Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Iron is a modifier of the phenotypes of JAK2-mutant myeloproliferative neoplasms.

JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.

Detection of disease-specific signatures in B cell repertoires of lymphomas using machine learning.

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.

The International Consensus Classification of Mature Lymphoid Neoplasms: a report from the Clinical Advisory Committee.

Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

Genomic profiling for clinical decision making in lymphoid neoplasms.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

Primary bone diffuse large B-cell lymphoma (PB-DLBCL): a distinct extranodal lymphoma of germinal centre origin, with a common EZB-like mutational profile and good prognosis.

AIMS: Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is not recognized as a separate entity by the current classification systems. Here we define and highlight its distinctive clinical presentation, morphology, phenotype, gene expression profile (GEP), and molecular genetics. METHODS: We collected 27 respective cases and investigated their phenotype, performed gDNA panel sequencing covering 172 genes, and carried out fluorescence in situ hybridization to evaluate MYC, BCL2, and BCL6 translocations. We attempted to genetically subclassify cases using the Two-step classifier and performed GEP for cell-of-origin subtyping and in silico comparison to uncover up- and downregulated genes as opposed to other DLBCL. RESULTS: Most cases (n = 22) were germinal centre B-cell-like (GCB) by immunohistochemistry and all by GEP. Additionally, PB-DLBCL had a mutational profile similar to follicular lymphoma and nodal GCB-DLBCL, with the exception of more frequent TP53 and B2M mutations. The GEP of PB-DLBCL was unique, and the frequency of BCL2 rearrangements was lower compared to nodal GCB-DLBCL. The Two-step classifier categorized eight of the cases as EZB, three as ST2, and one as MCD. CONCLUSION: This study comprehensively characterizes PB-DLBCL as a separate entity with distinct clinical and morpho-molecular features. These insights may aid in developing tailored therapeutic strategies and shed light on its pathogenesis.

Increased TIM-3 and galectin-9 serum levels in patients with advanced systemic mastocytosis.

BACKGROUND: Systemic mastocytosis is characterized by expansion of clonal mast cells in various tissues. Several biomarkers with diagnostic and therapeutic potential have recently been characterized in mastocytosis, such as the serum marker tryptase and the immune checkpoint molecule PD-L1. OBJECTIVE: We aimed to investigate whether serum levels of other checkpoint molecules are altered in systemic mastocytosis and whether these proteins are expressed in mastocytosis infiltrates in the bone marrow. METHODS: Levels of different checkpoint molecules were analyzed in serum of patients with different categories of systemic mastocytosis and healthy controls and correlated to disease severity. Bone marrow biopsies from patients with systemic mastocytosis were stained to confirm expression. RESULTS: Serum levels of TIM-3 and galectin-9 were increased in systemic mastocytosis, particularly in advanced subtypes, compared with healthy controls. TIM-3 and galectin-9 levels were also found to correlate with other biomarkers of systemic mastocytosis, such as serum tryptase and KIT D816V variant allele frequency in the peripheral blood. Moreover, we observed expression of TIM-3 and galectin-9 in mastocytosis infiltrates in bone marrow. CONCLUSIONS: Together, our results demonstrate for the first time that serum levels of TIM-3 and galectin-9 are increased in advanced systemic mastocytosis. Moreover, TIM-3 and galectin-9 are expressed in bone marrow infiltrates in mastocytosis. These findings provide a rationale for exploring TIM-3 and galectin-9 as diagnostic markers and eventually therapeutic targets in systemic mastocytosis, particularly in advanced forms.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Genetic Factors in Familial Manifestation of Primary Mediastinal Large B-Cell Lymphoma over Two Generations.

INTRODUCTION: Primary mediastinal large B-cell lymphoma (PMBL) is a rarely occurring lymphoid malignancy which typically affects young adults and presents itself as an anterior mediastinal mass. Gene expression profiling as well as somatic genetic analysis revealed that it is closely related to classical Hodgkin lymphoma, whereas morphologically, it tends to resemble diffuse large B-cell lymphoma. Familial clustering of PMBL is rare - only two reports have been published to date. While it is generally accepted that positive family history is associated with increased risk of developing a lymphoma, genetic risk factors which might predispose to PMBL are largely unknown. CASE PRESENTATION: We performed germline and tumor genetic analyses by whole-exome sequencing and array-CGH of a family, in which the father and the son both developed a PMBL. Germline investigations of both affected patients and of their two unaffected family members have not been able to provide a single risk factor associated with lymphoma predisposition. In addition, genes that were previously implicated in increased risk for PMBL, namely MLL (KMT2A) and TIRAP, were found to be intact in all investigated family members. Somatic genetic investigations identified known as well as novel genetic aberrations in tumors of the affected subjects. CONCLUSION: We conclude that predisposition to a PMBL might be inherited through a combination of low- or moderate-risk factors and provide a shortlist of the most likely selected candidates, which can be used in future studies.

