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Despite the effectiveness of immuno-chemotherapy, 40% of patients with diffuse large B-cell lymphoma (DLBCL) experience relapse or refractory disease. Longitudinal studies have previously focused on the mutational landscape of relapse but fell short of providing a consistent relapse-specific genetic signature. In our study, we have focused attention on the changes in GEP accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo patients with DLBCL. COO remained stable from diagnosis to relapse in 80% of patients, with only a single patient showing COO switching from activated B-cell–like (ABC) to germinal center B-cell–like (GCB). Analysis of the transcriptomic changes that occur following relapse suggest ABC and GCB relapses are mediated via different mechanisms. We developed a 30-gene discriminator for ABC–DLBCLs derived from relapse-associated genes that defined clinically distinct high- and low-risk subgroups in ABC–DLBCLs at diagnosis in datasets comprising both population-based and clinical trial cohorts. This signature also identified a population of

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.

Supplementary Table 4. Haloplex re-sequencing gene mutations

Supplementary Table 5. Sanger screening of NOTCH2 exon 34

Supplementary Figure 1. Additional gene maps

Supplementary Figure 2. ABSOLUTE

Supplementary Table 7. Therapies regimes use to treat SMZL patients in this study

Supplementary Table 6. Somatic data from 14 matched patients

Supplementary Tables 1-3. Supplementary Table 1: Sequential samples and clonal evolution Supplementary Table 2: Haloplex gene list Supplementary Table 3: Primer sequences

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively. Clin Cancer Res; 21(18); 4174–83. ©2015 AACR.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.

Angioimmunoblastic T‐cell lymphoma (AITL) is a neoplastic proliferation of T follicular helper cells with clinical and histological presentations suggesting a role of antigenic drive in its development. Genetically, it is characterized by a stepwise acquisition of somatic mutations, with early mutations involving epigenetic regulators (TET2, DNMT3A) and occurring in haematopoietic stem cells, with subsequent changes involving signaling molecules (RHOA, VAV1, PLCG1, CD28) critical for T‐cell biology. To search for evidence of potential oncogenic cooperation between genetic changes and intrinsic T cell receptor (TCR) signaling, we investigated somatic mutations and T‐cell receptor β (TRB) rearrangement in 119 AITL, 11 peripheral T‐cell lymphomas with T follicular helper phenotype (PTCL‐TFH), and 25 PTCL‐NOS using Fluidigm polymerase chain reaction (PCR) and Illumina MiSeq sequencing. We confirmed frequent TET2, DNMT3A, and RHOA mutations in AITL (72%, 34%, 61%) and PTCL‐TFH (73%, 36%, 45%) and showed multiple TET2 mutations (2 or 3) in 57% of the involved AITL and PTCL‐TFH. Clonal TRB rearrangement was seen in 76 cases with multiple functional rearrangements (2–4) in 18 cases (24%). In selected cases, we confirmed bi‐clonal T‐cell populations and further demonstrated that these independent T‐cell populations harboured identical TET2 mutations by using BaseScope in situ hybridization, suggesting their derivation from a common TET2 mutant progenitor cell population. Furthermore, both T‐cell populations expressed CD4. Finally, in comparison with tonsillar TFH cells, both AITL and PTCL‐TFH showed a significant overrepresentation of several TRB variable family members, particularly TRBV19*01. Our findings suggest the presence of parallel neoplastic evolutions from a common TET2 mutant haematopoietic progenitor pool in AITL and PTCL‐TFH, albeit to be confirmed in a large series of cases. The biased TRBV usage in these lymphomas suggests that antigenic stimulation may play an important role in predilection of T cells to clonal expansion and malignant transformation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p

