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Supplementary tables S1 to S6

Suppplementary Methods and Figures

Supplementary Methods, Supplementary Tables S1-S5, and Supplementary Figures S1-S15

Purpose:We analyzed the utility of cell-free DNA (cfDNA) in a prospective population-based cohort to determine the mutational profile, assess tumor burden, and estimate its impact in response rate and outcome in patients with diffuse large B-cell lymphoma (DLBCL).Experimental Design:A total of 100 patients were diagnosed with DLBCL during the study period. Mutational status of 112 genes was studied in cfDNA by targeted next-generation sequencing. Paired formalin-fixed, paraffin-embedded samples and volumetric PET/CT were assessed when available.Results:Appropriate cfDNA to perform the analyses was obtained in 79 of 100 cases. At least one mutation could be detected in 69 of 79 cases (87%). The sensitivity of cfDNA to detect the mutations was 68% (95% confidence interval, 56.2–78.7). The mutational landscape found in cfDNA samples was highly consistent with that shown in the tissue and allowed genetic classification in 43% of the cases. A higher amount of circulating tumor DNA (ctDNA) significantly correlated with clinical parameters related to tumor burden (elevated lactate dehydrogenase and β2-microglobulin serum levels, advanced stage, and high-risk International Prognostic Index) and total metabolic tumor volume assessed by PET/CT. In patients treated with curative intent, high ctDNA levels (>2.5 log hGE/mL) were associated with lower complete response (65% vs. 96%; P < 0.004), shorter progression-free survival (65% vs. 85%; P = 0.038), and overall survival (73% vs. 100%; P = 0.007) at 2 years, although it did not maintain prognostic value in multivariate analyses.Conclusions:In a population-based prospective DLBCL series, cfDNA resulted as an alternative source to estimate tumor burden and to determine the tumor mutational profile and genetic classification, which have prognostic implications and may contribute to a future tailored treatment.

The genetic mechanisms associated with splenic marginal zone lymphoma transformation (SMZL-T) are not well defined. We studied 41 SMZL patients that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in two groups: i) at diagnosis (SMZL, n=27 samples), and ii) at transformation (SMZL-T, n=32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell which acquired different genetic alterations in virtually all evaluable cases (12/13, 92%). Using whole genome sequencing from diagnostic and transformation samples in one patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype and international prognostic index at transformation predicted for a shorter survival from transformation (P=0.001, P=0.042, and P=0.007, respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event.

The chromatin activation landscape of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor immunoglobulin is currently unknown. Here, we report the results of a whole-genome chromatin profiling of histone 3 lysine 27 acetylation of 22 CLLs from major subsets which were compared against non-stereotyped CLLs and normal B cell subpopulations. Although subsets #1, #2, and #4 did not differ much from their non-stereotyped CLL counterparts, subset #8 displayed a remarkably distinct chromatin activation profile. In particular, we identified 209 de novo active regulatory elements in this subset, which showed similar patterns with U-CLLs undergoing Richter transformation. These regions were enriched for binding sites of 9 overexpressed transcription factors. In 78/209 regions, we identified 113 candidate overexpressed target genes, being 11 regions associated with more than two adjacent genes. These included blocks of up to 7 genes, suggesting a local co-upregulation within the same genome compartment. Our findings further underscore the uniqueness of subset #8 CLLs, notable for the highest risk of Richter's transformation amongst all CLL, and provide additional clues to decipher the molecular basis of its clinical behavior.

MALAT1is a long non-coding RNA with oncogenic roles in cancer but poorly studied in indolent B-cell neoplasms. Here,MALAT1expression was analyzed using RNA-seq, microarrays or qRT-PCR in primary samples from various clinico-biological subtypes of chronic lymphocytic leukemia (CLL, n=266) and follicular lymphoma (FL, n=61). In peripheral blood (PB) CLL samples, highMALAT1expression was associated with a significantly shorter time to treatment, independently from other known prognostic factors, such as IGHV mutational status. Coding genes whose expression levels were associated withMALAT1in CLL were predominantly related to oncogenic pathways stimulated in the lymph node (LN) microenvironment. Further analysis ofMALAT1expression by microarrays in paired CLL samples from PB/LN showed that its levels were maintained between both anatomical compartments, supporting that the clinical value ofMALAT1expression found in PB is mirroring expression differences already present in LN. Similarly, highMALAT1expression in FL predicted for a shorter progression-free survival, and its correlated expressed genes were associated with pathways promoting FL pathogenesis. In summary,MALAT1expression is related to pathophysiology and clinical behavior of indolent B-cell neoplasms. Particularly in CLL its levels could be a surrogate marker of the microenvironment stimulation and may contribute to refine the clinical management of these patients.

Mantle cell lymphoma (MCL), a rare and aggressive B cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T cells self-organize in disc-shaped structures, where B and T cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing towards personalized therapeutic approaches.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11− primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11− tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory T (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.

