The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21–derived heavy chains (HCs) with IGLV3-21–derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110–expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR–BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01–expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110–expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

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Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide riskadapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform. Supplementary informationThe online version of this article (https://

Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma subtype. This retrospective study elucidates the currently unknown genetic background of a large clinically well-annotated cohort of DLBCL with osseous localizations (O-DLBCL), including PB-DLBCL. 103 O-DLBCL patients were included and compared with 63 (extra)nodal non-osseous (NO)-DLBCLs with germinal center B-cell phenotype (NO-DLBCL-GCB). Cell-of-origin (COO) was determined by immunohistochemistry and gene-expression-profiling (GEP) using (extended)-NanoString/Lymph2Cx. Mutational profiles were identified with targeted next-generation deep-sequencing, including 52 B-cell lymphoma-relevant genes. O-DLBCLs, including 34 PB-DLBCL, were predominantly classified as GCB-phenotype based on immunohistochemistry (74%) and NanoString analysis (88%). Unsupervised hierarchical clustering of an extended-NanoString/Lymph2Cx demonstrated significantly different GEP-clusters for PB-DLBCL as opposed to NO-DLBCL-GCB (P<0.001). Expression levels of 23 genes of two different targeted GEP-panels, indicated a centrocyte-like phenotype for PB-DLBCL, whereas NO-DLBCL-GCB showed a centroblast-like constitution. PB-DLBCL had significantly more frequent mutations in four GCB-associated genes, i.e. B2M, EZH2, IRF8, and TNFRSF14, compared to NO-DLBCL-GCB (P=0.031, P=0.010, P=0.047, and P=0.003). PB-DLBCL with its corresponding specific mutational profile were significantly associated with a superior overall survival compared to equivalent Ann Arbor limited-stage I/II NO-DLBCL-GCB (P=0.011). This study is the first to demonstrate that PB-DLBCL is characterized by a GCB-phenotype, with a centrocyte-like GEP-pattern and a GCB-associated mutational profile (both involved in immune surveillance) and a favorable prognosis. These novel biology-associated features provide evidence that PB-DLBCL represents a distinct extranodal DLBCL entity and its specific mutational landscape holds potential for targeted therapies (e.g. EZH2-inhibitors).

Programmed cell death ligand-1 (PD-L1) expression may predict the response to both programmed cell death-1 and PD-L1 inhibitors in lung cancer. However, the extent of intratumoral heterogeneity of PD-L1 expression, which may cause false negative results, is largely unexplored. We aimed to assess the intratumoral heterogeneity of PD-L1 expression in surgically resected lung cancer specimens by applying a novel method of tissue microarray, namely Spiral Arrays, which enables us to observe the heterogeneity in spiral-shaped tissue cores. Adenocarcinoma and squamous cell carcinoma specimens were obtained from consecutive patients with lung cancer who had undergone surgical resection at Nagasaki University Hospital (Nagasaki, Japan) since 2009. Small cell lung cancer and large cell carcinoma specimens were selected from patients in the same archive who had undergone resection since 1998. Spiral Arrays were constructed of spiral-shaped cores, prepared from representative blocks of each case, which were subjected to immunohistochemistry using an anti-PD-L1 antibody. Each core was divided into 8 segments and each segment was classified as either PD-L1-positive or PD-L1-negative using thresholds of 1.0%, 5.0%, 10.0%, and 50.0%, respectively. In total, 138 specimens were selected, including 60 adenocarcinomas, 59 squamous cell carcinomas, 12 small cell lung cancers, and 7 large cell carcinomas. The majority of specimens with PD-L1-positive segments exhibited heterogeneous expression (i.e., had a mixture of PD-L1-positive and PD-L1-negative segments within a core) irrespective of the threshold (1.0%, 66.7%; 5.0%, 74.4%; 10.0%, 75.8%; and 50.0%, 85.7%]. Large variations in the ratios of PD-L1-positive segments were observed. At least 50.0% of the segments within a core were negative in no fewer than 50.0% (range, 50.0–76.0%) of cases with heterogeneous PD-L1 expression. In conclusion, intratumoral heterogeneity of PD-L1 expression was frequently observed in cases of lung cancer. Thus, multiple tumor biopsy specimens may be needed to accurately determine the PD-L1 expression status.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC.

Key Points Loss-of-function mutations in CD37 occur predominantly in diffuse large B-cell lymphoma at immune-privileged sites. CD37-mutated lymphoma B cells show impaired CD37 cell-surface localization, which may have implications for anti-CD37 therapies.