Introduction: Neuroendocrine tumours (NETs) are rare tumours with an increasing incidence. While low- and intermediate-grade pancreatic NET (PanNET) and small intestinal NET (siNET) are slow growing, they have a relatively high rate of metastasizing to the liver, leading to substantially worse outcomes. In many solid tumours, the outcome is determined by the quality of the antitumour immune response. However, the quality and significance of antitumour responses in NETs are incompletely understood. This study provides clinico-pathological analyses of the tumour immune microenvironment in PanNET and siNETs. Methods: Formalin-fixed paraffin-embedded tissue from consecutive resected PanNETs (61) and siNETs (131) was used to construct tissue microarrays (TMAs); 1-mm cores were taken from the tumour centre, stroma, tumour edge, and adjacent healthy tissue. TMAs were stained with antibodies against CD8, CD4, CD68, FoxP3, CD20, and NCR1. T-cell counts were compared with counts from lung cancers. Results: For PanNET, median counts were CD8+ 35.4 cells/mm2, CD4+ 7.6 cells/mm2, and CD68+ macrophages 117.7 cells/mm2. For siNET, there were CD8+ 39.2 cells/mm2, CD4+ 24.1 cells/mm2, and CD68+ 139.2 cells/mm2. The CD8+ cell density in the tumour and liver metastases were significantly lower than in the adjacent normal tissues, without evidence of a cell-rich area at the tumour edge that might have suggested immune exclusion. T-cell counts in lung cancer were significantly higher than those in PanNET and siNETs: CD8+ 541 cells/mm2 and CD4+ 861 cells/mm2 (p ≤ 0.0001). Conclusion: PanNETs and siNETs are immune cold with no evidence of T cell exclusion; the low density of immune infiltrates indicates poor antitumour immune responses.

Cancer development is an evolutionary process driven by the acquisition of stochastic mutations, some of which increase cellular fitness in a co-evolving microenvironment.[1][1] Evidence across multiple cancer types demonstrates that this process is highly protracted, likely beginning years or even

Both antigenic drive and genetic change play a critical role in the development of MALT lymphoma, but neither alone is sufficient for malignant transformation, and lymphoma development critically depends on their cooperation. However, which of these different events concur and how they cooperate in MALT lymphomagenesis is totally unknown. To explore this, we investigated somatic mutations of 17 genes and IGHV usage in 179 MALT lymphomas from various sites. We showed that: 1) there was a significant association between the biased usage of IGHV4-34 (binds to the carbohydrate I/i antigens) and inactivating mutation of TNFAIP3 (encoding a global negative regulator of the canonical NF-B pathway) in ocular adnexal MALT lymphoma; 2) IGHV1-69 was significantly overrepresented (54%) in MALT lymphoma of salivary gland, but not associated with mutation in any of the 17 genes investigated; and 3) MALT lymphoma lacked mutations frequently seen in other B-cell lymphomas characterised by constitutive NF-B activities, including CD79B, CARD11, MYD88, TNFRSF11A and TRAF3. Our findings show for the first time a significant association between biased usage of autoreactive IGHV and somatic mutation of NF-B regulators in MALT lymphoma, arguing for their cooperation in sustaining chronic BCR signalling and driving oncogenesis in lymphoma development.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.

e12533 Background: Obesity is associated with breast cancer development and worse survival. Obesity can initiate, promote, and maintain systemic inflammation via metabolic reprogramming of macrophages that encircle adipocytes, termed crown-like structures (CLS). In breast cancer patients, CLS are present in 36-50% of patients and have been associated with anthropometric parameters. Here we focus on HER2+ breast cancer. The role of adiposity in HER2+ breast cancer is conflicting which may be attributed to the tumour heterogeneity. Adiposity has also been shown to affect the local immune environment of solid tumours. However, the prognostic significance of CLS in HER2+ breast cancer is still unknown. Methods: We investigated the prognostic significance of CLS in a cohort of 219 patients with primary HER2+ breast cancer who were diagnosed between 1982 to 2012 in Southampton General Hospital. This cohort includes 76 HER2+ trastuzumab naïve patients and 143 HER2+ patients treated with adjuvant trastuzumab. We stained FFPE tumour samples for the expression of CD68, CD16 and CD32B on CLS and correlated these to clinical outcomes. CLS were defined as CLS within distant adipose tissue, CLS within the adipose-tumour border (B-CLS) and intratumoural CLS. CLS were quantified manually in full face sections by two independent scorers and descriptive and Cox regression analysis was carried out. Results: A total of 201 tumours were suitable for CLS analyses. The median follow-up was 34.74 months (range, 0.43-299.08). In the trastuzumab naive cohort, B-CLS≤1 and B-CLS > 1 were present in 37 (52.11%) and 34 (47.89%), respectively. In the trastuzumab treated cohort, B-CLS≤1 were identified in 69 (53.08%) and B-CLS > 1 were found in 61 (46.92%) of the tumours. CLS were more commonly found in the adipose-tumour border (60.89%) rather than in the distant adipose tissue (36.14%) or intratumorally (14.36%). The presence of any CLS was significantly associated with BMI≥25 kg/m2 (p = 0.018). There was strong evidence of association between CD68+CD32B+ B-CLS and BMI≥25 kg/m2 (p = 0.007). Co-expression of CD16 and CD32B by B-CLS was more frequent in patients with BMI≥25 kg/m2 (p = 0.036). Survival analysis showed shorter time to metastatic disease in patients with CD68+ B-CLS > 1 (p = 0.011) in the trastuzumab treated cohort. Subgroup analysis revealed that in the BMI≥25 kg/m2 group, patients with CD68+ B-CLS > 1 had shorter time to metastatic disease compared to patients with B-CLS≤1 (p = 0.004). Multivariate cox regression showed that B-CLS > 1 is an independent prognostic factor for shorter time to metastatic disease in patients with primary HER2+ breast cancer that received adjuvant trastuzumab (HR 6.81, 95%CI (1.38-33.54), p = 0.018). Conclusions: B-CLS can be potentially used as a predictive biomarker to optimize the stratification and personalisation of treatment in HER2-overexpressed breast cancer patients.

