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MOLECULAR CHARACTERIZATION CONTRIBUTES TO DIAGNOSIS AND PREDICTS OUTCOME IN PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMAS: A LYSA STUDY B Introduction: b Primary mediastinal large B-cell lymphoma (PMBL) is a unique entity with an unusually good prognosis, except for ~15% of refractory patients. Next, we applied Gene Expression Profiling (GEP) with a 137-genes RT-MLPA assay (LymphoSign signature) and Next-Generation Sequencing (NGS) with a 45-genes panel. [Extracted from the article]

Few data exist regarding the tumor B-cell receptor (BCR) repertoire and lymphoid microenvironment in primary mediastinal B-cell lymphoma (PMBL). We applied 5' rapid amplification of cDNA ends (5'RACE) to tumor RNA samples from 137 PMBL patients with available gene expression profiling and next-generation sequencing data. We obtained 5'RACE results for 75/137 (54.7%) patients, with clinical characteristics as follows: median [min-max] age, 33 [18-64] years; female, 53.3%; ECOG score 0-1, 86.7%; stage I-II, 57.3%; 1st-line treatment with anti-CD20 plus ACVBP, 72%; CHOP14, 14.7%; CHOP21, 13.3%. Among the 60 biopsies that expressed a productive BCR, we highlighted a strong somatic hypermutation profile with 58 (96.7%) patients carrying mutated IgVH, defined as <98% identity to the germline sequence. We then identified a subgroup of 12/75 patients (16%) with a worse prognosis (progression-free survival (PFS): HR [95% CI]=17 [3.2-88]; overall survival (OS): HR=21 [2.1-210]) associated with the highest clonal dominance status (HCD), defined by the dominant clonotype representing >81.1% and >78.6% of all CDR3 sequences for IgVH and IgVL, respectively. Compared to other patients, this subgroup had similar clinical characteristics but a greater median allele frequency for all somatic variants, decreased BCR diversity, and greater expression of PDL1/PDL2 and MS4A1 genes, suggesting a greater tumoral infiltration. According to a multivariate model integrating AID expression and BCR diversity, only HCD status was associated with outcome (PFS: HR=14.6 [2.46-86.8]; OS: HR=11.4 [1-128.8]). We confirmed this poorer prognosis in an independent cohort, in which 6/37 (16%) patients exhibited HCD (PFS: HR=12 [3-46]; OS: HR=17 [1.8-170])., (Copyright © 2024 American Society of Hematology.)

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., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Introduction: Follicular helper T-cell lymphomas (TFHL) have an aggressive course with a poor outcome. European and US guidelines recommend anthracycline-based chemotherapy as a first-line treatment, but the 5-year overall survival rate is still approximately 30%. We describe here the features of a cohort of TFHL patients who experienced prolonged survival despite the absence of specific treatment or the initiation of steroid-based therapy., Patients and Methods: In our study, we describe 15 adult patients who suffered from TFHL and had not received intensive chemotherapy at diagnosis for any reason. Biopsies of these cases were centrally reviewed, and the mutational pattern was determined using next-generation sequencing., Results: These 15 patients had the classic clinical, biological and pathological features of TFHL, angioimmunoblastic-type. TET2 mutations were found in 83% of patients; RHOA G17V, IDH2 R172 and DNMT3A mutations were found in 67%, 42% and 33% of the patients, respectively. Among the 15 patients, 8 did not receive any treatment, and 7 received steroid-based treatment. Ten patients had progression (5 in each group). Four patients died (3 of them from the progression of their lymphoma). The median follow-up in our cohort was 53 months. The 5-year OS was 66%, 100% for untreated patients and 29% for the others. In those 2 groups, the median time to treatment initiation was 22 months from diagnosis., Conclusion: We described a series of 15 well-characterized TFHL patients with an indolent outcome, suggesting that a watch-and-wait approach can be proposed in selected patients. Identifying factors predicting such evolution is warranted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Peripheral T-cell lymphomas (PTCL) comprised more than 30 rare heterogeneous entities, representing 10 to 15% of adult non-Hodgkin lymphomas. Although their diagnosis is still mainly based on clinical, pathological, and phenotypic features, molecular studies have allowed for a better understanding of the oncogenic mechanisms involved and the refinement of many PTCL entities in the recently updated classifications. The prognosis remains poor for most entities (5-year overall survival < 30%), with current conventional therapies based on anthracyclin-based polychemotherapy regimen, despite many years of clinical trials. The recent use of new targeted therapies appears to be promising for relapsed/refractory patients, such as demethylating agents in T-follicular helper (TFH) PTCL. However further studies are needed to evaluate the proper combination of these drugs in the setting of front-line therapy. In this review, we will summarize the oncogenic events for the main PTCL entities and report the molecular targets that have led to the development of new therapies. We will also discuss the development of innovative high throughput technologies that aid the routine workflow for the histopathological diagnosis and management of PTCL patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Drieux, Lemonnier and Gaulard.)

