ViCloD, an interactive web tool for visualizing B cell repertoires and analyzing intraclonal diversities: application to human B-cell tumors.

High throughput sequencing of adaptive immune receptor repertoire (AIRR-seq) has provided numerous human immunoglobulin (IG) sequences allowing specific B cell receptor (BCR) studies such as the antigen-driven evolution of antibodies (soluble forms of the membrane-bound IG part of the BCR). AIRR-seq data allows researchers to examine intraclonal differences caused primarily by somatic hypermutations in IG genes and affinity maturation. Exploring this essential adaptive immunity process could help elucidate the generation of antibodies with high affinity or broadly neutralizing activities. Retracing their evolutionary history could also clarify how vaccines or pathogen exposition drive the humoral immune response, and unravel the clonal architecture of B cell tumors. Computational methods are necessary for large-scale analysis of AIRR-seq properties. However, there is no efficient and interactive tool for analyzing intraclonal diversity, permitting users to explore adaptive immune receptor repertoires in biological and clinical applications. Here we present ViCloD, a web server for large-scale visual analysis of repertoire clonality and intraclonal diversity. ViCloD uses preprocessed data in the format defined by the Adaptive Immune Receptor Repertoire (AIRR) Community. Then, it performs clonal grouping and evolutionary analyses, producing a collection of useful plots for clonal lineage inspection. The web server presents diverse functionalities, including repertoire navigation, clonal abundance analysis, and intraclonal evolutionary tree reconstruction. Users can download the analyzed data in different table formats and save the generated plots as images. ViCloD is a simple, versatile, and user-friendly tool that can help researchers and clinicians to analyze B cell intraclonal diversity. Moreover, its pipeline is optimized to process hundreds of thousands of sequences within a few minutes, allowing an efficient investigation of large and complex repertoires.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Immunoglobulin Gene Mutational Status Assessment by Next Generation Sequencing in Chronic Lymphocytic Leukemia.

B cell receptor (BcR) immunoglobulins (IG) display a tremendous diversity due to complex DNA rearrangements, the V(D)J recombination, further enhanced by the somatic hypermutation process. In chronic lymphocytic leukemia (CLL), the mutational load of the clonal BcR IG expressed by the leukemic cells constitutes an important prognostic and predictive biomarker. Here, we provide a reliable methodology capable of determining the mutational status of IG genes in CLL using high-throughput sequencing, starting from leukemic cell DNA or RNA.

Cytogenetic and molecular abnormalities in Waldenström's macroglobulinemia patients: Correlations and prognostic impact.

While Waldenström macroglobulinemia (WM) is characterized by an almost unifying mutation in MYD88, clinical presentation at diagnosis and response to therapy can be widely different among WM patients. Current prognostic tools only partially address this clinical heterogeneity. Limited data compiling both molecular and cytogenetic information have been used in risk prognostication in WM. To investigate the clinical impact of genetic alterations in WM, we evaluated cytogenetic and molecular abnormalities by chromosome banding analyses, FISH and targeted NGS in a retrospective cohort of 239 WM patients, including 187 patients treated by first-line chemotherapy or immunochemotherapy. Most frequent mutations were identified in MYD88 (93%), CXCR4 (29%), MLL2 (11%), ARID1A (8%), TP53 (8%), CD79A/B (6%), TBL1XR1 (4%) and SPI1 (4%). The median number of cytogenetic abnormalities was two (range, 0-22). Main cytogenetic abnormalities were 6q deletion (del6q) (27%), trisomy 4 (tri4) (12%), tri18 (11%), del13q (11%), tri12 (7.5%) and del17p (7%). Complex karyotype (CK) was observed in 15% (n = 31) of cases, including 5% (n = 12) of highly CK (high-CK). TP53 abnormalities (TP53abn) were present in 15% of evaluable patients. TP53abn and del6q were associated with CK/high-CK (p < .05). Fifty-three percent of patients with hyperviscosity harbored CXCR4 mutations. Cytogenetic and molecular abnormalities did not significantly impact time to first treatment and response to therapy. Prognostic factors associated with shorter PFS were del6q (p = .01), TP53abn (p = .002) and high-CK (p = .01). These same factors as well as IPSSWM, tri4, CXCR4 frameshift and SPI1 mutations were significantly associated with lower OS (p < .05). These results argue for integration of both cytogenetic and molecular screening in evaluation of first-line WM patients.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

High frequency of clonal hematopoiesis in Erdheim-Chester disease.