Intravascular large B-cell lymphoma (IVLBCL) is an uncommon lymphoma with an aggressive clinical course characterized by selective growth of tumor cells within the vessels. Its pathogenesis is still uncertain and there is little information on the underlying genomic alterations. In this study, we performed a clinicopathologic and next-generation sequencing analysis of 15 cases of IVLBCL using a custom panel for the detection of alterations in 68 recurrently mutated genes in B-cell lymphomagenesis. Six patients had evidence of hemophagocytic syndrome. Four patients presented concomitantly a solid malignancy. Tumor cells outside the vessels were observed in 7 cases, 2 with an overt diffuse large B-cell cell lymphoma. In 4 samples, tumor cells infiltrated lymphatic vessel in addition to blood capillaries. Programmed death-ligand 1 (PD-L1) was positive in tumor cells in 4 of 11 evaluable samples and in macrophages intermingled with tumor cells in 8. PD-L1 copy number gains were identified in a higher proportion of cases expressing PD-L1 than in negative tumors. The most frequently mutated gene was PIM1 (9/15, 60%), followed by MYD88L265P and CD79B (8/15, 53% each). In 6 cases, MYD88L265P and CD79B mutations were detected concomitantly. We also identified recurrent mutations in IRF4, TMEM30A, BTG2, and ETV6 loci (4/15, 27% each) and novel driver mutations in NOTCH2, CCND3, and GNA13, and an IRF4 translocation in 1 case each. The mutational profile was similar in patients with and without evidence of hemophagocytic syndrome and in cases with or without dissemination of tumor cells outside the vessels. Our results confirm the relevance of mutations in B-cell receptor/nuclear factor-κB signaling and immune escape pathways in IVLBCL and identify novel driver alterations. The similar mutational profile in tumors with extravascular dissemination suggests that these cases may also be considered in the spectrum of IVLBCL.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history which is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy chain variable region of immunoglobulin genes (IGHV). To build the ‘CLL map,’ we integrated genomic, transcriptomic, and epigenomic data from 1148 patients. We identified 202 candidate genetic drivers of CLL (109 novel) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic, and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Context.— Despite their stromal origin, follicular dendritic cells (FDCs) share many functions with hematopoietic system cells. FDC neoplasms are currently classified by the World Health Organization along with those of a histiocytic nature. However, the molecular alterations driving oncogenesis in FDC sarcomas (FDCSs) are beginning to be unveiled, and do not seem to concur with those described in histiocytic neoplasms, namely MAPK pathway activation. Objective.— To identify molecular alterations driving tumorigenesis in FDCS. Design.— We investigated the role of MYC and TP53 in FDC-derived tumor oncogenesis and assessed comprehensively the status of the MAPK pathway in 16 FDCSs, 6 inflammatory pseudotumor (IPT)–like FDCSs, and 8 IPTs. Results.— MYC structural alterations (both amplifications and rearrangements) were identified in 5 of 14 FDCSs (35.7%), all associated with MYC overexpression. TP53 mutations were identified in 4 of 14 FDCSs (28.6%), all of which displayed intense and diffuse p53 expression. None of these alterations were identified in any IPT-like FDCS or in IPT cases. No MAPK pathway gene alterations were identified in any of the cases studied. Conclusions.— The presence of MYC and TP53 alterations and the lack of association with Epstein-Barr virus segregate classical FDCS from IPT-like FDCS, pointing at different oncogenic mechanisms in both entities. Our results suggest a possible oncogenic role of MYC and TP53 alterations in FDCS. The absence of MAPK pathway alterations confirms the lack of a significant role of this pathway in the oncogenesis of FDC-derived neoplasms.

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11– MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

Serum soluble CD23 (sCD23) levels have been acknowledged as a prognostic factor in patients with chronic lymphocytic leukemia (CLL), but their potential relevance has not been analyzed in recent times. We retrospectively studied 338 CLL, small lymphocytic lymphoma, or CLL-type monoclonal B-cell lymphocytosis patients from a single institution, with available sCD23 levels at diagnosis. Baseline features and outcomes were compared between patients with sCD23 ≤/1000 UI/L. The 140 patients (41%) who had sCD23 1000 UI/L showed adverse-risk clinical and biological characteristics. High sCD23 levels were predictive of a shorter time to first treatment (5-year probability of requiring treatment: 60 vs. 20%, p 0.0001; hazard ratio (HR) = 1.72, p = 0.003 in a multivariable model also including the CLL International Prognostic Index and the absolute lymphocyte count), and a poorer 5-year overall survival (70 vs. 82%, p = 0.0009). These data suggest the potential of sCD23 to predict treatment-free survival and to shed light on mechanisms of activity and resistance to CD23-directed therapies.

