Abstract: One of the most common secondary resistance mechanisms to ibrutinib (IBR, a first-generation, irreversible Bruton's tyrosine kinase [BTK] inhibitor) in CLL is the development of mutations in BTK involving Cys481, leading to impaired drug binding (Woyach et al, NEJM 2014; Furman et al, NEJM 2014). The mechanisms of resistance to second generation BTK inhibitors are currently unknown. We aimed to assess the spectrum of acquired BTK mutations in patients with CLL progression on zanubrutinib (ZANU), a second-generation, irreversible inhibitor of BTK. We identified 38 CLL patients, treated with ZANU on clinical trials (NCT02343120, NCT02569476, NCT03336333, NCT02795182) at three centres, for whom serial samples were available. Four of 38 patients (10.5%) had CLL progression on ZANU (time to progression 5, 26, 29 and 48 months) and underwent amplicon next generation sequencing (NGS) of BTK (exon 11, 15, 16) and PLCG2 (exon 16, 19-20, 24, 27-28). Remarkably, we detected a BTK kinase domain mutation, BTK Leu528Trp (NM_000061.2:c.1583T & gt;G), in all four patients progressing on ZANU. In addition, all four patients had detectable Cys481 mutations at lower variant allele frequency (VAF) than the BTK Leu528Trp (median BTK Leu528Trp 34.9% vs BTK Cys481 9.1%). Analysis of sequence reads from amplicon NGS and RNA-sequencing data demonstrated that BTK Leu528Trp and BTK Cys481 mutations were present on different alleles. Assessment of the BTK Leu528Trp and BTK Cys481 mutations with high sensitivity droplet digital PCR (ddPCR) confirmed the absence of both mutations prior to ZANU exposure in all patients (sensitivity 0.1% VAF). Longitudinal analysis of the four patients with the BTK Leu528Trp mutation demonstrated the appearance of the Leu528Trp coincident with rising measurable disease and subsequent clinical CLL progression. We then went on to test patients on ZANU without disease progression but with persistent measurable disease (n=34) by ddPCR and detected three further patients harbouring low level BTK Leu528Trp mutations (VAF & lt;1%). These mutations were first detected after a median of 40 months on ZANU therapy. The BTK Leu528Trp mutation has been described only once previously in a patient in the context of IBR resistance (who transformed with Richter's syndrome) where it co-occurred with Cys481 mutations (Maddocks et al, JAMA Oncol 2015). As the prevalence of BTK Leu528Trp among progressive disease samples in our cohort exceeds all prior reports in IBR-treated patients, we sought to further understand the specificity of BTK Leu528Trp for ZANU progression. Targeted sequencing in a cohort of 49 patients progressing on IBR from the European Research Initiative on CLL (ERIC) did not detect the BTK Leu528Trp in any patients (sensitivity 1% VAF). We went on to perform biochemical and cellular studies on the BTK Leu528Trp mutation. Assessment of enzymatic activity of BTKLeu528Trp demonstrated a significant loss of activity compared to both BTKWT and BTKCys481Ser. This was further confirmed by assessing BTK autophosphorylation in HEK293 cells. Autophosphorylation at BTK Tyr223 was markedly reduced in HEK293 cells stably expressing BTKLeu528Trp compared to both BTKCys481Ser and BTKWT. In addition, a crystal structure of apo-BTKLeu528Trp was solved to understand effects of BTKLeu528Trp on ZANU binding to BTK. The alignment of the crystal structure of apo-BTKLeu528Trp with that of BTKWT-ZANU or the modeled structure of BTK-ATP suggested potential steric clashes between BTKLeu528Trp and ZANU (Figure 1A), as well as BTKLeu528Trp and ATP (Figure 1B). In conclusion, we have described the novel enrichment of BTK Leu528Trp mutations occurring in patients with CLL progressing on ZANU and both structural and experimental data consistent with this mutation resulting in interference with both ATP and ZANU binding to BTK. These findings emphasize the potential for agent-specific resistance mutations with second generation BTK inhibitors and the need to include these mutations in diagnostic screening for BTK resistance in the clinic. SH/CPST co-first authors, CT/PB co-senior authors Disclosures Handunnetti: Abbvie: Other: Travel Grant; Gilead: Honoraria. Zhou:Beigene: Employment. Sun:Beigene: Employment. Xing:Beigene: Employment. Zhang:Beigene: Employment. Guo:Beigene: Employment. Sutton:Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy; Novartis: Research Funding; Juno/Celgene: Consultancy, Honoraria. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Seymour:Takeda: Consultancy; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Janssen: Research Funding; BeiGene: Research Funding. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Liu:Beigene: Employment. Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Tam:Janssen: Honoraria, Research Funding; BeiGene: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; Novartis: Honoraria; AbbVie: Honoraria, Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy.

Abstract: Motivation: An ever-increasing body of evidence supports the importance of B cell receptor immunoglobulin (BcR IG) sequence restriction, alias stereotypy, in chronic lymphocytic leukemia (CLL). This phenomenon accounts for ∼30% of studied cases, one in eight of which belong to major subsets, and extends beyond restricted sequence patterns to shared biologic and clinical characteristics and, generally, outcome. Thus, the robust assignment of new cases to major CLL subsets is a critical, and yet unmet, requirement. Results: We introduce a novel application, ARResT/AssignSubsets, which enables the robust assignment of BcR IG sequences from CLL patients to major stereotyped subsets. ARResT/AssignSubsets uniquely combines expert immunogenetic sequence annotation from IMGT/V-QUEST with curation to safeguard quality, statistical modeling of sequence features from more than 7500 CLL patients, and results from multiple perspectives to allow for both objective and subjective assessment. We validated our approach on the learning set, and evaluated its real-world applicability on a new representative dataset comprising 459 sequences from a single institution. Availability and implementation: ARResT/AssignSubsets is freely available on the web at http://bat.infspire.org/arrest/assignsubsets/ Contact:  nikos.darzentas@gmail.com Supplementary information:  Supplementary data are available at Bioinformatics online.