Erdheim-Chester disease (ECD) is a clonal hematopoietic disorder characterized by the accumulation of foamy histiocytes within organs (in particular, frequent retroperitoneal involvement) and a high frequency of BRAFV600E mutations. Although ECD is not commonly recognized to have overt peripheral blood (PB) or bone marrow (BM) disease, we recently identified that ECD patients have a high frequency of a concomitant myeloid malignancy. We thus conducted a systematic clinical and molecular analysis of the BM from 120 ECD patients. Surprisingly, 42.5% of ECD patients (51 of 120) had clonal hematopoiesis whereas 15.8% of patients (19 of 120) developed an overt hematologic malignancy (nearly all of which were a myeloid neoplasm). The most frequently mutated genes in BM were TET2, ASXL1, DNMT3A, and NRAS. ECD patients with clonal hematopoiesis were more likely to be older (P < .0001), have retroperitoneal involvement (P = .02), and harbor a BRAFV600E mutation (P = .049) than those without clonal hematopoiesis. The presence of the TET2 mutation was associated with a BRAFV600E mutation in tissue ECD lesions (P = .0006) and TET2-mutant ECD patients were more likely to have vascular involvement than TET2 wild-type ECD patients. Clonal hematopoiesis mutations in ECD were detected in cells derived from CD34+CD38- BM progenitors and PB monocytes but less frequently present in PB B and T lymphocytes. These data identify a heretofore unrecognized high frequency of clonal hematopoiesis in ECD patients, reaffirm the development of additional high risk of myeloid neoplasms in ECD, and provide evidence of a BM-based precursor cell of origin for many patients with ECD.

Identification of 2 DNA methylation subtypes of Waldenström macroglobulinemia with plasma and memory B-cell features.

Epigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients.

A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium.

T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.

Optimization of CSF biological investigations for CNS lymphoma diagnosis.

Diagnosis of lymphoma leptomeningeal dissemination is challenging and relies on a wide array of methods. So far, no consensus biological guidelines are available. This increases the chance of intra- and interpractice variations, despite the shared concern to perform the minimum amount of tests while preserving clinically relevant results.We evaluated a training cohort of 371 cerebrospinal fluid (CSF) samples from patients with putative lymphomatous central nervous system (CNS) localization using conventional cytology (CC), flow cytometry (FCM), molecular clonality assesment by PCR and cytokine quantification (CQ). This led us to propose a biological algorithm, which was then verified on a validation cohort of 197 samples. The samples were classified according to the clinical context and the results of each technique were compared. Using all four techniques was not useful for exclusion diagnosis of CNS lymphoma (CNSL), but they proved complementary for cases with suspected CNSL. This was particularly true for CQ in primary CNSL. Overall, diagnosis can be obtained with a two-step approach. The first step comprises CC and FCM, as results are available quickly and FCM is a sensitive method. Both PCR and CQ can be postponed and performed in a second step, depending on the results from the first step and the clinical context.The proposed algorithm missed none of the CNSL samples of the validation cohort. Moreover, applying this algorithm would have spared 30% of PCR tests and 20% of CQ over a one-year period, without compromising clinical management.

A Recurrent Activating Missense Mutation in Waldenström Macroglobulinemia Affects the DNA Binding of the ETS Transcription Factor SPI1 and Enhances Proliferation.

The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.This article is highlighted in the In This Issue feature, p. 681.

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors.See related commentary by Shingleton and Dave, p. 1515.This article is highlighted in the In This Issue feature, p. 1494.

Pathology of Nerve Biopsy and Diagnostic Yield of PCR-Based Clonality Testing in Neurolymphomatosis.

Infiltration of the peripheral nervous system (PNS) by lymphoma, called neurolymphomatosis, is a rare condition among the spectrum of lymphoma-associated neuropathies; its diagnosis is challenging. Cerebrospinal fluid (CSF) analysis is of great value, but nerve biopsy (NB) may be necessary to prove invasion by malignant cells. Clonality polymerase chain reaction (PCR)-based analysis is a validated method in the diagnosis of hematological malignancies, but there are very little data on its diagnostic yield on NB samples. We explored the contribution of NB with clonality analysis to the diagnosis of neurolymphomatosis in 15 patients with negative CSF analysis. Moreover, we assessed the performance of clonality testing in a case-control manner, using patients with inflammatory infiltrates on NB as controls. Neurolymphomatosis was the first manifestation of lymphoma in 60% and could be diagnosed on routine histology alone in 40%. Clonality testing showed monoclonal rearrangement in 86.7% and was unsuccessful in 8.1%. Performance of clonality testing was as follows: 92.9% positive predictive value, 90% negative predictive value, 86.7% sensitivity, 94.7% specificity. This study confirms the diagnostic challenge of neurolymphomatosis, the usefulness of NB in patients with negative CSF analysis, and highlights the high yield of PCR-based clonality testing to assess the malignant nature of PNS lymphoid infiltrates.

