Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and in genetically modified mouse models, and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Stress is a well-known risk factor to develop a functional neurological disorder, a frequent neuropsychiatric medical condition in which patients experience a variety of disabling neurological symptoms. Only little is known about biological stress regulation, and how it interacts with predisposing biological and psychosocial risk factors. Dysregulation of the hypothalamic-pituitary-adrenal axis in patients with functional neurological disorders has been postulated but its relationship to preceding psychological trauma and brain anatomical changes remains to be elucidated. We set out to study the hypothalamic-pituitary-adrenal axis analysing the cortisol awakening response and diurnal baseline cortisol in 86 patients with mixed functional neurological symptoms compared to 76 healthy controls. We then examined the association between cortisol regulation and the severity and duration of traumatic life events. Finally, we analysed volumetric brain alterations in brain regions particularly sensitive to psychosocial stress, acting on the assumption of the neurotoxic effect of prolonged cortisol exposure. Overall, patients had a significantly flatter cortisol awakening response (P < 0.001) and reported longer (P = 0.01) and more severe (P < 0.001) emotional neglect as compared to healthy controls. Moreover, volumes of the bilateral amygdala and hippocampus were found to be reduced in patients. Using a partial least squares correlation, we found that in patients, emotional neglect plays a role in the multivariate pattern between trauma history and hypothalamic-pituitary-adrenal axis dysfunction, whilst cortisol did not relate to reduced brain volumes. This suggests that psychological stress acts as a precipitating psychosocial risk factor, whereas a reduced brain volume rather represents a biological predisposing trait marker for the disorder. Contrarily, an inverse relationship between brain volume and cortisol was found in healthy controls, representing a potential neurotoxic effect of cortisol. These findings support the theory of reduced subcortical volumes representing a predisposing trait factor in functional neurological disorders, rather than a state effect of the illness. In summary, this study supports a stress-diathesis model for functional neurological disorders and showed an association between different attributes of trauma history and abnormalities in hypothalamus-pituitary-adrenal axis function. Moreover, we suggest that reduced hippocampal- and amygdalar volumes represent a biological ‘trait marker’ for functional neurological disorder patients, which might contribute to a reduced resilience to stress.

Introduction. The majority of SMZLs display an indolent course, however the disease is still incurable and a significant proportion of patients (~25-30%) experience poor outcomes surviving <5 years. Molecular alterations of SMZL are promising biomarkers, which might improve risk stratification of patients. The main objective of the study is to test the impact of molecular aspects on disease-specific survival prognostication in newly diagnosed SMZL. Methods. IELSG46 is a multicenter, international, retrospective, observational study in which already existing and coded health-related personal and biological material is used. The study included adults, who received a diagnosis of SMZL on spleen histology with a follow up >5 years, and for whom tumor material collected before initiation of medical therapy was available. Mutation analysis was performed by CAPP-seq targeted deep next generation sequencing of tumor genomic DNA. A stringent bioinformatic pipeline was applied to suppress the background noise allowing to call variants with a sensitivity of 5x10-2 in FFPE derived DNA. Copy number variations (CNVs) were identified by using the sequencing reads-based GATK4-CNV algorithm. IGHV rearrangements were obtained by using LymphoTrack® IGH FR1 Assay Panel kit. Molecular clusters were identified by an iterative algorithm that maximizes genetic distinctiveness of subgroups by reassigning patients between clusters that are created a priori based on the co-occurrence of genetic lesions. Relative survival, defined as the ratio between actuarial survival observed in the SMZL cohort and expected survival of the general population matched to patients by geographical origin, sex, age and calendar year of diagnosis, was calculated using the Ederer II method. Results. The analysis included 303 patients with a SMZL diagnosis confirmed on spleen histology. The sample size allowed to identify 30% differences in survival for molecular subgroups comprising at least 5% of cases with a statistical power between 80-100%. Median follow-up was 9.2 years. At 10 years, 85% of patients were alive, consistent with the known indolent behavior of this lymphoma. Genes recurrently affected by non-synonymous somatic mutations in >10% of SMZL included KLF2 (24%), NOTCH2 (19%), KMT2D (15%), TNFAIP3 (13%), EP300 and TP53 (10%). Deletion 7q was documented in 25% of cases and IGHV1-2*04 usage in 32%. By cluster analysis, three major molecular subgroups were identified, each of them characterized by a NOTCH pathway mutated gene (Fig. 1A). The first cluster was defined by NOTCH2 and/or KLF2 mutations and was enriched in TNFAIP3 mutations and IGHV1-2*04 gene usage (Fig. 1A). The second cluster was defined by SPEN mutations, and was enriched in KMT2D and other epigenetic gene mutations (Fig. 1A). The third cluster was enriched in NOTCH1 mutations (Fig. 1A). By relative survival analysis, the NOTCH2/KLF2 cluster showed a lower survival compared to the matched general population, indicating a significant impact of the disease on patients' expected survival (Fig. 1B). Conclusions. The large sample size and inclusion of SMZL confirmed by spleen histopathology review allowed for precise estimation of the prevalence of KLF2 and NOTCH2 mutations in this lymphoma. Three molecular clusters were identified in SMZL, each of them containing a NOTCH pathway gene, supporting the relevance of NOTCH signaling in the pathogenesis of SMZL. Patients belonging to the NOTCH2/KLF2 cluster had a lower relative survival compared to the matched general population. Disclosures Traverse-Glehen: Astra Zeneca: Other: Travel; Takeda: Research Funding. Gomes da Silva:Roche: Other: Institution's payment for consultancy, Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Other: Travelling support; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Ladetto:Celgene: Honoraria; Jannsen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria; Roche: Honoraria. Rambaldi:Pfizer: Consultancy; Novartis: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy; Celgene: Consultancy. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Zinzani:MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Salles:Servier: Honoraria; Abbvie: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria. Zucca:Celltrion: Consultancy; AstraZeneca: Consultancy.