Molecular characterization and genetic subclassification comparison of diffuse large B-cell lymphoma (DLBCL) - real-life experience with 74 cases.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity. Lately, several algorithms achieving therapeutically and prognostically relevant DLBCL subclassification have been published. METHODS: A cohort of 74 routine DLBCL cases was broadly characterized by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) of the BCL2, BCL6 and MYC loci, and comprehensive high throughput sequencing (HTS). Based on the genetic alterations found, cases were reclassified using two probabilistic tools - LymphGen and Two-step classifier, allowing for comparison of the two models. RESULTS: Hans and Tally's overall IHC-based subclassification success rate was 96% and 82%, respectively. HTS and FISH data allowed the LymphGen algorithm to successfully classify 11/55 cases, (1 - BN2, 7 - EZB, 1 - MCD, and 2 - genetically composite EZB/N1). The total subclassification rate was 20%. On the other hand, the Two-step classifier categorized 36/55 cases, with 65.5% success (9 - BN2, 12 - EZB, 9 - MCD, 2 - N1, and 4 - ST2). Clinical correlations highlighted MCD as an aggressive subtype associated with higher relapse and mortality. CONCLUSIONS: The Two-step algorithm has a better success rate at subclassifying DLBCL cases based on genetic differences. Further improvement of the classifiers is required to increase the number of classifiable cases and thus prove their applicability in routine diagnostics.

Optimized antiangiogenic reprogramming of the tumor microenvironment potentiates CD40 immunotherapy.

Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Deciphering the genetic landscape of pulmonary lymphomas.

Pulmonary lymphoid malignancies comprise various entities, 80% of them are pulmonary marginal zone B-cell lymphomas (PMZL). So far, little is known about point mutations in primary pulmonary lymphomas. We characterized the genetic landscape of primary pulmonary lymphomas using a customized high-throughput sequencing gene panel covering 146 genes. Our cohort consisted of 28 PMZL, 14 primary diffuse large B-cell lymphomas (DLBCL) of the lung, 7 lymphomatoid granulomatoses (LyG), 5 mature small B-cell lymphomas and 16 cases of reactive lymphoid lesions. Mutations were detected in 22/28 evaluable PMZL (median 2 mutation/case); 14/14 DLBCL (median 3 mutations/case) and 4/7 LyG (1 mutation/case). PMZL showed higher prevalence for mutations in chromatin modifier-encoding genes (44% of mutant genes), while mutations in genes related to the NF-κB pathway were less common (24% of observed mutations). There was little overlap between mutations in PMZL and DLBCL. MALT1 rearrangements were more prevalent in PMZL than BCL10 aberrations, and both were absent in DLBCL. LyG were devoid of gene mutations associated with immune escape. The mutational landscape of PMZL differs from that of extranodal MZL of other locations and also from splenic MZL. Their landscape resembles more that of nodal MZL, which also show a predominance of mutations of chromatin modifiers. The different mutational composition of pulmonary DLBCL compared to PMZL suggests that the former probably do not present transformations. DLBCL bear more mutations/case and immune escape gene mutations compared to LyG, suggesting that EBV infection in LyG may substitute for mutations.

Short regimen of rituximab plus lenalidomide in follicular lymphoma patients in need of first-line therapy.

The SAKK 35/10 phase 2 trial, developed by the Swiss Group for Clinical Cancer Research and the Nordic Lymphoma Group, compared the activity of rituximab vs rituximab plus lenalidomide in untreated follicular lymphoma patients in need of systemic therapy. Patients were randomized to rituximab (375 mg/m2 IV on day 1 of weeks 1-4 and repeated during weeks 12-15 in responding patients) or rituximab (same schedule) in combination with lenalidomide (15 mg orally daily for 18 weeks). Primary end point was complete response (CR)/unconfirmed CR (CRu) rate at 6 months. In total, 77 patients were allocated to rituximab monotherapy and 77 to the combination (47% poor-risk Follicular Lymphoma International Prognostic Index score in each arm). A significantly higher CR/CRu rate at 6 months was documented in the combination arm by the investigators (36%; 95% confidence interval [CI], 26%-48% vs 25%; 95% CI, 16%-36%) and confirmed by an independent response review of computed tomography scans only (61%; 95% CI, 49%-72% vs 36%; 95% CI, 26%-48%). After a median follow-up of 4 years, significantly higher 30-month CR/CRu rates and longer progression-free survival (PFS) and time to next treatment (TTNT) were observed for the combination. Overall survival (OS) rates were similar in both arms (≥90%). Toxicity grade ≥3 was more common in the combination arm (56% vs 22% of patients), mainly represented by neutropenia (23% vs 7%). Addition of lenalidomide to rituximab significantly improved CR/CRu rates, PFS, and TTNT, with expected higher, but manageable toxicity. The excellent OS in both arms suggests that chemotherapy-free strategies should be further explored. This trial was registered at www.clinicaltrials.gov as #NCT01307605.

JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Cytokeratin expression in plasmablastic lymphoma - a possible diagnostic pitfall in the routine work-up of tumours.

AIMS: Plasmablastic lymphoma (PBL) is a rare aggressive B-cell lymphoma that frequently arises at extranodal sites in the setting of immunosuppression. The diagnosis of PBL is complex, owing to a frequent solid or cohesive growth pattern, and an often unusual immunophenotype. Several case reports have described cytokeratin (CK) expression in PBL, introducing a diagnostic pitfall. The aim of this study was to determine the frequency of CK expression in PBL in the largest series available to date. METHODS AND RESULTS: By using immunohistochemistry in a cohort of 72 PBLs, we identified CK8/18 positivity in 11 of 72 cases (15%) and AE1/3 positivity in six of 65 cases (9%), clearly contrasting with a control series of non-PBL aggressive B-cell lymphomas (one of 96 diffuse large B-cell lymphomas), as well as with data in the literature describing only occasional CK expression in haematological neoplasms. CONCLUSIONS: Our data indicate CK expression in a substantial number (15%) of PBLs. In view of the particular morphological features of PBL and its frequent negativity for the common leukocyte antigen and B-cell markers, this feature represents a pitfall in the routine diagnostic work-up of PBL, and requires more extensive immunohistochemical and molecular characterisation of cases entering the differential diagnosis.

The tumor microenvironment of lymphomas: Insights into the potential role and modes of actions of checkpoint inhibitors.

The tumor microenvironment (TME) - a term comprising non-neoplastic cells and extracellular matrix as well as various cytokines, chemokines, growth factors, and other substances in the vicinity of tumor cells - is an integrative part of most tumors including lymphomas. Interactions between lymphoma cells and the TME are vital for survival and proliferation of the former. In addition, lymphoma cells often reprogram the TME to protect them from defense mechanisms of the host's immune system. In this review, we will introduce the role of the tumor microenvironment (TME) for lymphoma cells looking at direct cell-cell interactions as well as cytokine-related communications. The immunomodulative/immunosuppressive role of the TME is more and more coming into the focus of potential new targeted therapies, and thus a special attention will be given to the interactions of immune checkpoints such as programed cell death protein 1 and L1 (PD-1/PD-L1), T-cell immunoglobulin and mucin-domain containing protein-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), and cytotoxic T-lymphocyte-associated protein-4 (CTLA4) with the TME, as well as their expression by both lymphoma cells and cells of the TME. Aspects of the TME will be discussed for indolent and aggressive B-cell lymphomas, Hodgkin lymphomas, and T-cell lymphomas. In addition, the potential influence of other immunomodulators such as lenalidomide will be briefly touched. The complex role of the TME is in the focus of new therapeutic options. In order to exploit its full therapeutic potential, however, a thorough understanding of TME biology and interaction between lymphoma cells and the TME, as well as the host's immune system and the TME is necessary.

Prognostic implications of the microenvironment for follicular lymphoma under immunomodulation therapy.

Follicular lymphoma (FL) constitutes a significant proportion of lymphomas and shows frequent relapses. Beyond conventional chemotherapy, new therapeutic approaches have emerged, focussing on the interplay between lymphoma cells and the microenvironment. Here we report the immunophenotypic investigation of the microenvironment of a clinically well-characterized prospective cohort (study SAKK35/10, NCT01307605) of 154 treatment-naïve FL patients in need of therapy, who have been treated with rituximab only or a combination of rituximab and the immunomodulatory drug lenalidomide/Revlimid® A high ratio of CD4- to CD8-positive T cells (P = 0·009) and increased amounts of PD1+ tumour-infiltrating T cells (P = 0·007) were associated with inferior progression-free survival in the whole cohort. Interestingly, the prognostic impact of PD1+ T cells and the CD4/CD8 ratio lost its significance in the subgroup treated with R2 . In the latter group, high amounts of GATA3+ T helper (Th2) equivalents were associated with better progression-free survival (P < 0·001). We identified tumour microenvironmental features that allow prognostic stratification with respect to immuno- and combined immuno- and immunomodulatory therapy. Our analysis indicates that lenalidomide may compensate the adverse prognostic implication of higher amounts of CD4+ and, particularly, PD1+ T cells and that it has favourable effects mainly in cases with higher amounts of Th2 equivalents. [Correction added on 11 February 2020, after online publication: The NCT-trial number was previously incorrect and has been updated in this version].