Treatment of HER2+ breast cancer with trastuzumab is effective and combination anti-HER2 therapies have demonstrated benefit over monotherapy in the neoadjuvant and metastatic settings. This study investigated the therapeutic potential of targeting the BAG-1 protein co-chaperone in trastuzumab-responsive or -resistant cells. In the METABRIC dataset, BAG-1 mRNA was significantly elevated in HER2+ breast tumors and predicted overall survival in a multivariate analysis (HR = 0.81; p = 0.022). In a breast cell line panel, BAG-1 protein was increased in HER2+ cells and was required for optimal growth as shown by siRNA knockdown. Overexpression of BAG-1S in HER2+ SKBR3 cells blocked growth inhibition by trastuzumab, whereas overexpression of a mutant BAG-1S protein (BAG-1S H3AB), defective in binding HSC70, potentiated the effect of trastuzumab. Injection of a Tet-On SKBR3 clone, induced to overexpress myc-BAG-1S into the mammary fat pads of immunocompromised mice, resulted in 2-fold larger tumors compared to uninduced controls. Induction of myc-BAG-1S expression in two Tet-On SKBR3 clones attenuated growth inhibition by trastuzumab in vitro. Targeting endogenous BAG-1 by siRNA enhanced growth inhibition of SKBR3 and BT474 cells by trastuzumab, while BAG-1 protein-protein interaction inhibitor (Thio-S or Thio-2) plus trastuzumab combination treatment synergistically attenuated growth. In BT474 cells this reduced protein synthesis, caused G1/S cell cycle arrest and targeted the ERK and AKT signaling pathways. In a SKBR3 subpopulation with acquired resistance to trastuzumab BAG-1 targeting remained effective and either Thio-2 or BAG-1 siRNA reduced growth more compared to trastuzumab-responsive parental cells. In summary, targeting BAG-1 function in combination with anti-HER2 therapy might prove beneficial.

Highlights • Unique case of recurrent constrictive pericarditis associated with rheumatoid arthritis, requiring two pericardiectomies. • Recurrent constrictive “pericarditis” is a misleading term. • Rheumatoid arthritis cannot be demonstrated to be a risk factor for recurrent pericardial fibrosis due to the rare association., Introduction Constrictive pericarditis is an uncommon disease characterized by impaired diastolic filling of the ventricles, encased in a fibrotic pericardium resulting from an inflammatory process. Rheumatoid arthritis is a rare cause of constrictive pericarditis, usually due to a concomitant acute or chronic serositis. Presentation of case This paper presents a unique case of recurrent constrictive pericarditis associated with seropositive rheumatoid arthritis, requiring pericardiectomy and complicated three years later by recurrent fibrosis, demanding a second pericardiectomy. Discussion Defining this entity as recurrent constrictive “pericarditis” might be a mistake, given that a total pericardiectomy was performed in first instance. No risk factors for recurrent fibrosis have been identified in the current medical literature, apart from partial pericardiectomy. We cannot demonstrate exclusively that rheumatoid arthritis is a risk factor for recurrent pericardial/epicardial fibrosis due to the rare nature of the disease. Conclusion Further large scale studies are necessary to identify the risk factors for recurrence.

