Speakers Bureau.

Abstract Abstract 3967 Poster Board III-903 Background Follicular lymphoma (FL) is considered an indolent but incurable lymphoma. Treatment with cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) provides complete or partial responses in most patients. The BCL2 translocation t(14;18) is present in 85% of the cases, but additional genomic alterations must occur to induce overt FL. We have sequenced the genome and transcriptome of a cytogenetically normal FL sample taken from a patient (pt 1) that had an unusually aggressive clinical course with the aim of identifying genomic alterations that could contribute to FL pathogenesis. We identified a mutation in FAS/CD95, a key component of the extrinsic apoptotic pathway. We hypothesized that FAS mutations may contribute to treatment resistance in FL by inhibiting apoptosis. This study investigates the prevalence and clinical outcome of patients with FL harbouring mutations in the exons coding for the functional “death domain” of the FAS gene. Methods The initial FL sample was subjected to cytogenetic analysis and whole genome tiling array comparative genomic hybridization followed by next-generation sequencing of the tumour genome and tumor transcriptome following the manufacturer's protocol (Illumina). FAS emerged as a potential candidate that could explain the unusually aggressive FL. We performed PCR amplification of exons 7,8,9 and the 3'UTR of the FAS gene with universal M13F(-21) and M13 primer extensions on pre-treatment FL samples derived from 214 patients including 33 diffuse large B cell lymphoma samples that had evolved from a prior FL (i.e. paired FL and DLBCL). PCR products were purified using AMPpure magnetic beads and bi-directionally sequenced using BigDye® Terminator v3.1 and an ABI 3730 XL sequencer. Analysis was performed using Mutation Surveyor. Mutations were considered present if they were observed in both forward and reverse reads. Results Patient 1 had a t(14;18) negative, grade 1 FL, that progressed rapidly despite CVP-R and second line chemotherapy but is now in complete remission following an allogeneic stem cell transplant. The FL immunophenotype was CD19+, CD10+, BCL2+, BCL6+ and lambda clonal. Minimal genomic gains and losses were observed by aCGH. After filtering known single nucleotide polymorphisms (SNPs), a total of 320 candidate novel protein-altering changes were identified (affecting 298 genes) in the tumor genome and transcriptome sequence data. Validation of the mutated genes by Sanger sequencing revealed a novel, somatic and coding mutation in FAS at genomic position chr10:90764005, changing C to T, resulting in a premature truncation of the protein. We then sequenced exons 7, 8, 9 and the 3'UTR of the FAS gene from 214 FL patients. Ten novel FAS mutations were detected, of these six were coding, three of which produced a truncated protein. Of the six coding mutations, two were observed in the transformed DLBCL sample. Coding mutations in FAS appeared to be associated with an aggressive clinical course (median time to progression=12 months for coding mutations (n=6) vs 34 months for non-coding or wild type (n=208), P=0.06). 2 of the 6 patients developed early transformation to DLBCL, 2 had treatment resistant FL that required allogeneic bone marrow transplant and 2 have already died due to progressive treatment refractory FL. Conclusion Next generation sequencing technology revealed novel somatic mutations in a cytogenetically normal FL sample, one of which was a mutation in the FAS gene. Sanger sequencing of a large cohort of FL samples revealed that 5% of cases harboured novel mutations in the death domain of FAS (2% coding, 3% non-coding). Coding mutations were rare but when present were associated with atypically aggressive disease. Disclosures: Connors: Roche Canada: Research Funding. Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.