Prognostic models integrating quantitative parameters from baseline and interim positron emission computed tomography in patients with diffuse large B-cell lymphoma: post-hoc analysis from the SAKK38/07 clinical trial.

Positron emission computed tomography (PET/CT) in patients with diffuse large B-cell lymphoma (DLBCL) enrolled in a prospective clinical trial were reviewed to test the impact of quantitative parameters from interim PET/CT scans on overall (OS) and progression-free (PFS) survival. We centrally reviewed baseline and interim PET/CT scans of 138 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone given every 14 days (R-CHOP14) in the SAKK38/07 trial (ClinicalTrial.gov identifier: NCT00544219). Cutoff values for maximum standardized uptake value (SUVmax ), metabolic tumor volume (MTV), total lesion glycolysis (TLG) and metabolic heterogeneity (MH) were defined by receiver operating characteristic analysis. Responses were scored using the Deauville scale (DS). Patients with DS 5 at interim PET/CT (defined by uptake >2 times higher than in normal liver) had worse PFS (P = 0.014) and OS (P < 0.0001). A SUVmax reduction (Δ) greater than 66% was associated with longer PFS (P = 0.0027) and OS (P < 0.0001). Elevated SUVmax , MTV, TLG, and MH at interim PET/CT also identified patients with poorer outcome. At multivariable analysis, ΔSUVmax and baseline MTV appeared independent outcome predictors. A prognostic model integrating ΔSUVmax and baseline MTV discriminated three risk groups with significantly (log-rank test for trend, P < 0.0001) different PFS and OS. Moreover, the integration of MH and clinical prognostic indices could further refine the prediction of OS. PET metrics-derived prognostic models perform better than the international indices alone. Integration of baseline and interim PET metrics identified poor-risk DLBCL patients who might benefit from alternative treatments.

High throughput sequencing reveals high specificity of TNFAIP3 mutations in ocular adnexal marginal zone B-cell lymphomas.

The majority of ocular adnexal (OA) lymphomas (OAL) are extranodal marginal zone lymphomas (MZL). First high throughput sequencing (HTS) studies on OA-MZL showed inconsistent results and the distribution of mutations in reactive lymphoid lesions of this anatomic region has not yet been sufficiently addressed. We characterized OAL and lymphoid lesions of the OA by targeted HTS. The study included 34 OA-MZL, 11 chronic conjunctivitis, five mature small cell B-cell lymphomas spreading to the OA, five diseases with increase of IgG4+ plasma cells, three Burkitt lymphomas (BL), three diffuse large B-cell lymphomas (DLBCL), three mantle cell lymphomas, three idiopathic orbital inflammations/orbital pseudo tumors (PT), and three OA lymphoid hyperplasia. All cases were negative for Chlamydia. The mutational number was highest in BL and lowest in PT. The most commonly (and exclusively) mutated gene in OA-MZL was TNFAIP3 (10 of 34 cases). Altogether, 20 out of 34 patients harbored mutually exclusive mutations of either TNFAIP3, BCL10, MYD88, ATM, BRAF, or NFKBIE, or nonexclusive mutations of IRF8, TNFRSF14, KLHL6, and TBL1XR1, all encoding for NK-κB pathway compounds or regulators. Thirteen patients (38%) had, to a great part, mutually exclusive mutations of chromatin modifier-encoding genes: KMT2D, CREBBP, BCL7A, DNMT3A, EP300, or HIST1H1E. Only four patients harbored co-occurring mutations of genes encoding for NK-κB compounds and chromatin modifiers. Finally, PTEN, KMT2D, PRDM1, and HIST1H2BK mutations were observable in reactive lymphoid lesions too, while such instances were devoid of NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes. In conclusion, 80% of OA-MZL display mutations of either NK-κB compounds or chromatin modifiers. Lymphoid lesions of the OA bearing NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes highly likely represent lymphomas.