ALK-negative anaplastic large cell lymphoma (ALCL) comprises subgroups harboring rearrangements of DUSP22 (DUSP22- R) or TP63 (TP63-R). Two studies reported 90% and 40% 5-year overall survival (OS) rates in 21 and 12 DUSP22-R/TP63- not rearranged (NR) patients, respectively, making the prognostic impact of DUSP22-R unclear. Here, 104 newly diagnosed ALK-negative ALCL patients (including 37 from first-line clinical trials) from the LYSA TENOMIC database were analyzed by break-apart fluorescence in situ hybridization assays for DUSP22-R and TP63-R. There were 47/104 (45%) DUSP22-R and 2/93 (2%) TP63-R cases, including one DUSP22-R/TP63-R case. DUSP22-R tumors more frequently showed CD3 expression (62% vs. 35%, P=0.01), and less commonly a cytotoxic phenotype (27% vs. 82%; P<0.001). At diagnosis, DUSP22- R ALCL patients more frequently had bone involvement (32% vs. 13%, P=0.03). The patient with DUSP22-R/TP63-R ALCL had a rapidly fatal outcome. After a median follow-up of 4.9 years, 5-year progression-free survival (PFS) and OS rates of 84 patients without TP63-R treated with curative-intent anthracycline-based chemotherapy were 41% and 53%, respectively. According to DUSP22 status, 5-year PFS was 57% for 39 DUSP22-R versus 26% for 45 triple-negative (DUSP22-NR/TP63-NR/ALK-negative) patients (P=0.001). The corresponding 5-year OS rates were 65% and 41%, respectively (P=0.07). In multivariate analysis, performance status and DUSP22 status significantly affected PFS, and distinguished four risk groups, with 4-year PFS and OS ranging from 17% to 73% and 21% to 77%, respectively. Performance status but not DUSP22 status influenced OS. The use of brentuximab vedotin in relapsed/refractory patients improved OS independently of DUSP22 status. Our findings support the biological and clinical distinctiveness of DUSP22- R ALK-negative ALCL. Its relevance to outcome in patients receiving frontline brentuximab vedotin remains to be determined.

Background: The Banff Classification for Allograft Pathology recommendations for the diagnosis of kidney transplant rejection includes molecular assessment of the transplant biopsy. However, implementation of molecular tools in clinical practice is still limited, partly due to the required expertise and financial investment. The reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay is a simple, rapid, and inexpensive assay that permits simultaneous evaluation of a restricted gene panel using paraffin-embedded tissue blocks. The aim of this study was to develop and validate a RT-MLPA assay for diagnosis and classification of rejection., Methods: A retrospective cohort of 220 kidney transplant biopsies from two centers, which included 52 antibody-mediated rejection, 51 T-cell-mediated rejection, and 117 no-rejection controls, was assessed. A 17-gene panel was identified on the basis of relevant pathophysiological pathways. A support vector machine classifier was developed. A subset of 109 biopsies was also assessed using the Nanostring Banff Human Organ Transplant panel to compare the two assays., Results: The support vector machine classifier train and test accuracy scores were 0.84 and 0.83, respectively. In the test cohort, the F1 score for antibody-mediated rejection, T-cell-mediated rejection, and control were 0.88, 0.86, and 0.69, respectively. Using receiver-operating characteristic curves, the area under the curve for class predictions was 0.96, 0.89, and 0.91, respectively, with a weighted average at 0.94. Classifiers' performances were highest for antibody-mediated rejection diagnosis with 94% correct predictions, compared with 88% correct predictions for control biopsies and 60% for T-cell-mediated rejection biopsies. Gene expression levels assessed by RT-MLPA and Nanostring were correlated: r = 0.68, P < 0.001. Equivalent gene expression profiles were obtained with both assays in 81% of the samples., Conclusions: The 17-gene panel RT-MLPA assay, developed here for formalin-fixed paraffin-embedded kidney transplant biopsies, classified kidney transplant rejection with an overall accurate prediction ratio of 0.83., Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023&#95;04&#95;10&#95;CJN10100822.mp3., (Copyright © 2023 by the American Society of Nephrology.)

