ALBERT

Description: Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

Description: When the WHO defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" entity has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than co-occurring translocations - i.e. copy number variations (CNVs). While the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a shared underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma (DLBCL) morphology. While BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, while MYC IHC has been proposed as a screening tool for FISH testing, two mechanisms were observed that uncoupled MYC rearrangement from IHC positivity. 1) low MYC mRNA expression and 2) false-negative immunohistochemistry (IHC) staining mediated by a single nucleotide polymorphism resulting in an asparagine to serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.

Description: Pediatric AML is a heterogeneous disease in which treatment resistance remains an unsolved problem that is responsible for most deaths (Yeung and Radich 2017). Recently we have come to learn that resistance may be driven by mechanisms that extend beyond somatic mutations and DNA methylation changes (Ghasemi et al. 2020; van Galen et al. 2019; Bell et al. 2019). Transcriptional changes within specific primitive and committed cell types in AML tumours, which may be accompanied by alterations in chromatin structure and topology, can also contribute to disease progression (Ghasemi et al. 2020). To study such changes at the single-cell level, we analyzed single-cell RNA-seq (scRNA-seq) and matched scATAC-seq data from primary, remission and/or relapse samples obtained from three pediatric AML patients enrolled in the AAML1031 clinical trial (Alpenc et al. 2016) (Figure 1). Using the 10X Genomics single-cell platforms, we profiled a total of 39,738 cells using scRNA-seq (~4,826 cells per sample, 1,571 genes per cell), and 46,580 cells and 197,128 peaks using scATAC-seq (~6,718 cells per sample, 5,628 unique reads per cell). We then integrated these data types to determine the extent to which these two modalities corroborated and/or complemented each other in analyses of these longitudinally-obtained samples. Cell subpopulations detected in scRNA-seq through Leiden clustering on a k-nearest neighbor graph were generally consistent with recent observations of malignant and normal cell types detected in the bone marrow and peripheral blood compartments (van Galen et al. 2019; Hay et al. 2018). Malignant-like subpopulations at primary and relapse stages exhibited similar levels of cell type diversity along the myeloid lineage. These included hematopoietic stem-like cells, progenitors, granulocyte-monocyte progenitors, monocytes and dendritic cell-like subpopulations. Remission samples appeared to contain normal blood cell types including natural killers (NK), B and T cells, platelets and erythrocytes, consistent with the clearance of blasts. However, we also observed putative malignant-like conventional dendritic cell subpopulations at remission (50% and 16% in the respective samples), noting that these cells displayed increased expression of genes involved in antigen presentation and lysosomal protein processing. To integrate scATAC-seq with scRNA-seq data we performed clustering of transformed and reduced scATAC-seq data through iterative latent semantic indexing (Granja et al. 2020), and aligned cells in scATAC-seq to cells from scRNA-seq data using canonical correlation analysis (Stuart et al. 2019). We observed similar patterns of T cell expansion, presence of monocyte-like populations and NK cells at remission in the scATAC-seq data. However, scRNA-seq subpopulations dominated by malignant-like cells showed variability in mapping to distinctive chromatin states, with a few notable exceptions (Figures 2 and 3). One such exception is a subpopulation in scRNA-seq, found mostly at relapse, marked by high expression of genes involved in proliferation and growth factor-mediated cellular processes such as YBX3 (binds to GM-CSF promoter), CYTL1, and EGFL7 (regulator of vasculogenesis) (Figures 3 and 4). Cells within this subpopulation mapped to two scATAC-seq clusters whose significantly more highly accessible regions were enriched for functional processes such as blood vessel remodeling and neutrophil/granulocyte activation (Figure 4). These observations are consistent with recent evidence that AML tumour cells can activate the immune system to acquire resistance (Melgar et al. 2020). The scRNA-seq subpopulation, however, did not display high expression of myeloid/granulocyte factors such as CD15, ELANE, and MPO (Figure 4), perhaps consistent with the notion that such transcriptional programs may be primed but not yet activated within these malignant cells. We thus evaluated the potential of scATAC-seq to complement scRNA-seq in understanding transcriptional changes within cell types in AML tumours. We observed that normal cell types and specific malignant cell states could occupy distinctive chromatin states. Through integrative analyses, we conclude that scATAC-seq results can add additional information to complement scRNA-seq data, including identifying nascent transcriptional programs that may be poised for activation within malignant cells. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) are treated with standard frontline immunochemotherapy (R-CHOP). However, for cases where R-CHOP fails (relapsed-refractory DLBCL, rrDLBCL), prognosis is extremely poor, with 2-year overall survival of 20-40%. The successful development of new therapies may be hampered by our limited understanding of the genetic and molecular mechanisms underpinning treatment resistance. For example, recent data from our group has highlighted novel mutations that emerge following treatment with R-CHOP. The contribution of copy-number variations (CNVs) towards treatment resistance has not yet been thoroughly explored. A more complete characterization of these genetic alterations may lead to new prognostic biomarkers or treatment strategies. Methods We analyzed exome sequencing data from 59 rrDLBCL cases derived from either tissue biopsies or liquid biopsies collected after relapse, including both unpublished and previously published cases (Schmitz et al. (2018) NEJM 378:1396-1407 and Morin et al. (2016) Clin Can Res 22(9)). We separately performed low-pass whole-genome sequencing (lpWGS, 0.1-1x coverage) on 45 rrDLBCL liquid biopsies with ctDNA levels insufficient for exome-based analysis, for a total of 104 cases with copy-number information. We identified CNVs from exome and lpWGS data using Sequenza and ichorCNA, respectively. Next, we identified significant peaks of recurrent gains and losses using GISTIC2. Comparison of these peaks to CNVs in a previously published diagnostic DLBCL cohort (Schmitz et al. (2018) NEJM 378:1396-1407) enabled the identification of events that were significantly more prevalent in rrDLBCL. Results Overall, the landscape of CNVs in rrDLBCL is reminiscent of diagnostic DLBCL, with recurrent amplifications of chromosome 7 (43/104, 41.3%) and 18q (42/104, 40.4%) and recurrent deletions of 6q (25/104, 24.0%) and 17p13 (39/104, 37.5%). We identified nine regions enriched for recurrent amplifications or deletions among rrDLBCLs. These include deletions of 17p13.1 (20.4% in diagnostic biopsies vs 41.3% of rrDLBCLs, q=8.53x10-5) and recurrent amplifications of 8q24 (18.5% vs 42.3%, q=5.72x10-7) and 7p22 (27.2% vs 57.9%, q=6.29x10-8). Many of these peaks represent focal events that are exceedingly rare in diagnostic DLBCL and do not contain established lymphoma-associated genes, including amplifications affecting 700kb of 6p11.2 (2.03% vs 7.69%, q=0.0178) and 500kb of 19p13.3 (6.7% vs 31.7%, q=9.99x10-10). Notably, the 6p11.2 amplifications were associated with inferior progression-free survival following R-CHOP (p=0.02), with most tumors harboring this alteration relapsing within 12 months. We also identified a novel, recurrent deletion affecting a 20mb region of 5q (2.78% vs 10.6%, q=0.00604) which was significantly deleted in rrDLBCL. For tumors with additional samples collected prior to R-CHOP and following salvage therapy, deletions of 5q appeared to emerge following frontline therapy and persisted after subsequent treatments, suggesting they may contribute to treatment resistance. Discussion The 17p13.1 deletion enriched in rrDLBCL encompasses TP53, which is a common target of somatic point mutations in rrDLBCL and associated with inferior treatment outcomes. The amplification of 8q24 and 7p22 include MYC and GNA12/CARD11, respectively, although these large events encompass numerous additional genes which may be the target of such events. Curiously, the focal 6p11.2 amplification only overlaps a handful of genes including miR_598, which has been predicted to target CD27 and CD38 and whose expression is upregulated in B-cell cell lines (Lawrie et al. (2008) Leukemia 22:1440-2446). Further investigation and validation of these events and their corresponding targets will provide insight into the biology of rrDLBCL and may reveal novel therapeutic targets. Disclosures Michaud: Epizyme: Current Employment. Daigle:Epizyme: Current Employment. Jain:Kite/Gilead: Consultancy; Novartis: Consultancy. Kuruvilla:Merck: Consultancy, Honoraria; Bristol-Myers Squibb Company: Consultancy; Celgene Corporation: Honoraria; AstraZeneca Pharmaceuticals LP: Honoraria, Research Funding; AbbVie: Consultancy; Gilead: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria; Antengene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; TG Therapeutics: Honoraria. Crump:Servier: Consultancy; Roche: Consultancy; Kite/Gilead: Consultancy. Assouline:BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; Pfizer: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Steidl:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy; Bayer: Consultancy; Curis Inc: Consultancy. Johnson:AbbVie: Research Funding; Roche/Genentech, Merck: Honoraria; Roche/Genentech, Merck, Bristol-Myers Squibb, AbbVie: Consultancy. Scott:NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Janssen: Consultancy, Research Funding; Roche/Genentech: Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Celgene: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Morin:Celgene: Consultancy.

Description: The thickness of the tibial polyethylene (PE) insert is a critical parameter to ensure optimal soft-tissue balancing in the intraoperative decision-making procedure of total knee arthroplasty (TKA). However, there is a paucity of information about the kinetic response to PE insert thickness variations in the tibiofemoral (TF) joint, and subsequently, the secondary effects on the patellofemoral (PF) biomechanics. Therefore, the purpose of this study was to investigate the influence of varying PE insert thickness on the ligament and TF compressive forces, as well as on the PF forces and kinematics, after a cruciate-retaining TKA. A previous patient-specific musculoskeletal model of TKA was adapted to simulate a chair-rising motion in which PE insert thickness was varied with 2 mm increments or decrements compared to the reference case (9 mm), from 5 mm up to 13 mm. Greater PE insert thickness resulted in higher ligament forces and concurrently increased the TF compressive force by 21% (13 mm), but slightly unloaded the PF joint with 7% (13 mm) while shifting the patella distally in the trochlear groove, compared to the reference case. Thinner PE inserts showed an opposite trend. Our findings suggest that the optimal PE insert thickness selection is a trade-off between the kinetic outcomes of the TF and PF joints.