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TESTICULAR LARGE B-CELL LYMPHOMA IS GENETICALLY SIMILAR TO PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA AND DISTINCT FROM NODAL DIFFUSE LARGE B-CELL LYMPHOMA B Introduction: b Testicular large B-cell lymphoma (TLBCL) is an infrequent and aggressive lymphoma presenting in an immune-privileged site, recently recognized as a distinct entity from diffuse large B-cell lymphoma (DLBCL). Compared with nodal DLBCL, localized and disseminated TLBCL have less CNA complexity ( I P i = 0.01 and I P i < 0.04, respectively) but showed a higher number of variants ( I P i = 0.01 and I P i < 0.001, respectively). [Extracted from the article]

Testicular large B-cell lymphoma (TLBCL) is an infrequent and aggressive lymphoma arising in an immune-privileged site and has recently been recognized as a distinct entity from diffuse large B-cell lymphoma (DLBCL). We describe the genetic features of TLBCL and compare them with published series of nodal DLBCL and primary large B-cell lymphomas of the CNS (PCNSL). We collected 61 patients with TLBCL. We performed targeted next-generation sequencing, copy number arrays, and fluorescent in situ hybridization to assess chromosomal rearrangements in 40 cases with available material. Seventy percent of the cases showed localized stages. BCL6 rearrangements were detected in 36% of cases, and no concomitant BCL2 and MYC rearrangements were found. TLBCL had fewer copy number alterations ( p  < 0.04) but more somatic variants ( p  < 0.02) than nodal DLBCL and had more frequent 18q21.32-q23 ( BCL2 ) gains and 6q and 9p21.3 ( CDKN2A/B ) deletions. PIM1 , MYD88 L265P , CD79B , TBL1XR1 , MEF2B , CIITA , EP300, and ETV6 mutations were more frequent in TLBCL, and BCL10 mutations in nodal DLBCL. There were no major genetic differences between TLBCL and PCNSL. Localized or disseminated TLBCL displayed similar genomic profiles. Using LymphGen, the majority of cases were classified as MCD. However, we observed a subgroup of patients classified as BN2, both in localized and disseminated TLBCL, suggesting a degree of genetic heterogeneity in the TLBCL genetic profile. TLBCL has a distinctive genetic profile similar to PCNSL, supporting its recognition as a separate entity from DLBCL and might provide information to devise targeted therapeutic approaches., Competing Interests: Ferran Nadeu has received honoraria from Janssen, AbbVie, AstraZeneca, and SOPHiA GENETICS for speaking at educational activities; has received research support from Gilead; and has licensed the use of the protected IgCaller algorithm to Diagnóstica Longwood. Tycho Baumann has received consulting fees or honoraria from Janssen, Roche, Novartis, Merck, Gilead/Kite, Incyte, Lilly, Abbvie, AstraZeneca, and BeiGene. Alejandro Martin García‐Sancho has received consulting fees or honoraria from Janssen, Roche, BMS/Celgene, Kyowa Kirin, Clinigen, EUSAPharma, Novartis, Gilead/Kite, Incyte, Lilly, Takeda, ADC Therapeutics America, Miltenyi, Ideogen, Abbvie, and BeiGene. Elías Campo has been a consultant for GenMab, and Takeda; has received research support from AstraZeneca; received honoraria from Janssen, EUSPharma, Takeda, and Roche for speaking at educational activities; and is an inventor on a Lymphoma and Leukemia Molecular Profiling Project patent “Method for subtyping lymphoma subtypes by means of expression profiling” (PCT/US2014/64161) and a bioinformatic tool (IgCaller) licensed to Diagnostic Longwood. Eva Giné has received honoraria or consulting fees from Gilead, Kite Pharma, Janssen, Genmab, Miltenyi, and Lilly; has received research support from Janssen and travel expenses from Gilead and Kite Pharma. Armando López Guillermo served on the advisory board of Roche, Celgene, Novartis, and Gilead/Kite, received grants from Celgene and Gilead/Kite, and travel expenses from Kite/Gilead. The remaining authors declare no competing interests., (© 2024 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

