Analysis of immunoglobulin/T-cell receptor repertoires by high-throughput RNA sequencing reveals a continuous dynamic of positive clonal selection in follicular lymphoma.

Follicular lymphoma (FL) course is highly variable, making its clinical management challenging. In this incurable and recurring pathology, the interval between relapses tends to decrease while aggressiveness increases, sometimes resulting in the transformation to higher-grade lymphoma. These evolutions are particularly difficult to anticipate, resulting from complex clonal evolutions where multiple subclones compete and thrive due to their capacity to proliferate and resist therapies. Here, to apprehend further these processes, we used a high-throughput RNA sequencing approach to address simultaneously the B-cell immunoglobulin repertoires and T-cell immunoglobulin repertoires repertoires of lymphoma cells and their lymphoid microenvironment in a large cohort of 131 FL1/2-3A patients. Our data confirm the existence of a high degree of intra-clonal heterogeneity in this pathology, resulting from ongoing somatic hyper-mutation and class switch recombination. Through the evaluation of the Simpson ecological-diversity index, we show that the contribution of the cancerous cells increases during the course of the disease to the detriment of the reactive compartment, a phenomenon accompanied by a concomitant decrease in the diversity of the tumoral population. Clonal evolution in FL thus contrasts with many tumors, where clonal heterogeneity steadily increases over time and participates in treatment evasion. In this pathology, the selection of lymphoma subclones with proliferative advantages progressively outweighs clonal diversification, ultimately leading in extreme cases to transformation to high-grade lymphoma resulting from the rapid emergence of homogeneous subpopulations.

High PDL1/PDL2 gene expression correlates with worse outcome in primary mediastinal large B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is an uncommon entity of aggressive B-cell lymphoma with an unusually good prognosis, except for 10-15% of chemotherapy-refractory cases. To identify earlier these higher risk patients, we performed molecular characterization of a retrospective multicenter cohort of patients treated with firstline immunochemotherapy. The traits of the patients with gene-expression profiling data (n = 120) were as follows: median age of 34 years (range, 18-67 years); female sex, 58.3%; elevated lactate dehydrogenase, 82.5%; Eastern Cooperative Oncology Group performance status score of 0 to 1, 85.7%; Ann Arbor stage I/II, 55%; International Prognostic Index score of 1 to 2, 64.4%; and median metabolic tumor volume, 290.4 cm3 (range, 15.7-1147.5 cm3). Among all 137 markers tested for correlation with survival data, only programmed death-ligand (PDL) 1 and PDL2 expression showed a prognostic impact. Overall, both PDL1 and PDL2 genes were highly expressed in 37 patients (30.8%; PDL1high/PDL2high). The baseline clinical characteristics of patients with PDL1high/PDL2high were similar to those of other patients. In univariate analysis, PDL1high/PDL2high status was associated with poor progression-free survival (PFS) (hazard ratio [HR], 4.292) and overall survival (OS; HR, 8.24). In multivariate analysis, PDL1high/PDL2high status was an independent prognostic factor of adverse outcomes (PFS: HR, 5.22; OS: HR, 10.368). We validated these results in an independent cohort of 40 patients and confirmed the significant association between PDL1high/PDL2high status and inferior PFS (HR, 6.11). High PDL1/PDL2 gene expression defines a population with strong immune privilege and poorer outcomes from standard chemotherapy who might benefit from firstline checkpoint inhibitor therapy.

5' Rapid amplification of cDNA ends (5'RACE): A simpler method to analyze immunoglobulin genes and discover the value of the light chain in chronic lymphocytic leukemia.