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare aggressive T-cell lymphoma most reported in Asia. We performed a comprehensive clinical, pathological and genomic study of 71 European MEITL patients (36 males, 35 females, median age 67 years). The majority presented with gastrointestinal involvement and had emergency surgery, and 40% had stage IV disease. The tumors were morphologically classified into two groups: typical (58%) and atypical (i.e., non-monomorphic or with necrosis, angiotropism or starry-sky pattern) (42%), sharing a homogeneous immunophenotypic profile (CD3+ [98%] CD4- [94%] CD5- [97%] CD7+ [97%] CD8+ [90%] CD56+ [86%] CD103+ [80%] cytotoxic marker+ [98%]) with more frequent expression of TCRgd (50%) than TCRab (32%). MYC expression (30% of cases) partly reflecting MYC gene locus alterations, correlated with non-monomorphic cytology. Almost all cases (97%) harbored deleterious mutation(s) and/or deletion of the SETD2 gene and 90% had defective H3K36 trimethylation. Other frequently mutated genes were STAT5B (57%), JAK3 (50%), TP53 (35%), JAK1 (12.5%), BCOR and ATM (11%). Both TP53 mutations and MYC expression correlated with atypical morphology. The median overall survival (OS) of 63 patients (43/63 only received chemotherapy after initial surgery) was 7.8 months. Multivariate analysis found a strong negative impact on outcome of MYC expression, TP53 mutation, STAT5B mutation and poor performance status while aberrant B-cell marker expression (20% of cases) correlated with better survival. In conclusion, MEITL is an aggressive disease with resistance to conventional therapy, predominantly characterized by driver gene alterations deregulating histone methylation and JAK/STAT signaling and encompasses genetic and morphologic variants associated with very high clinical risk.