The genetic landscape underlying the transformation of splenic diffuse red pulp small B-cell lymphoma (SDRPL) is not well understood. The present study aimed to unravel the genomic alterations involved in the progression and transformation of SDRPL. We performed genetic studies on both SDRPL and subsequent or synchronous diffuse large B cell lymphoma (DLBCL) samples in three SDRPL patients who eventually developed DLBCL. Our findings revealed that SDRPL cases progressing to DLBCL acquired genomic alterations in genes related to the cell cycle ( CDKN2A/B, TP53, MYC and CCND3 ) and B cell development ( BCL6 )., Competing Interests: Elias Campo has been a consultant for GenMab, and Takeda; has received research support from AstraZeneca; received honoraria from Janssen, EUSPharma, Takeda and Roche for speaking at educational activities; and is an inventor on a Lymphoma and Leukemia Molecular Profiling Project patent ‘Method for subtyping lymphoma subtypes by means of expression profiling’ (PCT/US2014/64161) and a bioinformatic tool (IgCaller) licenced to Diagnostic Longwood. Armando López‐Guillermo. served on the advisory board of Roche, Celgene, Novartis and Gilead/Kite, and received grants from Celgene and Gilead/Kite. Ferran Nadeu has received honoraria from Janssen, AbbVie, AstraZeneca and SOPHiA GENETICS for speaking at educational activities; has received research support from Gilead; and has licensed the use of the protected IgCaller algorithm to Diagnóstica Longwood. The remaining authors declare no competing financial interests., (© 2024 The Author(s). eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Competing Interests: Declaration of competing interest All authors report no relevant potential conflicts of interest.

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL., (© 2024. The Author(s).)

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with a heterogeneous clinical behavior. In 5-10% of patients the disease transforms into a diffuse large-B cell lymphoma known as Richter transformation (RT), which is associated with dismal prognosis. Here, we aimed to establish patient-derived xenograft (PDX) models to study the molecular features and evolution of CLL and RT. We generated two PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs were morphologically and phenotypically similar to RT. Whole-genome sequencing analysis at different time points of the PDX evolution revealed a genomic landscape similar to RT tumors from both patients and uncovered an unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. In PDX12, the transformed cells expanded from a very small subclone already present at the CLL stage. Transcriptomic analysis of PDXs showed a high oxidative phosphorylation (OXPHOS) and low B-cell receptor (BCR) signaling similar to the RT in the patients. IACS-010759, an OXPHOS inhibitor, reduced proliferation, and circumvented resistance to venetoclax. In summary, we have generated new RT-PDX models, one of them from CLL cells that mimicked the evolution of CLL to RT uncovering intrinsic features of RT cells of therapeutical value., (© 2023. The Author(s).)

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil., Competing Interests: Declaration of interests H.H. is co-founder of Omniscope, SAB member of Nanostring and MiRXES, and consultant to Moderna and Singularity. J.C.N. is consultant to Omniscope., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Epstein-Barr virus-positive Inflammatory follicular dendritic cell/fibroblastic reticular cell tumour (EBV-IFDC/FRCT) is a rare neoplasm that occurs almost exclusively in the liver or spleen. Extra-hepatosplenic presentation is infrequent and exceptional cases have been described arising in the gastrointestinal tract or in the pharynx. However, EBV-IFDC/FRCT cases have not been previously reported in the larynx. This report describes a case of a 32-year-old woman who arrived to the emergency department due to progressive dyspnea with associated inspiratory stridor and non-productive cough. Direct laryngoscopy showed a nodular tumour arising on the left posterior subglottic mucosa obstructing 90% of the airway. A preoperative dual energy contrast enhanced computed tomography (CECT) was performed demonstrating a low attenuation lesion on virtual non-contrast (VNC) images and vivid iodine uptake on the iodine map. The tumour was excised and the histopathological analysis led to the diagnosis of an EBV-IFDC/FRCT. A fibre-optic laryngoscopy six months after the surgery did not show any abnormalities. Although the vast majority of EBV-IFDC/FRCT occur in the liver or spleen, some extra hepatosplenic tumours have been reported affecting the head and neck region. We describe here the first case arising in the larynx, as well as the usefulness of preoperative dual energy imaging techniques to assess these lesions, thus providing information that could have management implications., Competing Interests: Competing InterestsThe authors have no relevant financial or non-financial interests to disclose., (© Association of Otolaryngologists of India 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