The mutational status of the variable region of the immunoglobulin heavy chain gene (IGHV) is a very important biomarker for chronic lymphocytic leukemia (CLL) patients. However, the routine detection of IGHV mutational status is time-consuming and costly. Therefore, we performed 5' Rapid amplification of cDNA ends (5' RACE) in 81 CLL patients who previously underwent detection using Biomed-2. The agreement rate of these two methods was 93.8 %. Regarding the discordant cases, 5' RACE was more sensitive to identify unproductive and multiple rearrangements. Furthermore, 5' RACE can also be used to simultaneously sequence light chains. In most CLL cases, the mutational statuses of heavy and light chains are concordant, except in IGLV3-21. Most IGLV3-21 (24/25) rearrangement shared a similar LCDR3 (QVWDSSSDHPWV) and harbored a single point mutation, namely, IGLV3-21R110. Compared to mutated-CLL non IGLV3-2R110, IGLV3-21R110-CLL exhibited a shorter overall survival (OS) and time to first treatment (TTFT) (p = 0.05, p < 0.0001, respectively) even though 75 % (18/24) of these patients expressed mutated heavy chains. Altogether, IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and emphasizes the important value of the light chain. This study is the first to use 5' RACE to detect the mutational status of IGH in CLL. Here, 5' RACE was a reliable and effective method to test the mutational status of heavy and light chains. In addition, 5' RACE can be combined with other assays in the NGS workflow to obtain more detailed insight into subclonal architecture and intraclonal diversity.

UMI-Varcal: A Low-Frequency Variant Caller for UMI-Tagged Paired-End Sequencing Data.

The rapid transition from traditional sequencing methods to Next-Generation Sequencing (NGS) has allowed for a faster and more accurate detection of somatic variants (Single-Nucleotide Variant (SNV) and Copy Number Variation (CNV)) in tumor cells. NGS technologies require a succession of steps during which false variants can be silently added at low frequencies. Filtering these artifacts can be a rather difficult task especially when the experiments are designed to look for very low frequency variants. Recently, adding unique molecular barcodes called UMI (Unique Molecular Identifier) to the DNA fragments appears to be a very effective strategy to specifically filter out false variants from the variant calling results (Kukita et al. DNA Res 22(4):269-277, 2015; Newman et al. Nat Biotechnol 34(5):547-555, 2016; Schmitt et al. Proc Natl Acad Sci U S A 109(36):14508-14513). Here, we describe UMI-VarCal (Sater et al. Bioinformatics 36:2718-2724, 2020), which can use the UMI information from UMI-tagged reads to offer a faster and more accurate variant calling analysis.

Circulating tumor DNA in primary mediastinal large B-cell lymphoma versus classical Hodgkin lymphoma: a retrospective study.

Few data exist concerning circulating tumor DNA (ctDNA) relevance in primary mediastinal B-cell lymphoma (PMBL). To explore this topic, we applied a 9-gene next-generation sequencing pipeline to samples from forty-four PMBL patients (median age 36.5 years). The primary endpoint was a similarity between paired biopsy/plasma mutational profiles. We detected at least one variant in 32 plasma samples (80%). The similarity between the biopsy and ctDNA genetic profiles for the 30 patients with paired mutated biopsy/plasma samples was greater than or equal to 80% in 19 patients (63.3%). We then compared PMBL ctDNA features with those of a cohort of Hodgkin lymphoma patients (n = 60). The top three mutated genes were SOCS1, TNFAIP3, and B2M in both lymphoma types. PMBL displayed more alterations in TNFAIP3 (71.9% vs. 46.3%, p = 0.029) and GNA13 (46.9% vs. 17.1%, p = 0.013) than cHL. Our 9-gene set may delineate tumor genotypes using ctDNA samples from both lymphoma types.

Concomitant occurrence of genetically distinct Hodgkin lymphoma and primary mediastinal lymphoma.

Synchronous Hodgkin Lymphoma and Primary Mediastinal B-cell Lymphoma is possible, with molecular analyses proving the absence of clonal filiation between both entities. This suggests a common etiology but the existence of two divergent clones.

cfDNA Sequencing: Technological Approaches and Bioinformatic Issues.

In the era of precision medicine, it is crucial to identify molecular alterations that will guide the therapeutic management of patients. In this context, circulating tumoral DNA (ctDNA) released by the tumor in body fluids, like blood, and carrying its molecular characteristics is becoming a powerful biomarker for non-invasive detection and monitoring of cancer. Major recent technological advances, especially in terms of sequencing, have made possible its analysis, the challenge still being its reliable early detection. Different parameters, from the pre-analytical phase to the choice of sequencing technology and bioinformatic tools can influence the sensitivity of ctDNA detection.

Improving high-resolution copy number variation analysis from next generation sequencing using unique molecular identifiers.