MotivationThe cost reduction in sequencing and the extensive genomic characterization of a wide variety of cancers is expanding the use of tumor sequencing approaches to a wide number of research groups and to the clinical practice. Although specific pipelines have been generated for the identification of somatic mutations, their results usually differ considerably, and a common approach in many projects is to use several callers to achieve a more reliable set of mutations. This procedure is computationally very expensive and time-consuming, and it suffers from the same limitations in sensitivity and specificity as other approaches. Expert revision of mutant calls is therefore required to verify calls that might be used for clinical diagnosis. Machine learning techniques provide a useful approach to incorporate expert-reviewed information for the identification of somatic mutations.ResultsWe have developed RFcaller, a pipeline based on machine learning algorithms, for the detection of somatic mutations in tumor-normal paired samples. RFcaller shows high accuracy for the detection of substitutions and indels from whole genome or exome data. It allows the detection of mutations in driver genes missed by other approaches, and has been validated by comparison to deep sequencing and Sanger sequencing. The pipeline is able to analyze a whole genome in a small period of time, and with a small computational footprint.Availability and implementationRFcaller is available at GitHub repository (https://github.com/xa-lab/RFcaller) and DockerHub (https://hub.docker.com/repository/docker/labxa/rfcaller).Contactxspuente@uniovi.esSupplementary informationSupplementary data is available online.

Mutational signatures have emerged as powerful biomarkers in cancer patients, with prognostic and therapeutic implications. Wider clinical utility requires access to reproducible algorithms, which allow characterization of mutational signatures in a given tumor sample. Here, we show how mutational signature fitting can be applied to hematological cancer genomes to identify biologically and clinically important mutational processes, highlighting the importance of careful interpretation in light of biological knowledge. Our newly released R package mmsig comes with a dynamic error-suppression procedure that improves specificity in important clinical and biological settings. In particular, mmsig allows accurate detection of mutational signatures with low abundance, such as those introduced by APOBEC cytidine deaminases. This is particularly important in the most recent mutational signature reference (COSMIC v3.1) where each signature is more clearly defined. Our mutational signature fitting algorithm mmsig is a robust tool that can be implemented immediately in the clinic., Rustad et al. present a software package for the R statistical environment for the accurately quantify of somatic mutational signatures in hematological malignancies

Frailty and concurrent medical conditions are crucial factors in the management of follicular lymphoma (FL). We evaluated the impact of age and comorbidity on survival, causes of death, histological transformation (HT), and second malignancies (SM) in a large single-center series of grade 1-3A FL. We studied 414 patients diagnosed in the rituximab era, categorized into three age groups (≤60, 61-70,70 years) and two comorbidity groups (Charlson Comorbidity Index, CCI, 0-1 and ≥2). Despite a similar cumulative incidence of relapse, older and comorbid patients had a lower 10-year overall survival (OS, 88, 65, and 41% for patients ≤60 years, 61-70 years, and70 years, P0.0001; and 76 vs. 51% for CCI 0-1 and ≥2, P0.0001). In a multivariate analysis for OS, comorbidity retained its prognostic impact (HR=2.5, P=0.0003). The proportion of patients dying due to FL was higher among those ≤60 years (74%) and those with a CCI 0-1 (67%). Furthermore, 10-year excess mortality (survival reduction) was more prominent for patients70 years (30%) and those with a CCI ≥2 (32%). Patients with a CCI ≥2 also had a higher incidence of SM. These data encourage a comprehensive pre-treatment evaluation and a tailored therapeutic approach for all FL patients.

Purpose: Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression. Cyclin D1 is a major cell-cycle regulator and also regulates transcription, but the impact of cyclin D1–mediated transcriptional dysregulation on MCL pathogenesis remains poorly understood. The aim of this study was to define a cyclin D1–dependent gene expression program and analyze its prognostic value. Experimental Design: We integrated genome-wide expression analysis of cyclin D1–silenced and overexpressing cells with cyclin D1 chromatin-binding profiles to identify a cyclin D1–dependent transcriptional program in MCL cells. We analyzed this gene program in two MCL series of peripheral blood samples (n = 53) and lymphoid tissues (n = 106) to determine its biological and clinical relevance. We then obtained a simplified signature of this program and evaluated a third series of peripheral blood MCL samples (n = 81) by NanoString gene expression profiling to validate our findings. Results: We identified a cyclin D1–dependent transcriptional program composed of 295 genes that were mainly involved in cell-cycle control. The cyclin D1–dependent gene program was overexpressed in MCL tumors directly proportional to cyclin D1 levels. High expression of this program conferred an adverse prognosis with significant shorter overall survival of the patients. These observations were validated in an independent cohort of patients using a simplified 37-gene cyclin D1 signature. The cyclin D1–dependent transcriptional program was also present in multiple myeloma and breast tumors with cyclin D1 overexpression. Conclusions: We identified a cyclin D1–dependent transcriptional program that is overexpressed in MCL and predicts clinical outcome.