Context: Bone marrow biopsy (BMB) is a common procedure in haematology used for the diagnosis and evaluation of response treatment. Because the procedure is difficult for haematologists to perform, patients often experience pain and stress. On Control, a system device, was introduced in the 2000s and uses a drill-powered needle to perform BMB., Objective: The aim of this study was to compare the quality of BMB, based on the length of the trephine, the number of interosseous spaces and the interpretability of the examination, obtained from manual BMB vs. drill-powered BMB. The secondary objectives were to evaluate the patient's pain and anxiety, and the haematologist's perceived difficulty in performing BMB., Design: This was a retrospective study conducted between June 2016 and June 2017 in the Henri Becquerel Cancer Centre in Rouen, France., Results: A total of 439 patients were included in the study; the sex ratio (M:F) was 1.34 and 70.2% underwent a drill-powered BMB. A significant difference was observed concerning trephine length (14.30 ± 5.58 mm with the drill-powered system vs. 11.18 ± 4.43 mm with manual BMB, p < 0.0001) and the number of interosseous spaces (9.49 ± 5.35 vs. 7.93 ± 4.01, respectively, p = 0.01). The interpretability of the examination did not differ between the two procedures (p = 0.9)., Conclusions: On Control, the drill-powered system for BMB, is widely distributed in North America and Europe, but this procedure is not yet generally applied. Although this procedure is costly, the ongoing development of this technique, because of its performance, is beneficial especially to obese patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nodal peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) with cytotoxic phenotype is overall rare, with most reports coming from Asia. Given its elusive pathobiology, we undertook a clinicopathological and molecular study of 54 Western patients diagnosed with PTCL, NOS expressing cytotoxic molecules, within a lymph node. More commonly males (M/F-2,6/1) with median age of 60 years were affected. Besides lymphadenopathy, 87% of patients had ≥1 involved extranodal site. High-stage disease (III-IV), International Prognostic Index >2, B symptoms, LDH level, and cytopenia(s) were observed in 92, 63, 67, 78, and 66% of cases, respectively. Ten patients had a history of B-cell malignancies, one each of myeloid neoplasm, breast or prostate cancer, and 4 others had underlying immune disorders. Most patients (70%) died, mostly of disease, with a median overall survival of 12.7 months. Immunophenotypically, the neoplastic lymphocytes were T-cell receptor (TCR) αβ + (47%), TCR-silent (44%) or TCRγδ+ (10%), commonly CD8 + (45%) or CD4-CD8- (32%). All except one had an activated cytotoxic profile, and 95% were subclassified into PTCL-TBX21 subtype based on CXCR3, TBX21, and GATA3 expression pattern. Seven patients (13%) disclosed EBER + tumor cells. Targeted DNA deep-sequencing (33 cases) and multiplex ligation-dependent reverse transcription-polymerase chain reaction assay (43 cases) identified frequent mutations in epigenetic modifiers (73%), including TET2 (61%) and DNMT3A (39%), recurrent alterations affecting the TCR (36%) and JAK/STAT (24%) signaling pathways and TP53 mutations (18%). Fusion transcripts involving VAV1 were identified in 6/43 patients (14%). Patients with nodal cytotoxic PTCL, NOS have an aggressive behavior and frequently present in a background of impaired immunity, although the association with Epstein-Barr virus is rare. The recurrent alterations in genes involved in DNA methylation together with genes related to cytokine or TCR signaling, suggest that co-operation of epigenetic modulation with cell-signaling pathways plays a critical role in the pathogeny of these lymphomas., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Background: The pathology-based diagnosis of cardiac antibody-mediated rejection (AMR) relies on the 2013 International Society for Heart and Lung Transplantation Working Formulation, in which microvascular inflammation (MVI) is considered as present or absent regardless of its extent. This work assessed the biological and clinical value of a semiquantitative evaluation of the extent of MVI in endomyocardial biopsies (EMBs)., Methods: We retrospectively graded the extent of MVI in 291 EMB from 291 patients according to a 4-point scale in which MVI scores of 0, 1, 2, and 3 represented 0%, 1%-10%, 11%-50%, and >50% of the myocardial area, respectively. We analyzed the association between the MVI score and tissue rejection molecular activity assessed by microarrays or reverse transcriptase multiplex ligation-dependent probe amplification, current pathology classification (pathologic AMR [pAMR]), anti-HLA donor-specific antibodies, and graft dysfunction., Results: Overall, 172 (59.1%), 33 (11.4%), 42 (14.4%), and 44 (15.1%) EMB were given MVI scores of 0, 1, 2, and 3, respectively. pAMR1(H+) and pAMR2/3 categories were found to be heterogeneous in terms of MVI score. Acute cellular rejection grades did not influence the MVI score. In both molecular approaches, we observed a stepwise increase in the expression of AMR-related transcripts with increasing MVI scores, independent of the C4d or CD68 status (P < 0.001). Both the frequency and mean fluorescence intensity of donor-specific antibodies gradually increased with the MVI score (P < 0.001). Acute graft dysfunction was more frequent in MVI score 3 (P < 0.001)., Conclusions: The intensity of MVI in EMB, based on a semiquantitative evaluation of its extent, has biological and clinical importance., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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.