BACKGROUND: Recently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct. RESULTS: Here, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization. CONCLUSION: We provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

Targeted genotyping of circulating tumor DNA for classical Hodgkin lymphoma monitoring: a prospective study.

The relevance of circulating tumor DNA (ctDNA) analysis as a liquid biopsy and minimal residual disease tool in the management of classical Hodgkin Lymphoma (cHL) patients was demonstrated in retrospective settings and remains to be confirmed in a prospective setting. We developed a targeted Next-Generation sequencing (NGS) panel for fast analysis (AmpliSeq technology) of nine commonly mutated genes in biopies and ctDNA of cHL patients. We then conducted a prospective trial to assess ctDNA follow up at diagnosis and after 2 cycles of chemotherapy (C2). Sixty cHL patients treated by first line conventional chemotherapy (BEACOPPescalated [21.3%], ABVD/ABVD-like [73.5%] and other regimens [5.2%, for elderly patients] were assessed in this non-interventional study. Median age of the patients was 33.5 years (range 20-86). Variants were identified in 42 (70%) patients. Mutations of NFKBIE, TNFAIP3, STAT6, PTPN1, B2M, XPO1, ITPKB, GNA13 and SOCS1 were found in 13.3%, 31.7%, 23.3%, 5%, 33.3%, 10%, 23.3%, 13.3% and 50% of patients, respectively. ctDNA concentration and genotype are correlated with clinical characteristics and presentation. Regarding early therapeutic response, 45 patients (83%, NA=6) had a negative positron emission tomography (PET) after C2 (Deauville Score 1-3). Mean of DeltaSUVmax after C2 was -78.8%. We analyzed ctDNA after C2 for 54 patients (90%). ctDNA became rapidly undetectable in all cases after C2. Variant detection in ctDNA is suitable to depict the genetic features of cHL at diagnosis and may help to assess early treatment response, in association with PET. Clinical Trial reference: NCT02815137.

XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-cell Lymphoma.

The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR-Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin ß1 modified the nuclear export/import dynamics of relevant cargoes.

Correlations between baseline 18F-FDG PET tumour parameters and circulating DNA in diffuse large B cell lymphoma and Hodgkin lymphoma.

BACKGROUND: 18F-FDG PET/CT is a standard for many B cell malignancies, while blood DNA measurements are emerging tools. Our objective was to evaluate the correlations between baseline PET parameters and circulating DNA in diffuse large B cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). METHODS: Twenty-seven DLBCL and forty-eight cHL were prospectively included. Twelve PET parameters were analysed. Spearman's correlations were used to compare PET parameters each other and to circulating cell-free DNA ([cfDNA]) and circulating tumour DNA ([ctDNA]). p values were controlled by Benjamini-Hochberg correction. RESULTS: Among the PET parameters, three different clusters for tumour burden, fragmentation/massiveness and dispersion parameters were observed. Some PET parameters were significantly correlated with blood DNA parameters, including the total metabolic tumour surface (TMTS) describing the tumour-host interface (e.g. ρ = 0.81 p < 0.001 for [ctDNA] of DLBLC), the tumour median distance between the periphery and the centroid (medPCD) describing the tumour's massiveness (e.g. ρ = 0.81 p < 0.001 for [ctDNA] of DLBLC) and the volume of the bounding box including tumours (TumBB) describing the disease's dispersion (e.g. ρ = 0.83 p < 0.001 for [ctDNA] of DLBLC). CONCLUSIONS: Some PET parameters describing tumour burden, fragmentation/massiveness and dispersion are significantly correlated with circulating DNA parameters of DLBCL and cHL patients. These results could help to understand the pathophysiology of B cell malignancies.

Combining gene expression profiling and machine learning to diagnose B-cell non-Hodgkin lymphoma.