Background: The anti-CD20 monoclonal antibody (mAb), obinutuzumab (G), has shown improved outcomes versus rituximab (R) in indolent lymphomas; however, no improvement was seen in diffuse large B-cell lymphoma (DLBCL), and the molecular basis is unclear. The inhibitory Fc gamma receptor IIB (Fc γRIIB), expressed on lymphoma cells, can impair the effects of direct targeting mAbs, such as R, by binding and internalizing them, reducing opsonization and limiting FcγR-mediated killing. We hypothesized that FcγRIIB expression on cellular effectors and/or the lymphoma confers treatment resistance in some patients (pts) and evaluated if outcomes differ for therapies involving a non-internalized mAb (G). Methods: We evaluated correlates between FCGR2B mRNA and/or FcγRIIB protein expression and pt outcomes in two discovery cohorts of de novo DLBCL treated with R-CHOP (Arthur et al. 2018, n=372; Schmitz et al. 2018, n=234), and the phase III GOYA trial (NCT01287741; n=552), which compared R-CHOP with G-CHOP in pts with previously untreated DLBCL. FCGR2B mRNA expression was assessed by RNA-Seq, and protein expression was assessed in evaluable cohorts by immunohistochemistry, using tissue microarrays with macrophages identified by CD68. FCGR2B expression was also measured by a NanoString assay (Arthur cohort). Cox regression analyzed the impact of FcγRIIB/FCGR2B expression on progression-free survival (PFS), with univariate and multivariate models adjusted for International Prognostic Index (IPI), cell of origin (COO), and BCL2 protein expression. Results: In the discovery cohorts, a higher FCGR2B expression was significantly associated with shorter PFS (Arthur: HR 1.09 [95% CI: 1.01–1.19], P=0.036; Schmitz: HR 1.13 [95% CI: 1.02–1.26], P=0.0243). Expression by NanoString strongly correlated with RNA-Seq, confirming the association with shorter PFS (HR 1.13 [95% CI: 1.04–1.23], P=0.0048). In GOYA, a significant association between PFS and FCGR2B was observed in the R arm (HR 1.26 [95% CI: 1.00–1.58], P=0.0455), with no prognostic effect observed for G (HR 0.91 [95% CI: 0.69–1.20], P=0.5). Pts with high FCGR2B expression appeared to benefit more from G than R (HR 0.67 [95% CI: 0.44–1.02], P=0.0622), in contrast to pts with low FCGR2B expression (HR 1.58 [95% CI: 1.00–2.50], P=0.0503). In both Arthur and GOYA cohorts, FCGR2B expression by RNA-Seq was associated with FcγRIIB on the tumor, which correlated with a shorter PFS for R (HR 2.17 [95% CI: 1.04–4.50], P=0.03), but not G (HR 1.37 [95% CI: 0.66–2.87], P=0.4). This prognostic effect on PFS was independent of established prognostic biomarkers, IPI, COO and BCL2. Conclusion: High FcγRIIB/FCGR2B expression in pts with DLBCL has prognostic value in those treated with R and may confer differential responsiveness to R or G. Citation Format: Laura K. Hilton, Malgorzata Nowicka, Margaret Ashton-Key, Chantal E. Hargreaves, Chern Lee, Russell Foxall, Matthew J. Carter, Stephen A. Beers, Kathleen N. Potter, Christopher R. Bolen, Christian Klein, Andrea Knapp, Farheen Mir, Matthew Rose-Zerilli, Cathy Burton, Wolfram Klapper, David W. Scott, Laurie H. Sehn, Umberto Vitolo, Maurizio Martelli, Marek Trneny, Graham W. Slack, Pedro Farinha, Jonathan C. Strefford, Mikkel Z. Oestergaard, Ryan D. Morin, Mark S. Cragg. Prognostic significance of Fc gamma receptor IIB expression in the response of previously untreated diffuse large B-cell lymphomas to anti-CD20 monoclonal antibodies: Differing impact of rituximab and obinutuzumab [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-26.