While some follicular lymphoma (FL) patients do not require treatment or experience prolonged responses, others relapse early, and little is known about genetic alterations specific to patients with a particular clinical behavior. We selected 56 grade 1-3A FL patients according to their need of treatment or timing of relapse: never treated (n = 7), non-relapsed (19), late relapse (14), early relapse or POD24 (11), and primary refractory (5). We analyzed 56 diagnostic and 12 paired relapse lymphoid tissue biopsies and performed copy number alteration (CNA) analysis and next generation sequencing (NGS). We identified six focal driver losses (1p36.32, 6p21.32, 6q14.1, 6q23.3, 9p21.3, 10q23.33) and 1p36.33 copy-neutral loss of heterozygosity (CN-LOH). By integrating CNA and NGS results, the most frequently altered genes/regions were KMT2D (79%), CREBBP (67%), TNFRSF14 (46%) and BCL2 (40%). Although we found that mutations in PIM1, FOXO1 and TMEM30A were associated with an adverse clinical behavior, definitive conclusions cannot be drawn, due to the small sample size. We identified common precursor cells harboring early oncogenic alterations of the KMT2D, CREBBP, TNFRSF14 and EP300 genes and 16p13.3-p13.2 CN-LOH. Finally, we established the functional consequences of mutations by means of protein modeling (CD79B, PLCG2, PIM1, MCL1 and IRF8). These data expand the knowledge on the genomics behind the heterogeneous FL population and, upon replication in larger cohorts, could contribute to risk stratification and the development of targeted therapies., (© 2023 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

Posttransplant lymphoproliferative disorders (PTLDs) represent a broad spectrum of lymphoid proliferations, frequently associated with Epstein-Barr virus (EBV) infection. The molecular profile of pediatric monomorphic PTLDs (mPTLDs) has not been elucidated, and it is unknown whether they display similar genetic features as their counterpart in adult and immunocompetent (IMC) pediatric patients. In this study, we investigated 31 cases of pediatric mPTLD after solid organ transplantation, including 24 diffuse large B-cell lymphomas (DLBCLs), mostly classified as activated B cell, and 7 cases of Burkitt lymphoma (BL), 93% of which were EBV positive. We performed an integrated molecular approach, including fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. Overall, PTLD-BL carried mutations in MYC, ID3, DDX3X, ARID1A, or CCND3 resembling IMC-BL, higher mutational burden than PTLD-DLBCL, and lesser CN alterations than IMC-BL. PTLD-DLBCL showed a very heterogeneous genomic profile with fewer mutations and CN alterations than IMC-DLBCL. Epigenetic modifiers and genes of the Notch pathway were the most recurrently mutated in PTLD-DLBCL (both 28%). Mutations in cell cycle and Notch pathways correlated with a worse outcome. All 7 patients with PTLD-BL were alive after treatment with pediatric B-cell non-Hodgkin lymphoma protocols, whereas 54% of patients with DLBCL were cured with immunosuppression reduction, rituximab, and/or low-dose chemotherapy. These findings highlight the low complexity of pediatric PTLD-DLBCL, their good response to low-intensity treatment, and the shared pathogenesis between PTLD-BL and EBV-positive IMC-BL. We also suggest new potential parameters that could help in the diagnosis and the design of better therapeutic strategies for these patients., (© 2023 by The American Society of Hematology.)