Non-Hodgkin B-cell lymphomas (B-NHLs) are a highly heterogeneous group of mature B-cell malignancies. Their classification thus requires skillful evaluation by expert hematopathologists, but the risk of error remains higher in these tumors than in many other areas of pathology. To facilitate diagnosis, we have thus developed a gene expression assay able to discriminate the seven most frequent B-cell NHL categories. This assay relies on the combination of ligation-dependent RT-PCR and next-generation sequencing, and addresses the expression of more than 130 genetic markers. It was designed to retrieve the main gene expression signatures of B-NHL cells and their microenvironment. The classification is handled by a random forest algorithm which we trained and validated on a large cohort of more than 400 annotated cases of different histology. Its clinical relevance was verified through its capacity to prevent important misclassification in low grade lymphomas and to retrieve clinically important characteristics in high grade lymphomas including the cell-of-origin signatures and the MYC and BCL2 expression levels. This accurate pan-B-NHL predictor, which allows a systematic evaluation of numerous diagnostic and prognostic markers, could thus be proposed as a complement to conventional histology to guide the management of patients and facilitate their stratification into clinical trials.

[Low-grade dural extranodal marginal zone lymphoma]. / Lymphome de la zone marginale localisé à la dure-mère.

Primary low-grade dural marginal zone lymphoma is an indolent low grade lymphoma occurring especially among middle-aged immunocompetent women, and is not associated to an infectious process, contrary to gastric or intestinal marginal zone lymphomas. Dural location is rare since only 105 cases have been reported so far. We report herein on two additional cases, a 72-year-old woman and a 36-year-old man whose lymphoma was revealed by partial seizures and headaches. Morphological analysis of surgical specimens displayed a tumoral proliferation made of small lymphocytes arranged in sheets or in nodules with CD20, CD79a and BCL2-immunopositivity, but CD5 and CD10 negativity. Molecular analysis using a panel of 34 genes involved in lymphomagenesis disclosed a deletion of SOCS1 and TNFAIP3 genes, implicated in the JAK/STAT and NFκB pathways respectively in the first patient that could explain unfavourable prognosis despite complementary radiotherapy. No anomaly was identified in the second patient who is alive with no recurrence or progression seven years after the diagnosis. Currently, there are no standardized treatment schedules, but the vast majority of patients are treated by surgery, then radiotherapy followed by adjuvant chemotherapy using methotrexate alone or in combination with rituximab. Literature review indicates that five-year survival has been estimated at 96.7%, suggesting a better prognosis compared to other locations.

UMI-VarCal: a new UMI-based variant caller that efficiently improves low-frequency variant detection in paired-end sequencing NGS libraries.

MOTIVATION: Next-generation sequencing has become the go-to standard method for the detection of single-nucleotide variants in tumor cells. The use of such technologies requires a PCR amplification step and a sequencing step, steps in which artifacts are introduced at very low frequencies. These artifacts are often confused with true low-frequency variants that can be found in tumor cells and cell-free DNA. The recent use of unique molecular identifiers (UMI) in targeted sequencing protocols has offered a trustworthy approach to filter out artefactual variants and accurately call low-frequency variants. However, the integration of UMI analysis in the variant calling process led to developing tools that are significantly slower and more memory consuming than raw-reads-based variant callers. RESULTS: We present UMI-VarCal, a UMI-based variant caller for targeted sequencing data with better sensitivity compared to other variant callers. Being developed with performance in mind, UMI-VarCal stands out from the crowd by being one of the few variant callers that do not rely on SAMtools to do their pileup. Instead, at its core runs an innovative homemade pileup algorithm specifically designed to treat the UMI tags in the reads. After the pileup, a Poisson statistical test is applied at every position to determine if the frequency of the variant is significantly higher than the background error noise. Finally, an analysis of UMI tags is performed, a strand bias and a homopolymer length filter are applied to achieve better accuracy. We illustrate the results obtained using UMI-VarCal through the sequencing of tumor samples and we show how UMI-VarCal is both faster and more sensitive than other publicly available solutions. AVAILABILITY AND IMPLEMENTATION: The entire pipeline is available at https://gitlab.com/vincent-sater/umi-varcal-master under MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Refining diffuse large B-cell lymphoma subgroups using integrated analysis of molecular profiles.