Abstract: Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes.

Abstract: Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes ( BIRC3 , EGR2 , MYD88, NFKBIE , NOTCH1 , POT1 , SF3B1, TP53 , and XPO1 ) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3–9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53 , BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Abstract: Until quite recently, the prevailing view, adopted by the WHO 2008 Classification, was that mantle-cell lymphoma (MCL) originates from a peripheral B cell located within the inner mantle zone, an area comprised of naïve pre-germinal center (GC) type B cells. However, this notion has been challenged by molecular and functional evidence. Indeed, MCL is characterized by a skewed repertoire of immunoglobulin heavy variable (IGHV) genes and by some imprint of somatic hypermutation (SHM) in the clonotypic IGHV genes of the great majority (~70%) of cases, indicating antigen selection. Furthermore, both relapsed/refractory and treatment-naïve patients with MCL exhibit remarkable responses to B-cell receptor signaling inhibitors, strongly supporting a role for microenvironmental triggering in the natural history of MCL. In the present study, we sought to obtain additional insight into MCL ontogeny through a combined morphologic, immunohistochemical and immunogenetic analysis of 230 patients with a diagnosis of MCL according to the 2008 WHO Classification criteria. The study group included 139 nodal, 32 extranodal, 18 primary splenic MCLs as well as 41 bone marrow biopsies (BMB) infiltrated by MCL. Morphologically, 144/206 (70%) cases were ascribed to the common variety, while 48/206 (23.3%) and 14/206 (6.7%) were characterized as blastoid or pleomorphic variant, respectively. The immunohistochemical analysis (on paraffin sections) focused on CD27, DBA.44 and IRF4 (MUM1), markers not normally expressed by the naïve pre-germinal centre B-cell of the inner mantle zone. The results were as follows: (i) 117/214 (54.7%) cases positive for CD27 expression; (ii) 18/176 (10.2%) cases positive for DBA.44; (iii) 53/98 (54%) cases positive for IRF4. Amongst CD27+ cases, 10/86 (11.6%) were also positive for DBA.44, whereas 27/51 (52.9%) were also positive for IRF4. Immunogenetic information regarding IGHV-IGHD-IGHJ gene rearrangements was available for 167 cases of the study. Fifty of 167 cases (30%) carried IGHV genes with no SHM (100% identity to the germline, GI), whereas the remaining 117 cases (70%) bore some imprint of SHM: in particular, 95/167 cases (56.8%) carried IGHV genes with 97-99.9% GI, while 22/167 cases (13.2%) carried IGHV genes with 〈 97% GI. In keeping with the literature, the IGHV gene repertoire of the present cohort was remarkably biased, with the IGHV3-21, IGHV4-34, IGHV3-23, IGHV1-8 and IGHV5-51 genes accounting for 51% of cases (85/167). The following noteworthy observations were made from the combined assessment of morphological, immunohistochemical and immunogenetic results. (1) DBA.44 was not detected in any of the 21 extranodal MCL cases analyzed, was rare in nodal MCL (5/108, 4.6%), whereas, in contrast, was significantly enriched among primary splenic MCL (6/14, 42.8%; p 〈 0.01 for both comparisons). (2) Unexpectedly, CD27 expression was more prevalent among cases with minimal/borderline SHM (56/91, 61.5%) or no SHM (100% GI: 23/46, 50%), whereas it was less frequent among cases with a significant SHM load ( 〈 97% GI: 6/20, 30%; p=0.01 for comparison to 100% GI cases). (3) CD27 expression was significantly (p 〈 0.05) more frequent amongst cases classified as pleomorphic (11/14 cases, 78.6%) versus either blastoid (38/46 cases, 60.8%) or common (65/131 cases, 49.6%). (4) IRF4 was detected in cases from all SHM categories: 10/16 (62.5%) cases with 100 GI%, 14/30 (46.6%) cases with 97-99.9% GI and 3/6 (50%) cases with 〈 97% GI. (5) Blastoid cytology was less frequent in primary splenic MCL (2/18 cases, 11.1%) compared to either nodal (33/126 cases, 26.2%) or extranodal MCL (8/28 cases, 28.5%), however the difference did not reach significance likely due to small numbers. In conclusion, we document the remarkable immunohistochemical and immunogenetic heterogeneity of MCL. Certain profiles, identified here for the first time, are in sharp contrast to those of the naïve pre-germinal centre B-cell of the inner mantle zone (IG-unmutated, CD27-, IRF4-, DBA.44-), which is the postulated MCL progenitor according to the WHO 2008 classification. These findings strongly support antigen drive in a significant fraction of MCL cases. Furthermore, they raise the intriguing possibility that many ontogenetic pathways may give rise to MCL or, alternatively, that several types of normal B cells may serve as MCL progenitors. Disclosures Stamatopoulos: Janssen Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.