CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

Introduction: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Methods: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. Results: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Discussion: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1.

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis.

BACKGROUND: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). METHODS: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. RESULTS: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. CONCLUSIONS: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.

PARP14 is a novel target in STAT6 mutant follicular lymphoma.

The variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of tumor cells and complex interactions within the tumor microenvironment (TME). IL-4 producing follicular helper T cells (TFH) are critical components of the FL TME. Binding of IL-4 to IL-4R on FL cells activates JAK/STAT signaling. We identified STAT6 mutations (STAT6MUT) in 13% of FL (N = 33/258), all clustered within the DNA binding domain. Gene expression data and immunohistochemistry showed upregulation of IL-4/STAT6 target genes in STAT6MUT FL, including CCL17, CCL22, and FCER2 (CD23). Functionally, STAT6MUT was gain-of-function by serial replating phenotype in pre-B CFU assays. Expression of STAT6MUT enhanced IL-4 induced FCER2/CD23, CCL17 and CCL22 expression and was associated with nuclear accumulation of pSTAT6. RNA sequencing identified PARP14 -a transcriptional switch and co-activator of STAT6- among the top differentially upregulated genes in IL-4 stimulated STAT6MUT lymphoma cells and in STAT6MUT primary FL cells. Quantitative chromatin immunoprecipitation (qChIP) demonstrated binding of STAT6MUT but not STAT6WT to the PARP14 promotor. Reporter assays showed increased IL-4 induced transactivation activity of STAT6MUT at the PARP14 promotor, suggesting a self-reinforcing regulatory circuit. Knock-down of PARP14 or PARP-inhibition abrogated the STAT6MUT gain-of-function phenotype. Thus, our results identify PARP14 as a novel therapeutic target in STAT6MUT FL.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Patient specific real-time PCR in precision medicine - Validation of IG/TR based MRD assessment in lymphoid leukemia.

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

Molecular characterization of Burkitt lymphoma in the breast or ovary.

Breast and ovary have been described as rare but typical sites of presentation of Burkitt lymphoma (BL) in females, particularly after puberty. We revised a historic series of 44 lymphomas of the breast or the ovary in women diagnosed between 1973 and 2014 as BL. Fluorescence in situ hybridization (FISH) was applied to all, and array-based copy number analysis as well as expression profiling to a subset of those cases. Of the 42 cases evaluable for FISH, 19 cases showed an IG-MYC translocation but only 9 of those fulfilled the criteria of the current WHO classification for the diagnosis of BL. Those nine cases resembled BL of other sites with regard to molecular features. Our findings along with literature data suggest that breast and ovarian BL (1) seem to be rarer than hitherto assumed, (2) share typical molecular features with other BL, and (3) predominantly affect women in the fertile age.

Double-hit lymphoma of the male breast: a case report.

BACKGROUND: Whereas lymphoma of the female breast is already rare, lymphoma of the male breast has only anecdotally been reported. Within a study of 32 lymphoma of the breast reported between 1973 and 2014 as Burkitt lymphoma, we observed a single male case, which we report here. CASE PRESENTATION: A 72-years-old Caucasian man presented with a mass in his left breast. Clinical history included prior basal cell carcinoma, leiomyosarcoma, and administration of spironolactone. The reference pathology diagnosis at presentation was Burkitt lymphoma according to the Kiel Classification. The present re-investigation using fluorescence in situ hybridization revealed an IGH-MYC translocation and a break in the BCL2 locus in the tumor cells. Thus, in light of the current WHO classification, the diagnosis was revised to high-grade B-cell lymphoma with MYC and BCL2 rearrangement, Burkitt morphology (so-called "double-hit" lymphoma). Genome-wide chromosomal imbalance mapping revealed a complex pattern of aberrations in line with this diagnosis. The aberrations, including copy-number gains in chromosomes 3q and 18 and focal homozygous loss in 9p21.3, resembled typical changes of lymphomas affecting "immune-privileged" sites. CONCLUSION: The present case adds to the understanding of the pathogenesis of male breast lymphomas, about which hardly any molecular characterization has been published yet.

Platform independent protein-based cell-of-origin subtyping of diffuse large B-cell lymphoma in formalin-fixed paraffin-embedded tissue.