Context.—: Despite their stromal origin, follicular dendritic cells (FDCs) share many functions with hematopoietic system cells. FDC neoplasms are currently classified by the World Health Organization along with those of a histiocytic nature. However, the molecular alterations driving oncogenesis in FDC sarcomas (FDCSs) are beginning to be unveiled and do not seem to concur with those described in histiocytic neoplasms, namely MAPK pathway activation., Objective.—: To identify molecular alterations driving tumorigenesis in FDCS., Design.—: We investigated the role of MYC and TP53 in FDC-derived tumor oncogenesis and assessed comprehensively the status of the MAPK pathway in 16 FDCSs, 6 inflammatory pseudotumor (IPT)-like FDCSs, and 8 IPTs., Results.—: MYC structural alterations (both amplifications and rearrangements) were identified in 5 of 14 FDCSs (35.7%), all associated with MYC overexpression. TP53 mutations were identified in 4 of 14 FDCSs (28.6%), all of which displayed intense and diffuse p53 expression. None of these alterations were identified in any IPT-like FDCSs or in IPT cases. No MAPK pathway gene alterations were identified in any of the cases studied., Conclusions.—: The presence of MYC and TP53 alterations and the lack of association with Epstein-Barr virus segregate classical FDCS from IPT-like FDCS, pointing at different oncogenic mechanisms in both entities. Our results suggest a possible oncogenic role of MYC and TP53 alterations in FDCS. The absence of MAPK pathway alterations confirms the lack of a significant role of this pathway in the oncogenesis of FDC-derived neoplasms., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2023 College of American Pathologists.)

The genetic mechanisms associated with splenic marginal zone lymphoma (SMZL) transformation are not well defined. We studied 41 patients with SMZL that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained either only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in 2 groups: (1) at diagnosis (SMZL, n = 27 samples), and (2) at transformation (SMZL-T, n = 32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains, and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses, and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell that acquired different genetic alterations in virtually all evaluable cases (92%, 12 of 13 cases). Using whole-genome sequencing of diagnostic and transformation samples in 1 patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype, and International Prognostic Index score at transformation were predictive of a shorter survival from transformation (P = .001; P = .042; and P = .007; respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event., (© 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.)

Background: Animal models of acute respiratory distress syndrome (ARDS) do not completely resemble human ARDS, struggling translational research. We aimed to characterize a porcine model of ARDS induced by pneumonia-the most common risk factor in humans-and analyze the additional effect of ventilator-induced lung injury (VILI)., Methods: Bronchoscopy-guided instillation of a multidrug-resistant Pseudomonas aeruginosa strain was performed in ten healthy pigs. In six animals (pneumonia-with-VILI group), pulmonary damage was further increased by VILI applied 3 h before instillation and until ARDS was diagnosed by PaO 2 /FiO 2  < 150 mmHg. Four animals (pneumonia-without-VILI group) were protectively ventilated 3 h before inoculum and thereafter. Gas exchange, respiratory mechanics, hemodynamics, microbiological studies and inflammatory markers were analyzed during the 96-h experiment. During necropsy, lobar samples were also analyzed., Results: All animals from pneumonia-with-VILI group reached Berlin criteria for ARDS diagnosis until the end of experiment. The mean duration under ARDS diagnosis was 46.8 ± 7.7 h; the lowest PaO 2 /FiO 2 was 83 ± 5.45 mmHg. The group of pigs that were not subjected to VILI did not meet ARDS criteria, even when presenting with bilateral pneumonia. Animals developing ARDS presented hemodynamic instability as well as severe hypercapnia despite high-minute ventilation. Unlike the pneumonia-without-VILI group, the ARDS animals presented lower static compliance (p = 0.011) and increased pulmonary permeability (p = 0.013). The highest burden of P. aeruginosa was found at pneumonia diagnosis in all animals, as well as a high inflammatory response shown by a release of interleukin (IL)-6 and IL-8. At histological examination, only animals comprising the pneumonia-with-VILI group presented signs consistent with diffuse alveolar damage., Conclusions: In conclusion, we established an accurate pulmonary sepsis-induced ARDS model., (© 2023. The Author(s).)