Dennis H Wright was appointed as the foundation professor of pathology at the University of Southampton in 1972. Although he retired in 1996, he remained active in diagnosis until very recently, regularly attending the weekly lymphoma review panel in Southampton, and was still researching a further hypothesis regarding Burkitt lymphoma. His last international presentation was on the pathogenesis of endemic Burkitt lymphoma, at the meeting of the European Association of Haematopathology in Basel in September 2016 at the age of 85, which received a standing ovation. His final publication was the third edition of his book Diagnostic Lymph Node Pathology , published in 2016, which was awarded “highly commended” in the 2017 BMA book awards. Dennis grew up in Norfolk and attended the City of Norwich School with a county scholarship. He was subsequently awarded the University of Bristol open scholarship to read medicine. He qualified with honours and a substantial collection of academic prizes, including the Bristol Royal Hospital gold …

MALT lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterised. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin mediated receptor desensitization and internalisation. Screening of these newly identified mutations, together with previously identified genetic changes, identified distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterised by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic cooperation between receptor signalling and genetic changes. ispartof: Haematologica vol:103 issue:8 pages:1329-1336 ispartof: location:Italy status: published

Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (

Aims Immunoglobulin (Ig)G4-related disease (IgG4-RD) is an increasingly recognized fibroinflammatory condition that commonly exhibits multisystem involvement, with localized (e.g. inflammatory pseudotumours that can mimic malignancy) or diffuse (leading to organ dysfunction) patterns of tissue involvement. The 2012 Boston criteria have standardized the histopathological approach to the diagnosis of IgG4-RD and require one or more of the cardinal morphological features with prominence of IgG4+ plasma cells and an IgG4+/IgG+ plasma cell ratio of at least 40%. The relative prevalence of the morphological criteria varies between anatomical sites, but granulomas are rarely found and, indeed, their presence would usually deter a pathologist from making this diagnosis. The aim was to characterize two cases of IgG4-RD in which granulomas were present and to highlight this as an unusual feature of the condition. Methods and results We describe two cases in which the features of IgG4-RD were present within lymph nodes, together with granulomas. This is a recognized but rare morphological pattern of IgG4-RD. Conclusions While an unusual finding, the presence of granulomas should not preclude a diagnosis of IgG4-RD in the appropriate clinicopathological context.

Both antigenic drive and genetic change play critical roles in the development of mucosa-associated lymphoid tissue (MALT) lymphoma, but neither alone is sufficient for malignant transformation, and lymphoma development critically depends on their cooperation. However, which of these different events concur and how they cooperate in MALT lymphomagenesis is totally unknown. To explore this, we investigated somatic mutations of 17 genes and immunoglobulin heavy chain variable region (IGHV) usage in 179 MALT lymphomas from various sites. We showed that: (1) there was a significant association between the biased usage of IGHV4-34 (binds to the carbohydrate I/i antigens) and inactivating mutation of TNFAIP3 [encoding a global negative regulator of the canonical nuclear factor-κB (NF-κB) pathway] in ocular adnexal MALT lymphoma; (2) IGHV1-69 was significantly overrepresented (54%) in MALT lymphoma of the salivary gland, but was not associated with mutation in any of the 17 genes investigated; and (3) MALT lymphoma lacked mutations that are frequently seen in other B-cell lymphomas characterized by constitutive NF-κB activities, including mutations in CD79B, CARD11, MYD88, TNFRSF11A, and TRAF3. Our findings show, for the first time, a significant association between biased usage of autoreactive IGHV and somatic mutation of NF-κB regulators in MALT lymphoma, arguing for their cooperation in sustaining chronic B-cell receptor signalling and driving oncogenesis in lymphoma development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