Background and Objectives: Glomerular C4d deposits are associated the severity and outcomes of IgA nephropathy. Whether this holds true in immunoglobulin A vasculitis (IgAV) is not known. The main objective of the study was to analyze the prognostic value of glomerular C4d immunolabelling on kidney impairment in adults with IgAV. Design, Setting, Participants, Measurements: This retrospective cohort study included 120 adults with IgAV and a kidney biopsy performed between 1995 and 2018 in two French university hospital centers. All paraffin-embedded biopsies were reassessed according to Oxford classification. Immunofluorescence for C4d was performed in all cases. For analysis, patients were grouped according to positivity for C4d in the glomerular area. The main outcome was a composite endpoint of 50% increase in 24 h-proteinuria, or eGFR decrease by 50%, or kidney replacement therapy. Results: The median follow-up was 28.3 months. Twenty-three patients met the composite endpoint, 12 for kidney replacement therapy, 6 for an eGFR decrease >50% and 5 for a >50% increase in proteinuria. At time of biopsy, the median proteinuria was 1.9 g/24 h and the median eGFR 73.5 mL/min/1.73 m 2 . Among the 102 patients evaluable for C4d, 24 were positive on >30% glomeruli, mainly with a parieto-mesangial pattern. In this group, the initial proteinuria was more frequently nephrotic than in the C4d- group (60% vs. 33%, P = 0.039). Mesangial hypercellularity was more frequent in the C4d+ group (42% vs. 13%; P = 0.006) whereas macroscopic hematuria was more frequent in the C4d- group (18% vs. 0%; P = 0.03). After a median follow-up of 28 months, kidney survival did not differ according to C4d status. Conclusion: In a population of adult IgAV patients, glomerular positivity for C4d was associated with the severity of the kidney disease at presentation, but not with subsequent renal function deterioration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Romero, Drieux, François, Dervaux, Xu, Titeca-Beauport, Bertrand and Guerrot.)

The genetic basis of peripheral T-cell lymphoma (PTCL) is complex and encompasses several recurrent fusion transcripts discovered over the past years by means of massive parallel sequencing. However, there is currently no affordable and rapid technology for their simultaneous detection in clinical samples. Herein, we developed a multiplex ligation-dependent RT-PCR-based assay, followed by high-throughput sequencing, to detect 33 known PTCL-associated fusion transcripts. Anaplastic lymphoma kinase (ALK) fusion transcripts were detected in 15 of 16 ALK-positive anaplastic large-cell lymphomas. The latter case was further characterized by a novel SATB1&#95;ALK fusion transcript. Among 239 other PTCLs, representative of nine entities, non-ALK fusion transcripts were detected in 24 samples, mostly of follicular helper T-cell (TFH) derivation. The most frequent non-ALK fusion transcript was ICOS&#95;CD28 in nine TFH-PTCLs, one PTCL not otherwise specified, and one adult T-cell leukemia/lymphoma, followed by VAV1 rearrangements with multiple partners (STAP2, THAP4, MYO1F, and CD28) in five samples (three PTCL not otherwise specified and two TFH-PTCLs). The other rearrangements were CTLA4&#95;CD28 (one TFH-PTCL), ITK&#95;SYK (two TFH-PTCLs), ITK&#95;FER (one TFH-PTCL), IKZF2&#95;ERBB4 (one TFH-PTCL and one adult T-cell leukemia/lymphoma), and TP63&#95;TBL1XR1 (one ALK-negative anaplastic large-cell lymphoma). All fusions detected by our assay were validated by conventional RT-PCR and Sanger sequencing in 30 samples with adequate material. The simplicity and robustness of this targeted multiplex assay make it an attractive tool for the characterization of these heterogeneous diseases., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Novel tools are needed to improve diagnostic accuracy and risk prediction in BK virus nephropathy (BKVN). We assessed the utility of intragraft gene expression testing for these purposes. Eight hundred genes were measured in 110 archival samples, including a discovery cohort of native kidney BKVN (n = 5) vs pure T cell-mediated rejection (TCMR; n = 10). Five polyomavirus genes and seven immune-related genes (five associated with BKVN and two associated with TCMR) were significantly differentially expressed between these entities (FDR < 0.05). These three sets of genes were further evaluated in samples representing a spectrum of BK infection (n = 25), followed by a multicenter validation cohort of allograft BKVN (n = 60) vs TCMR (n = 10). Polyomavirus 5-gene set expression reliably distinguished BKVN from TCMR (validation cohort AUC = 0.992), but the immune gene sets demonstrated suboptimal diagnostic performance (AUC ≤ 0.720). Within the validation cohort, no significant differences in index biopsy gene expression were identified between BKVN patients demonstrating resolution (n = 35), persistent infection (n = 14) or de novo rejection (n = 11) 6 months following a standardized reduction in immunosuppression. These results suggest that, while intragraft polyomavirus gene expression may be useful as an ancillary diagnostic for BKVN, assessment for concurrent TCMR and prediction of clinical outcome may not be feasible with current molecular tools., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Peripheral T-cell lymphoma comprises a heterogeneous group of mature non-Hodgkin lymphomas. Their diagnosis is challenging, with up to 30% of cases remaining unclassifiable and referred to as "not otherwise specified". We developed a reverse transcriptase-multiplex ligation-dependent probe amplification gene expression profiling assay to differentiate the main T-cell lymphoma entities and to study the heterogeneity of the "not specified" category. The test evaluates the expression of 20 genes, including 17 markers relevant to T-cell immunology and lymphoma biopathology, one Epstein-Barr virus-related transcript, and variants of RHOA (G17V) and IDH2 (R172K/T). By unsupervised hierarchical clustering, our assay accurately identified 21 of 21 ALK-positive anaplastic large cell lymphomas, 16 of 16 extranodal natural killer (NK)/T-cell lymphomas, 6 of 6 hepatosplenic T-cell lymphomas, and 13 of 13 adult T-cell leukemia/lymphomas. ALK-negative anaplastic lymphomas (n=34) segregated into one cytotoxic cluster (n=10) and one non-cytotoxic cluster expressing Th2 markers (n=24) and enriched in DUSP22 -rearranged cases. The 63 T FH -derived lymphomas divided into two subgroups according to a predominant T FH (n=50) or an enrichment in Th2 (n=13) signatures. We next developed a support vector machine predictor which attributed a molecular class to 27 of 77 not specified T-cell lymphomas: 17 T FH , five cytotoxic ALK-negative anaplastic and five NK/T-cell lymphomas. Among the remaining cases, we identified two cell-of-origin subgroups corresponding to cytotoxic/Th1 (n=19) and Th2 (n=24) signatures. A reproducibility test on 40 cases yielded a 90% concordance between three independent laboratories. This study demonstrates the applicability of a simple gene expression assay for the classification of peripheral T-cell lymphomas. Its applicability to routinely-fixed samples makes it an attractive adjunct in diagnostic practice., (Copyright© 2020 Ferrata Storti Foundation.)