BACKGROUND: Gene expression profiling (GEP), next-generation sequencing (NGS) and copy number variation (CNV) analysis have led to an increasingly detailed characterization of the genomic profiles of DLBCL. The aim of this study was to perform a fully integrated analysis of mutational, genomic, and expression profiles to refine DLBCL subtypes. A comparison of our model with two recently published integrative DLBCL classifiers was carried out, in order to best reflect the current state of genomic subtypes. METHODS: 223 patients with de novo DLBCL from the prospective, multicenter and randomized LNH-03B LYSA clinical trials were included. GEP data was obtained using Affymetrix GeneChip arrays, mutational profiles were established by Lymphopanel NGS targeting 34 key genes, CNV analysis was obtained by array CGH, and FISH and IHC were performed. Unsupervised independent component analysis (ICA) was applied to GEP data and integrated analysis of multi-level molecular data associated with each component (gene signature) was performed. FINDINGS: ICA identified 38 components reflecting transcriptomic variability across our DLBCL cohort. Many of the components were closely related to well-known DLBCL features such as cell-of-origin, stromal and MYC signatures. A component linked to gain of 19q13 locus, among other genomic alterations, was significantly correlated with poor OS and PFS. Through this integrated analysis, a high degree of heterogeneity was highlighted among previously described DLBCL subtypes. INTERPRETATION: The results of this integrated analysis enable a global and multi-level view of DLBCL, as well as improve our understanding of DLBCL subgroups.

Mutations of the B-Cell Receptor Pathway Confer Chemoresistance in Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

A recurrent clonally distinct Burkitt lymphoma case highlights genetic key events contributing to oncogenesis.

Burkitt lymphoma (BL) is characterized by a translocation of the MYC oncogene that leads to the upregulation of MYC expression, cell growth and proliferation. It is well-established that MYC translocation is not a sufficient genetic event to cause BL. Next-generation sequencing has recently provided a comprehensive analysis of the landscape of additional genetic events that contribute to BL lymphomagenesis. Refractory BL or relapsing BL are almost always incurable as a result of the selection of a highly chemoresistant clonally related cell population. Conversely, a few BL recurrence cases arising from clonally distinct tumors have been reported and were associated with a favorable outcome similar to that reported for first-line treatment. Here, we used an unusual case of recurrent but clonally distinct EBV+ BL to highlight the key genetic events that drive BL lymphomagenesis. By whole exome sequencing, we established that ID3 gene was targeted by distinct mutations in the two clonally unrelated diseases, highlighting the crucial role of this gene during lymphomagenesis. We also detected a heterozygous E1021K PIK3CD mutation, thus increasing the spectrum of somatic mutations altering the PI3K signaling pathway in BL. Interestingly, this mutation is known to be associated with activated phosphoinositide 3-kinase delta syndrome (APDS). Finally, we also identified an inherited heterozygous truncating c.5791CT FANCM mutation that may contribute to the unusual recurrence of BL.

Cyclin D1-positive Mediastinal Large B-Cell Lymphoma With Copy Number Gains of CCND1 Gene: A Study of 3 Cases With Nonmediastinal Disease.

Primary mediastinal large B-cell lymphoma (PMBL) is a mature large B-cell lymphoma of putative thymic B-cell origin involving the mediastinum with younger age distribution and better prognosis than diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Recently, based on gene expression profile analysis and morphologic findings, cases of PMBL without mediastinal involvement have been reported. In this study, we analyzed 3 cases of nodal DLBCL with morphologic features of PMBL presenting in submandibular or supraclavicular lymph nodes, in middle-aged to elderly patients, 2 of them without clinical or radiologic evidence of mediastinal involvement. The 3 patients presented with stage I/II disease and had excellent response to R-CHOP/R-EPOCH therapy. The 3 cases showed MAL expression and were positive for CD23 and/or CD30. All 3 cases expressed cyclin D1 with copy number gains of CCND1 gene but without rearrangement. There was no rearrangement of CIITA or PDL1/PDL2. Reverse transcriptase-multiplex ligation-dependent probe amplification, a mRNA-based gene expression profile analysis revealed high probability of PMBL (87.6%, 98.7%, and 99%) in these 3 cases. Targeted next-generation sequencing analysis showed SOCS1 mutations in the 3 cases, and TNFAIP3 and XPO1 mutations in one, further supporting the diagnosis of PMBL. In conclusion, we report 3 cases of nodal PMBL, 2 of them without mediastinal mass, and expression of cyclin D1 due to copy number gains of CCND1 gene, a diagnostic pitfall with mantle cell lymphoma and DLBCL, not otherwise specified.