Background: Pseudomonas aeruginosa pneumonia is commonly treated with systemic antibiotics to ensure adequate treatment of multidrug resistant (MDR) bacteria. However, intravenous (IV) antibiotics often achieve suboptimal pulmonary concentrations. We therefore aimed to evaluate the effect of inhaled amikacin (AMK) plus IV meropenem (MEM) on bactericidal efficacy in a swine model of monolateral MDR P. aeruginosa pneumonia., Methods: We ventilated 18 pigs with monolateral MDR P. aeruginosa pneumonia for up to 102 h. At 24 h after the bacterial challenge, the animals were randomized to receive 72 h of treatment with either inhaled saline (control), IV MEM only, or IV-MEM plus inhaled AMK (MEM + AMK). We dosed IV MEM at 25 mg/kg every 8 h and inhaled AMK at 400 mg every 12 h. The primary outcomes were the P. aeruginosa burden and histopathological injury in lung tissue. Secondary outcomes included the P. aeruginosa burden in tracheal secretions and bronchoalveolar lavage fluid, the development of antibiotic resistance, the antibiotic distribution, and the levels of inflammatory markers., Results: The median (25-75th percentile) P. aeruginosa lung burden for animals in the control, MEM only, and MEM + AMK groups was 2.91 (1.75-5.69), 0.72 (0.12-3.35), and 0.90 (0-4.55) log 10 CFU/g (p = 0.009). Inhaled therapy had no effect on preventing dissemination compared to systemic monotherapy, but it did have significantly higher bactericidal efficacy in tracheal secretions only. Remarkably, the minimum inhibitory concentration of MEM increased to > 32 mg/L after 72-h exposure to monotherapy in 83% of animals, while the addition of AMK prevented this increase (p = 0.037). Adjunctive therapy also slightly affected interleukin-1β downregulation. Despite finding high AMK concentrations in pulmonary samples, we found no paired differences in the epithelial lining fluid concentration between infected and non-infected lungs. Finally, a non-significant trend was observed for higher amikacin penetration in low-affected lung areas., Conclusions: In a swine model of monolateral MDR P. aeruginosa pneumonia, resistant to the inhaled AMK and susceptible to the IV antibiotic, the use of AMK as an adjuvant treatment offered no benefits for either the colonization of pulmonary tissue or the prevention of pathogen dissemination. However, inhaled AMK improved bacterial eradication in the proximal airways and hindered antibiotic resistance., (© 2023. The Author(s).)

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce. Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed. Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them. Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings., Competing Interests: The authors have no conflicts of interest that relate to the content of this article.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS) high -B cell receptor (BCR) low -signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT., (© 2022. The Author(s).)

High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60-78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases.

CART19 cells are emerging as an alternative therapy for patients with chronic lymphocytic leukemia (CLL). Here we report the outcome of nine consecutive patients with CLL treated with ARI-0001 CART19 cells, six of them with Richter's transformation (RT). One patient with RT never received therapy. The cytokine release syndrome rate was 87.5% (12.5% grade ≥3). Neurotoxicity was not observed in any patient. All patients experienced absolute B-cell aplasia, and seven (87.5%) responded to therapy. With a median follow-up of 5.6 months, two patients with RT experienced a CD19-negative relapse. In conclusion, ARI-0001 cell therapy was feasible, safe, and effective in patients with high-risk CLL or RT., Competing Interests: VO-M: Consultant or advisory role (Kite Gilead, Celgene, Novartis), travel grants (Kite Gilead, Celgene, Novartis, Roche, Takeda, Janssen), honoraria (Kite Gilead). EG: Consultant or advisory role (Kite Gilead, Janssen, Genmab), research funding (Kite Gilead, Janssen, Roche). AM-R: Consultant or advisory role (Bristol Myers Squibb, Abbvie), travel grants (Kite Gilead, Roche, Takeda, Janssen, Abbvie), honoraria (Abbvie). ML: Honoraria (Grifols, Fresenius Kabi), research funding (Terumo BCT, Maco-Pharma). JE: Consultant or advisory role (Abbvie, Novartis, Celgene, Astellas, Jazz, Daiichi Dankyo, Roche, Amgen, Pfizer), travel grants (Celgene, Roche, Astellas, Daiichi Dankyo), research funding (Novartis, Celgene). AL-G: Consultant or advisory role (Roche, Kite Gilead, Celgene/Bristol-Myers, Incyte), honoraria (Roche, Novartis, Takeda, Bayer, Sandoz, Kern), research grants (Roche, Kite Gilead, Celgene/Bristol-Myers, Novartis, Incyte, Janssen, Pfizer, Takeda). ÁU-I: Consultant or advisory role (Kite Gilead, Celgene, Miltenyi), travel grants (Kite Gilead, Celgene). MJ: Consultant or advisory role (Kite Gilead, Grifols), honoraria (Kite Gilead, Grifols). The remaining 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 © 2022 Ortiz-Maldonado, Frigola, Español-Rego, Balagué, Martínez-Cibrián, Magnano, Giné, Pascal, Correa, Martínez-Roca, Cid, Lozano, Villamor, Benítez-Ribas, Esteve, López-Guillermo, Campo, Urbano-Ispizua, Juan and Delgado.)