"Double-hit" chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain.

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p-) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p- and the additional aberrations influenced the prognosis in a series of 195 patients with 17p-CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment-free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this "double-hit" CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p- CLL.

An unusual case of acute leukemia.

We report the case of a 31 year-old man diagnosed with an atypical acute leukemia difficult to characterize cytologically. The immunophenotyping identified a blastic population co-expressing myeloid, lymphoid B and lymphoid T markers suggesting the diagnosis of either a mixed phenotype acute leukemia (MPAL) or an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Because of the poor prognosis linked to these leukemias, the patient benefited from chemotherapy targeting both myeloid and lymphoid components, followed by allogeneic hematopoietic stem cell transplantation. DNA-based techniques analyzing B and T-cell clonality identified partial rearrangements in immunoglobulin and TCR genes, allowing the monitoring of minimal residual disease. This observation highlights the difficulty to classify some atypical cases of acute leukemias. It emphasizes on the complementarity of cytomorphology, immunophenotyping by flow cytometry and molecular techniques in order to promptly characterize and treat these leukemias.

Hepatitis C virus - Associated marginal zone lymphoma.

The link between hepatitis C virus (HCV) infection and the development of B-cell non-Hodgkin lymphoma is now well established and based on a number of epidemiological studies. It is further supported by the observation of lymphoma regression after HCV eradication by antiviral treatment. The far most frequent entities are marginal zone lymphoma (MZL) and diffuse large B-cell lymphoma (DLBCL). MZL usually emerge on a background of mixed cryoglobulinemia, a low-grade lymphoproliferation, and often transform into DLBCL, thereby following a multistep oncogenesis process. The role of HCV in lymphomagenesis is not yet fully understood but several mechanisms have been proposed including (i) chronic external stimulation through the B-cell receptor and other surface receptors, and (ii) direct transformation by intracellular viral proteins, the former being probably predominant in MZL. Regression of HCV-associated MZL can be achieved with antiviral therapy and the novel generation of direct-acting antiviral agents appears highly effective and safe for the treatment of these lymphoma.

The CSF IL-10 concentration is an effective diagnostic marker in immunocompetent primary CNS lymphoma and a potential prognostic biomarker in treatment-responsive patients.

INTRODUCTION: We aimed to confirm the diagnostic value and to evaluate the pre- and post-therapeutic prognostic value of cerebrospinal fluid (CSF) concentrations of interleukin (IL)-10 and IL-6 in patients with diffuse large B-cell primary central nervous system lymphoma (PCNSL). PATIENTS AND METHODS: IL-10 and IL-6 concentrations were measured in 79 patients with PCNSL at diagnosis and in 40 control individuals. Fifty-four PCNSL patients underwent repeat assessments starting at diagnosis. RESULTS: The IL-10 concentration distinguished PCNSL from other neurologic diseases with a sensitivity of 88.6% and a specificity of 88.9% with a cutoff of 4 pg/ml. In a multivariate analysis of PCNSL patients, CSF involvement was associated with a higher IL-10 concentration (mean log (IL-10) of 4.4 versus 2.5 pg/ml, respectively, p = 0.0004). The pre-therapeutic IL-10 concentration had no prognostic impact on outcome. The IL-10 concentration decreased after treatment for most patients tested. Among patients with complete remission or partial remission, as evaluated by magnetic resonance imaging (MRI), a persistent detectable IL-10 level in the CSF at the end of treatment was associated with a negative impact on progression-free survival (PFS) (1-year PFS: 15%, 95% confidence interval [CI]: 2.5-38% versus 59%, 95% CI: 32-78%, respectively, p = 0.0004). CONCLUSION: Our study confirmed that IL-10 is a useful biomarker for the diagnosis of PCNSL. We highlight new findings showing that the IL-10 level in the CSF could be used as a surrogate marker for CSF involvement and that the post-treatment IL-10 concentration could complement standard MRI for therapeutic response assessment in PCNSL.