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.

Background: Molecular biology has emerged as a potential companion to histology for the diagnosis of rejection after heart transplantation. Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) is a technique of targeted gene expression analysis suitable for formalin-fixed paraffin-embedded (FFPE) biopsies. Our aim was to assess RT-MLPA for the diagnosis of allograft rejection in heart transplantation., Methods: We performed a cross-sectional, case-control, multicenter study. After the selection of a 14-transcript panel (endothelial burden, Natural killer cells, interferon-γ pathway, effector T-cells, and antigen presentation), RT-MLPA was applied to 183 FFPE endomyocardial biopsies (EMB), randomized into a training (n = 113) and a validation (n = 70) series. A two-step class prediction analysis was developed (Linear prediction score-LPS1: rejection vs non-rejection; LPS2: antibody-mediated rejection [AMR] vs acute cellular rejection [ACR]). A study of the agreement between pathology and RT-MLPA was performed., Results: Overall, 48 ACR, 82 AMR, 5 mixed rejection, and 48 non-rejection EMBs were analyzed. Three molecular clusters were delineated by unsupervised hierarchical analysis (molecular non-rejection, ACR, and AMR). AMR was characterized by the high expression of CCL4, GNLY, FCGR3, CXCL11 and ACR by the high expression of CCL18 and ADAMdec. RT-MLPA and histopathology agreed in the final diagnosis in 82.2%, 67.7%, and 76.8% of the EMB in the test, validation, and overall cohort, respectively. Disagreement cases were more common in the case of histologic low-grade rejection and early post-transplant EMB., Conclusions: RT-MLPA is a suitable technique for targeted gene expression analysis on FFPE EMB with a good overall agreement with the histologic diagnosis of heart allograft rejection., (Copyright © 2019. Published by Elsevier Inc.)

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., (© 2019 The Authors. Genes, Chromosomes & Cancer published by Wiley Periodicals, Inc.)