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11- primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11- tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory t (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies., (© 2021 by The American Society of Hematology.)

Streptococcus pneumoniae is the most common microbial cause of community-acquired pneumonia. Currently, there are no available models of severe pneumococcal pneumonia in mechanically ventilated animals to mimic clinical conditions of critically ill patients. We studied endogenous pulmonary flora in 4 healthy pigs and in an additional 10 pigs in which we intra-bronchially instilled S. pneumoniae serotype 19 A, characterized by its resistance to penicillin, macrolides and tetracyclines. The pigs underwent ventilation for 72 h. All pigs that were not challenged with S. pneumoniae completed the 72-h study, whereas 30% of infected pigs did not. At 24 h, we clinically confirmed pneumonia in the infected pigs; upon necropsy, we sampled lung tissue for microbiological/histological confirmation of pneumococcal pneumonia. In control pigs, Streptococcus suis and Staphylococcus aureus were the most commonly encountered pathogens, and their lung tissue mean ± s.e.m. concentration was 7.94 ± 20 c.f.u./g. In infected pigs, S. pneumoniae was found in the lungs of all pigs (mean ± s.e.m. pulmonary concentration of 1.26 × 10 5 ± 2 × 10 2 c.f.u./g). Bacteremia was found in 50% of infected pigs. Pneumococcal pneumonia was confirmed in all infected pigs at 24 h. Pneumonia was associated with thrombocytopenia, an increase in prothrombin time, cardiac output and vasopressor dependency index and a decrease in systemic vascular resistance. Upon necropsy, microbiological/histological pneumococcal pneumonia was confirmed in 8 of 10 pigs. We have therefore developed a novel model of penicillin- and macrolide-resistant pneumococcal pneumonia in mechanically ventilated pigs with bacteremia and severe hemodynamic compromise. The model could prove valuable for appraising the pathogenesis of pneumococcal pneumonia, the effects associated with macrolide resistance and the outcomes related to the use of new diagnostic strategies and antibiotic or complementary therapies., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Anti-programmed cell death (PD1)/ligand-1 (PD-L1) checkpoint inhibitors have improved the survival of non-small cell lung cancer (NSCLC) patients. Additionally, PD-L1 has emerged as a predictive biomarker of response. Our goal was to examine the histological features of all PD-L1 cases of NSCLC analyzed in our center between 2017 and 2020, as well as to correlate the expression values of the same patient in different tested samples. PD-L1 immunohistochemistry (IHC) was carried out on 1279 external and internal samples: 482 negative (tumor proportion score, TPS < 1%; 37.7%), 444 low-expression (TPS 1-49%; 34.7%) and 353 high-expression (TPS ≥ 50%; 27.6%). Similar results were observed with samples from our institution (N = 816). Significant differences were observed with respect to tumor histological type ( p = 0.004); squamous carcinoma was positive in a higher proportion of cases than other histological types. There were also differences between PD-L1 expression and the type of sample analyzed (surgical, biopsy, cytology; p < 0.001), with a higher frequency of negative cytology. In addition, there were cases with more than one PD-L1 determination, showing heterogeneity. Our results show strong correlation with the literature data and reveal heterogeneity between tumors and samples from the same patient, which could affect eligibility for treatment with immunotherapy.