Diffuse large B-cell lymphoma (DLBCL) is commonly classified by gene expression profiling according to its cell of origin (COO) into activated B-cell (ABC)-like and germinal center B-cell (GCB)-like subgroups. Here we report the application of label-free nano-liquid chromatography - Sequential Window Acquisition of all THeoretical fragment-ion spectra - mass spectrometry (nanoLC-SWATH-MS) to the COO classification of DLBCL in formalin-fixed paraffin-embedded (FFPE) tissue. To generate a protein signature capable of predicting Affymetrix-based GCB scores, the summed log2-transformed fragment ion intensities of 780 proteins quantified in a training set of 42 DLBCL cases were used as independent variables in a penalized zero-sum elastic net regression model with variable selection. The eight-protein signature obtained showed an excellent correlation (r = 0.873) between predicted and true GCB scores and yielded only 9 (21.4%) minor discrepancies between the three classifications: ABC, GCB, and unclassified. The robustness of the model was validated successfully in two independent cohorts of 42 and 31 DLBCL cases, the latter cohort comprising only patients aged >75 years, with Pearson correlation coefficients of 0.846 and 0.815, respectively, between predicted and NanoString nCounter based GCB scores. We further show that the 8-protein signature is directly transferable to both a triple quadrupole and a Q Exactive quadrupole-Orbitrap mass spectrometer, thus obviating the need for proprietary instrumentation and reagents. This method may therefore be used for robust and competitive classification of DLBCLs on the protein level.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

The A818-6 system as an in-vitro model for studying the role of the transportome in pancreatic cancer.

BACKGROUND: The human pancreatic cancer cell line A818-6 can be grown in vitro either as a highly malignant, undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS) simulating a benign, highly differentiated, duct-like pancreatic epithelial structure. This characteristic allowing A818-6 cells to switch from one phenotype to another makes these cells a unique system to characterize the cellular and molecular modifications during differentiation on one hand and malignant transformation on the other hand. Ion channels and transport proteins (transportome) have been implicated in malignant transformation. Therefore, the current study aimed to analyse the transportome gene expression profile in the A818-6 cells growing as a monolayer or as hollow spheres. METHODS & RESULTS: The study identified the differentially expressed transportome genes in both cellular states of A818-6 using Agilent and Nanostring arrays and some targets were validated via immunoblotting. Additionally, these results were compared to a tissue Affymetrix microarray analysis of pancreatic adenocarcinoma patients' tissues. The overall transcriptional profile of the ML and HS cells confirmed the formerly described mesenchymal features of ML and epithelial nature of HS which was further verified via high expression of E-cadherin and low expression of vimentin found in HS in comparison to ML. Among the predicted features between HS and ML was the involvement of miRNA-9 in this switch. Importantly, the bioinformatics analysis also revealed substantial number (n = 126) of altered transportome genes. Interestingly, three genes upregulated in PDAC tissue samples (GJB2, GJB5 and SLC38A6) were found to be also upregulated in ML and 3 down-regulated transportome genes (KCNQ1, TRPV6 and SLC4A) were also reduced in ML. CONCLUSION: This reversible HS/ML in vitro system might help in understanding the pathophysiological impact of the transportome in the dedifferentiation process in pancreatic carcinogenesis. Furthermore, the HS/ML model represents a novel system for studying the role of the transportome during the switch from a more benign, differentiated (HS) to a highly malignant, undifferentiated (ML) phenotype.

A novel lymphoma-associated macrophage interaction signature (LAMIS) provides robust risk prognostication in diffuse large B-cell lymphoma clinical trial cohorts of the DSHNHL.

Diffuse large B-cell lymphoma (DLBCL) is a disease with heterogeneous outcome. Stromal signatures have been correlated to survival in DLBCL. Their use, however, is hampered by the lack of assays for formalin-fixed paraffin-embedded material (FFPE). We constructed a lymphoma-associated macrophage interaction signature (LAMIS) interrogating features of the microenvironment using a NanoString assay applicable to FFPE. The clinical impact of the signature could be validated in a cohort of 466 patients enrolled in prospective clinical trials of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Patients with high expression of the signature (LAMIShigh) had shorter EFS, PFS, and OS. Multivariate analyses revealed independence from IPI factors in EFS (HR 1.7, 95% CI 1.2-2.4, p-value = 0.001), PFS (HR 1.8, 95% CI 1.2-2.5, p-value = 0.001) and OS (HR 1.8, 95% CI 1.3-2.7, p-value = 0.001). Multivariate analyses adjusted for the IPI factors showed the signature to be independent from COO, MYC rearrangements and double expresser status (DE). LAMIShigh and simultaneous DE status characterized a patient subgroup with dismal prognosis and early relapse. Our data underline the importance of the microenvironment in prognosis. Combined analysis of stromal features, the IPI and DE may provide a new rationale for targeted therapy.

A modular transcriptome map of mature B cell lymphomas.