Background: Although diffuse large B cell lymphoma (DLBCL) can be cured using immuno-chemotherapy, 40% of patients experience relapse or refractory disease. Large-scale profiling studies have mainly focused on DLBCL at diagnosis, resolving different outcome groups based on gene expression (e.g. cell-of-origin (COO) or molecular high grade), MYC/BCL2 translocations (double-hit lymphoma) or gene mutations and copy number aberrations (Schmitz et al, NEJM 2018; Chapuy et al, NatureMedicine 2018). In comparison, longitudinal studies have been hindered by the limited availability of sequential biopsy samples. To date, the relapse-specific gene mutations identified are limited and inconsistent across studies. In our study, we have focussed attention on the changes in gene expression profile (GEP) accompanying DLBCL relapse. Methods: We retrospectively collected archival paired diagnostic/relapse formalin fixed paraffin embedded tumor biopsies from 38 de novo DLBCL patients collected from multiple UK sites treated with rituximab-based immuno-chemotherapy, where partial or complete remission was reported following treatment. COO classification was performed by the Lymph2Cx assay on NanoString to distinguish activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subtypes. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit was used to measure the expression levels of > 20,000 genes on the paired samples. Results: COO remained stable from diagnosis to relapse in 17 ABC-ABC pairs, 11 GCB-GCB pairs and 4 unclassified (UNC)-UNC pairs. Frank COO switching was observed in 6 cases (1 ABC-GCB, 2 ABC-UNC, 2 GCB-UNC, 1 UNC-ABC). Pairs with stable COO were taken forward for further analysis. Gene expression analysis using the limma R package identified 163 and 136 genes as differentially expressed (DE) (p 1) between the diagnostic and relapse biopsies in ABC and GCB tumors respectively, with only a one gene overlap. Gene Set Enrichment Analysis further suggested that ABC and GCB relapses are mediated via different mechanisms, with tumor growth and proliferation signatures enriched in ABC relapses, whilst adaptive immunity-related signatures accompanied GCB relapses. Next, we aimed to utilise our relapse-specific genes to identify outcome predictors at diagnosis using publicly available GEP datasets. In order to increase our discovery power and accuracy, a larger set of DE genes from the paired differential analysis (796 genes in ABC pairs and 387 from GCB pairs) were selected (p The prognostic significance of this 30-gene discriminator was successfully validated using a linear predictor in two independent GEP datasets: 1) a population-based cohort (Lenz et al, NEJM 2008) with 93 R-CHOP-treated ABC cases identifying 47 low and 46 high-risk cases (HR=1.92, p=0.046, C-index=0.77; Fig1.C) and 2) a clinical trial dataset (REMoDL-B, Davies et al, Lancet Oncol 2019) with 255 ABC cases identifying 110 low and 145 high-risk ABC cases (HR=1.95, p=0.0051, C-index=0.70; Fig1.D). Conclusions: Here we describe a 30-gene discriminator in ABC-DLBCL, derived from genes differentially expressed between diagnosis and relapse, that allowed the definition of clinically distinct high and low risk subgroups in ABC-DLBCLs at diagnosis. The clinical translation of such a tool may be useful to guide therapy for this unfavourable subgroup of ABC-DLBCLs. Validation of this signature is currently underway in additional datasets and further study is required to understand the contribution of these genes in DLBCL pathology. Disclosures Korfi: Roche: Consultancy. Burton:Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rule:TG Therapeutics: Consultancy, Honoraria; Napp: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Astra-Zeneca: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Crosbie:Janssen: Honoraria. Scott:Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Roche/Genentech: Research Funding. Rimsza:NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution]. Davies:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Bayer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Acerta Pharma: Honoraria, Research Funding; ADCT Therapeutics: Honoraria, Research Funding; BioInvent: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gribben:Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Okosun:Gilead Sciences: Honoraria, Research Funding. Johnson:Epizyme: Honoraria, Research Funding; Novartis: Honoraria; Kite: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Boehringer Ingelheim: Honoraria; Takeda: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Incyte: Honoraria. Fitzgibbon:Epizyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Speakers Bureau.

Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.

Primary central nervous system (CNS) lymphoma is an aggressive B-cell tumor that is defined clinically by the absence of systemic disease. We have used immunoglobulin variable (V)–gene analysis to identify tumor cells at the CNS site in 12 cases and to probe the involvement of peripheral tissues in 3 patients. Clonal tracking revealed tumor cells in the bone marrow and/or blood for 3 of 3 cases, with evidence for increased V-gene mutational activity at peripheral sites. In 2 of 3 cases, intraclonal variant analysis revealed identity with the brain biopsy but detected additional variants unique to extracerebral sites. These findings suggest that peripheral tumor cells can undergo separate development locally with no reentry into the brain. Primary CNS lymphoma appears to have both CNS-specific and systemic components with limited interchange. The more malignant behavior of tumor cells in the CNS suggests either a local environmental influence or a less malignant phenotype of the peripheral clone.