The rising frequency of multidrug-resistant and extensively drug-resistant (MDR/XDR) pathogens is making more frequent the inappropriate empirical antimicrobial therapy (IEAT) in nosocomial pneumonia, which is associated with increased mortality. We aim to determine the short-term benefits of appropriate empirical antimicrobial treatment (AEAT) with ceftolozane/tazobactam (C/T) compared with IEAT with piperacillin/tazobactam (TZP) in MDR Pseudomonas aeruginosa pneumonia. Twenty-one pigs with pneumonia caused by an XDR P. aeruginosa strain (susceptible to C/T but resistant to TZP) were ventilated for up to 72 h. Twenty-four hours after bacterial challenge, animals were randomized to receive 2-day treatment with either intravenous saline (untreated) or 25 to 50 mg of C/T per kg body weight (AEAT) or 200 to 225 mg of TZP per kg (IEAT) every 8 h. The primary outcome was the P. aeruginosa burden in lung tissue and the histopathology injury. P. aeruginosa burden in tracheal secretions and bronchoalveolar lavage (BAL) fluid, the development of antibiotic resistance, and inflammatory markers were secondary outcomes. Overall, P. aeruginosa lung burden was 5.30 (range, 4.00 to 6.30), 4.04 (3.64 to 4.51), and 4.04 (3.05 to 4.88) log 10 CFU/g in the untreated, AEAT, and IEAT groups, respectively ( P  = 0.299), without histopathological differences ( P  = 0.556). In contrast, in tracheal secretions ( P  < 0.001) and BAL fluid ( P  = 0.002), bactericidal efficacy was higher in the AEAT group. An increased MIC to TZP was found in 3 animals, while resistance to C/T did not develop. Interleukin-1β (IL-1β) was significantly downregulated by AEAT in comparison to other groups ( P  = 0.031). In a mechanically ventilated swine model of XDR P. aeruginosa pneumonia, appropriate initial treatment with C/T decreased respiratory secretions' bacterial burden, prevented development of resistance, achieved the pharmacodynamic target, and may have reduced systemic inflammation. However, after only 2 days of treatment, P. aeruginosa tissue concentrations were moderately affected., (Copyright © 2021 American Society for Microbiology.)

Background: NANCI, an intergenic long non-coding RNA (lncRNA) is essential for buffering NKX2-1 expression during embryonic development and in adult tissue. We analyzed NANCI and NKX2-1 in human lung embryonic samples and adult lung tissues and evaluated their potential as prognostic markers in stage I non-small cell lung cancer (NSCLC)., Methods and Results: NANCI and NKX2-1 expression was assessed by TaqMan assays in 18 human embryonic samples from 8 to 13 weeks, 59 non-tumoral (NT) lung tissue samples, and 98 stage I NSCLC tumor samples. NANCI and NKX2-1 expression in embryonic and NSCLC samples were downregulated in comparison to adult NT tissue. Patients with low expression of NANCI had shorter disease-free survival (DFS) and overall survival (OS) than those with high levels (47.6 vs 69.3 months, P=0.032 and 57.7 vs 77.6 months, P=0.021, respectively). When the expression levels of NANCI and NKX2-1 were evaluated in combination, four groups were identified (high NANCI/high NKX2-1, low NANCI/high NKX2-1, high NANCI/low NKX2-1 and low NANCI/low NKX2-1) with differential impact on DFS (P=0.042) and OS (P=0.024). Interestingly, the high NANCI/high NKX2-1 duplex group had longer DFS and OS than the other three groups (71.25 vs 46.3 months, P=0.009 and 81.3 vs 56.1 months, P=0.004, respectively). In the multivariate analysis, the high NANCI/high NKX2-1 duplex was identified as an independent prognostic factor for longer DFS (HR 0.346, 95% CI, 0.169-0.709; P=0.004) and OS (HR 0.309, 95% CI, 0.121-0.786; P=0.014)., Conclusions: NANCI and the NANCI-NKX2-1 duplex impacts prognosis in stage I NSCLC patients., (Copyright © 2020 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.)

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D₄ (LTD₄) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD₄ on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD₄. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT₁ cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD₄, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT₁ receptor antagonist, blocked the LTD₄-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD₄-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD₄ and exogenous HB-EGF. These findings suggest LTD₄ induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.