BACKGROUND: Germinal center-derived B cell lymphomas are tumors of the lymphoid tissues representing one of the most heterogeneous malignancies. Here we characterize the variety of transcriptomic phenotypes of this disease based on 873 biopsy specimens collected in the German Cancer Aid MMML (Molecular Mechanisms in Malignant Lymphoma) consortium. They include diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt's lymphoma, mixed FL/DLBCL lymphomas, primary mediastinal large B cell lymphoma, multiple myeloma, IRF4-rearranged large cell lymphoma, MYC-negative Burkitt-like lymphoma with chr. 11q aberration and mantle cell lymphoma. METHODS: We apply self-organizing map (SOM) machine learning to microarray-derived expression data to generate a holistic view on the transcriptome landscape of lymphomas, to describe the multidimensional nature of gene regulation and to pursue a modular view on co-expression. Expression data were complemented by pathological, genetic and clinical characteristics. RESULTS: We present a transcriptome map of B cell lymphomas that allows visual comparison between the SOM portraits of different lymphoma strata and individual cases. It decomposes into one dozen modules of co-expressed genes related to different functional categories, to genetic defects and to the pathogenesis of lymphomas. On a molecular level, this disease rather forms a continuum of expression states than clearly separated phenotypes. We introduced the concept of combinatorial pattern types (PATs) that stratifies the lymphomas into nine PAT groups and, on a coarser level, into five prominent cancer hallmark types with proliferation, inflammation and stroma signatures. Inflammation signatures in combination with healthy B cell and tonsil characteristics associate with better overall survival rates, while proliferation in combination with inflammation and plasma cell characteristics worsens it. A phenotypic similarity tree is presented that reveals possible progression paths along the transcriptional dimensions. Our analysis provided a novel look on the transition range between FL and DLBCL, on DLBCL with poor prognosis showing expression patterns resembling that of Burkitt's lymphoma and particularly on 'double-hit' MYC and BCL2 transformed lymphomas. CONCLUSIONS: The transcriptome map provides a tool that aggregates, refines and visualizes the data collected in the MMML study and interprets them in the light of previous knowledge to provide orientation and support in current and future studies on lymphomas and on other cancer entities.

Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma.

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.

Non-leukemic pediatric mixed phenotype acute leukemia/lymphoma: Genomic characterization and clinical outcome in a prospective trial for pediatric lymphoblastic lymphoma.

Rare cases of hematological precursor neoplasms fulfill the diagnostic criteria of mixed phenotype acute leukemia (MPAL), characterized by expression patterns of at least two hematopoietic lineages, for which a highly aggressive behavior was reported. We present a series of 11 pediatric non-leukemic MPAL identified among 146 precursor lymphoblastic lymphomas included in the prospective trial Euro-LBL 02. Paraffin-embedded biopsies of 10 cases were suitable for molecular analyses using OncoScan assay (n = 7), fluorescence in situ hybridization (FISH; n = 7) or both (n = 5). Except for one case with biallelic KMT2A (MLL) breaks, all cases analyzed by FISH lacked the most common translocations defining molecular subsets of lymphoblastic leukemia/lymphomas. Two non-leukemic B-myeloid MPALs showed the typical genomic profile of hyperdiploid precursor B-cell lymphoblastic leukemia with gains of chromosomes 4, 6, 10, 14, 18, and 21. One B-T MPAL showed typical aberrations of T-cell lymphoblastic lymphoma, such as copy number neutral loss of heterozygosity (CNN-LOH) at 9p targeting a 9p21.3 deletion of CDKN2A and 11q12.2-qter affecting the ATM gene. ATM was also mutated in a T-myeloid MPAL case with additional loss at 7q21.2-q36.3 and mutation of NRAS, two alterations common in myeloid disorders. No recurrent regions of CNN-LOH were observed. The outcome under treatment was good with all patients being alive in first complete remission after treatment according to a protocol for precursor lymphoblastic lymphoma (follow-up 3-10 years, median: 4.9 years). In summary, the present series of non-leukemic MPALs widely lacked recurrently reported translocations in lymphoid/myeloid neoplasias and showed heterogeneous spectrum of chromosomal imbalances.

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma.

To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

Duodenal-type and nodal follicular lymphomas differ by their immune microenvironment rather than their mutation profiles.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.

Advanced patient age at diagnosis of diffuse large B-cell lymphoma is associated with molecular characteristics including ABC-subtype and high expression of MYC.

The incidence of diffuse large B-cell lymphoma (DLBCL) increases with age being patient age at diagnosis an adverse prognostic factor. However, elderly patients are often underrepresented in common studies. To investigate the effect between age and biological characteristics in DLBCL, we analyzed data of 1534 patients encompassing all adult age groups, enriched for the age ≥75 years. Follicular lymphoma (FL) grade 3B with histopathological characteristics of DLBCLs were included. Gender, centroblastic cytology, FL grade 3B morphology, CD10 expression, and ABC/non-GCB-subtype were significantly associated with age after correction for multiple testing and after adjusting for cohorts. Analysis of a subgroup points towards an association of MYC expression with age. Our data indicate that biological features of DLBCL and FL grade 3B are associated with increasing age among adult patients. The prevalence of the ABC/non-GCB-subtype in elderly patients suggests that therapies targeting this molecular subtype should be specifically explored in this subgroup.