// Bashar Zeidan 1 , Thomas R. Jackson 2, 5 , Samantha E.T. Larkin 1 , Ramsey I. Cutress 1 , Gary R. Coulton 3 , Margaret Ashton-Key 1 , Nick Murray 1 , Graham Packham 1 , Vassilis Gorgoulis 2, 4, 5 , Spiros D. Garbis 1 , Paul A. Townsend 1, 2, 5 1 Cancer Sciences Unit, University of Southampton, Southampton, UK 2 Faculty Institute for Cancer Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK 3 St George’s Medical Biomics Centre, St. George’s University of London, London, UK 4 Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece 5 Manchester Centre for Cellular Metabolism, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK Correspondence to: Paul A. Townsend, e-mail: p.a.townsend@manchester.ac.uk Keywords: annexin A3, migration, neoplasm, biomarker, breast cancer Received: February 06, 2015 Accepted: May 22, 2015 Published: June 04, 2015 ABSTRACT Breast cancers are the most common cancer-affecting women; critically the identification of novel biomarkers for improving early detection, stratification and differentiation from benign tumours is important for the reduction of morbidity and mortality. To identify and functionally characterise potential biomarkers, we used mass spectrometry (MS) to analyse serum samples representing control, benign breast disease (BBD) and invasive breast cancer (IDC) patients. Complementary and multidimensional proteomic approaches were used to identify and validate novel serum markers. Annexin A3 (ANX A3) was found to be differentially expressed amongst different breast pathologies. The diagnostic value of serum ANX A3 was subsequently validated by ELISA in an independent serum set representing the three groups. Here, ANX A3 was significantly upregulated in the benign disease group sera compared with other groups ( P < 0.0005). In addition, paired breast tissue immunostaining confirmed that ANX A3 was abundantly expressed in benign and to a lesser extent malignant neoplastic epithelium. Finally, we illustrated ANX A3 expression in cell culture lysates and conditioned media from neoplastic breast cell lines, and its role in neoplastic breast cell migration in vitro . This study confirms the novel role of ANX A3 as a mammary biomarker, regulator and therapeutic target.

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03). Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively. Clin Cancer Res; 21(18); 4174–83. ©2015 AACR.

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

We have compared light chain immunohistochemistry in reactive lymphoid tissue and a series of paraffin-embedded B-cell lymphomas using standard trypsin digestion with a heat mediated epitope retrieval method. Fifty-seven B-cell lymphomas (18 high grade, 29 low grade and 10 cases of nodular lymphocyte predominance Hodgkin's disease), two reactive lymph nodes and eight tonsils fixed for known times between 12 h and 2 years were studied. Paraffin-embedded tissue was stained with polyclonal anti-kappa and anti-lambda antibodies. For each antibody staining was performed on two sections, one treated with trypsin digestion and one with microwave heating. Sections were scored from 0 to + + + with 0 representing poor staining and + + + excellent staining. A score of ++ was considered satisfactory. Light chain restriction was recorded if present. Satisfactory staining was obtained in 34/59 cases using trypsin digestion and 56/59 cases using heat retrieval. Light chain restriction was demonstrated in 32/57 (56%) B-cell lymphomas using trypsin digestion and 52/57 (91%) using heat retrieval. Satisfactory staining was obtained in tonsils fixed for up to 48 h using trypsin digestion and up to 2 years using heat retrieval. We have shown that for light chain immunostaining a heat mediated epitope retrieval method produces more consistent and satisfactory results and is effective over a greater range of fixation times than traditional trypsin digestion.

AIMS: To investigate the occurrence of abnormal patterns of HLA-ABC and HLA-DR expression in enteropathy associated T cell lymphoma and to relate such abnormalities to the Epstein Barr virus (EBV) status of the tumours. METHODS: Eleven enteropathy associated T cell lymphomas were immunostained with HC10 (HLA-ABC heavy chain) and TAL 1B5 (HLA-DR alpha chain) monoclonal antibodies and polyclonal anti-beta 2 microglobulin (beta 2m, the HLA-ABC light chain) antibodies. In situ hybridisation for EBV using EBER probes was performed on all cases. RESULTS: Tumour cells of two of 11 patients were EBER positive. One of these showed partial, and the other, complete loss of beta 2m. HLA-DR expression was undetectable in both patients. Of the remaining nine EBER negative tumours, two were HLA-ABC heavy chain negative or showed only occasional positive cells and five of nine showed partial or complete loss of the HLA-ABC light chain, beta 2m. Seven of the nine cases were either negative for HLA-DR or showed weak expression in a proportion of tumour cells. CONCLUSIONS: These data show that low or absent HLA-ABC and HLA-DR antigen expression occurs commonly in enteropathy associated T cell lymphoma. These abnormal patterns of HLA expression may be associated with escape from immune attack which, in a minority of patients, could be directed against EBV antigens.