Chronic lymphocytic leukemia (CLL) may induce renal complications, which are becoming increasingly common, but in this context the occurrence of minimal change disease (MCD) remains rare. Monoclonal B lymphocytosis (MBL) is a precursor state of CLL and is currently under recognized. Since MBL is seen as a benign disorder that rarely evolves into CLL, screening for MBL is not standardized and does not require any treatment. When reviewing renal disease associated with MBL, there is very scant data in the literature and to date there is no case describing the association between MBL and MCD. Here, we describe the case of a 71-year old woman admitted for nephrotic syndrome (NS). We diagnosed a MBL. Kidney biopsy revealed MCD. Treatment with corticosteroids was introduced but no improvement was observed. Chemotherapy with rituximab and chlorambucil was thus started, leading to complete remission of both MBL and MCD. To our knowledge, this is the first description of the association of MBL and MCD. This case suggests that screening for MBL may have unexpected diagnostic and therapeutic implications in patients presenting with seemingly idiopathic NS.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It includes three major subtypes termed germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. With the emergence of novel targeted therapies, accurate methods capable of interrogating this cell-of-origin classification should soon become essential in the clinics. To address this issue, we developed a novel gene expression profiling DLBCL classifier based on reverse transcriptase multiplex ligation-dependent probe amplification. This assay simultaneously evaluates the expression of 21 markers, to differentiate primary mediastinal B-cell lymphoma, activated B-cell-like, germinal center B-cell-like, and also Epstein-Barr virus-positive DLBCLs. It was trained using 70 paraffin-embedded biopsies and validated using >160 independent samples. Compared with a reference classification established from Affymetrix U133 + 2 data, reverse transcriptase multiplex ligation-dependent probe amplification classified 85.0% samples into the expected subtype, comparing favorably with current diagnostic methods. This assay also proved to be highly efficient in detecting the MYD88 L265P mutation, even in archival paraffin-embedded tissues. This reliable, rapid, and cost-effective method uses common instruments and reagents and could thus easily be implemented into routine diagnosis workflows, to improve the management of these aggressive tumors., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Light chain proximal tubulopathy (LCPT) is a rare kidney disease associated with plasma cell dyscrasias, characterized by light chain deposits in the proximal tubular cells, with or without crystal formation. We describe an exceptional case of LCPT without crystal formation in a kidney allograft, in a patient who underwent two renal transplants for a light chain deposition disease (LCDD) complicating smoldering myeloma. This is the first description of this association in successive kidney allografts. We concisely describe pathology of LCDD and LCPT and discuss potential pathophysiological mechanisms relating these two conditions.

Background: Chronic lymphocytic leukemia and small lymphocytic lymphoma are 2 different presentations of the most common B-cell neoplasm in western countries (CLL/SLL). In this disease, kidney involvement is usually silent, and is rarely reported in the literature. This study provides a clinicopathological analysis of all-cause kidney disease in CLL/SLL patients., Methods: Fifteen CLL/SLL patients with kidney biopsy were identified retrospectively. Demographic, clinical, pathological and laboratory data were assessed at biopsy, and during follow-up., Results: At biopsy 11 patients presented impaired renal function, 7 patients nephrotic syndrome, 6 patients dysproteinemia, and 3 patients cryoglobulinemia. Kidney pathology revealed CLL/SLL-specific monoclonal infiltrate in 10 biopsies, glomerulopathy in 9 biopsies (5 membranoproliferative glomerulonephritis, 2 minimal change disease, 1 glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits, 1 AHL amyloidosis). Five patients presented interstitial granulomas attributed to CLL/SLL. After treatment of the hematological disease, improvement of renal function was observed in 7/11 patients, and remission of nephrotic syndrome in 5/7 patients. During follow-up, aggravation of the kidney disease systematically occurred in the absence of favorable response to hematological treatment., Conclusions: A broad spectrum of kidney diseases is associated with CLL/SLL. In this setting, kidney biopsy can provide important information for diagnosis and therapeutic guidance.

Sorafenib is a molecular-targeted therapy used in palliative treatment of advanced hepatocellular carcinoma (HCC) in Child-Pugh A patients. We describe the case of a patient who presented with a large HCC in the left liver associated with portal vein thrombosis (PVT). After 9 months of sorafenib treatment, reassessment showed that the tumors had decreased in size with recanalization of the portal vein. A lateral left hepatectomy was performed and pathology showed complete necrosis of the tumor. Sorafenib can downstage HCC in patients with cirrhosis allowing further surgical resection.