Follicular lymphoma-associated mutations in vacuolar ATPase ATP6V1B2 activate autophagic flux and mTOR.

The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to mTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the human v-ATPase subunit ATP6V1B2 (also known as Vma2 in yeast) activate autophagic flux and maintain mTOR/TOR in an active state. Engineered lymphoma cell lines and primary FL B cells carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutations. These data support the idea of mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a possible new therapeutically targetable pathway.

An open-label phase 2 trial of entospletinib in indolent non-Hodgkin lymphoma and mantle cell lymphoma.

Spleen tyrosine kinase (Syk) mediates B-cell receptor signalling in normal and malignant B cells. Entospletinib is an oral, selective Syk inhibitor. Entospletinib monotherapy was evaluated in a multicentre, phase 2 study of patients with relapsed or refractory indolent non-Hodgkin lymphoma or mantle cell lymphoma (MCL). Subjects received 800 mg entospletinib twice daily. Forty-one follicular lymphoma (FL), 17 lymphoplasmacytoid lymphoma/Waldenström macroglobulinaemia (LPL/WM), 17 marginal zone lymphoma (MZL) and 39 MCL patients were evaluated. The primary endpoint was a progression-free survival (PFS) rate (defined as not experiencing progression or death) at 16 weeks for patients with MCL and at 24 weeks for patients with FL, LPL/WM and MZL. The most common treatment-emergent adverse events were fatigue, nausea, diarrhoea, vomiting, headache and cough. Common laboratory abnormalities were anaemia, neutropenia and thrombocytopenia; aspartate transaminase, alanine transaminase, total bilirubin and serum creatinine were all increased. PFS at 16 weeks in the MCL cohort was 63·9% [95% confidence interval (CI) 45-77·8%]; PFS at 24 weeks in the FL, LPL/WM, MCL and MZL cohorts was 51·5% (95% CI 32·8-67·4%), 69·8% (95% CI 31·8-89·4%), 56·6% (95% CI 37·5-71·8%) and 46·2% (95% CI 18·5-70·2%), respectively. Entospletinib had limited single-agent activity with manageable toxicity in these patient populations.

Emerging EZH2 Inhibitors and Their Application in Lymphoma.

PURPOSE OF REVIEW: Enhancer of Zeste Homolog 2 (EZH2) is histone methyltransferase and catalyzes the methylation of histone 3 lysine 27, a mark of transcriptional repression. Various studies have elucidated the complex role of EZH2 in both normal biology and tumorigenesis. Here, we critically review the emerging role of EZH2 in malignancies, the development of small molecule inhibitors of EZH2, and their application in lymphoma. RECENT FINDINGS: Activating mutations and overexpression of EZH2 are found in non-Hodgkin lymphoma (NHL). As a result, several EZH2 inhibitors have been developed and entered clinical investigation. Tazemetostat, first-in-class EZH2 inhibitor, demonstrated enhanced clinical activity in mutant follicular lymphoma and diffuse large B cell lymphoma. With the early activity noted by tazemetostat in B cell lymphomas, the role of EZH2 inhibition in NHL is becoming more evident. This can be leveraged in future rationale combinations to enhance the activity of EZH2 inhibitors.

CD10 down expression in follicular lymphoma correlates with gastrointestinal lesion involving the stomach and large intestine.

Follicular lymphoma (FL) shows co-expression of B-cell lymphoma 2 (BCL2) and CD10, whereas downexpression of CD10 is occasionally experienced in gastrointestinal (GI) FL with unknown significance. Gastrointestinal FL is a rare variant of FL, and its similarity with mucosa-associated lymphoid tissue lymphoma was reported. We investigated the clinicopathological and genetic features of CD10 downexpressed (CD10down ) GI-FL. The diagnosis of CD10down FL was carried out with a combination of pathological and molecular analyses. The incidence of CD10down GI-FL was shown in 35/172 (20.3%) cases, which was more frequent than nodal FL (3.5%, P < 0.001). The difference was additionally significant between GI-FL and nodal FL when the analysis was confined to primary GI-FL (55.2% vs 3.5%, P < 0.001). Compared to CD10+ GI-FL, CD10down GI-FL significantly involved the stomach or large intestine (P = 0.015), and additionally showed the downexpression of BCL6 (P < 0.001). The follicular dendritic cell meshwork often showed a duodenal pattern in the CD10down group (P = 0.12). Furthermore, a lymphoepithelial lesion was observed in 5/12 (40%) gastric FL cases, which indicated caution in the differentiation of mucosa-associated lymphoid tissue lymphoma. Molecular analyses were undertaken in seven cases of CD10down GI-FL, and an identical clone was found between CD10down follicles and CD10+ BCL2+ neoplastic follicles. In the diagnosis of cases with CD10down BCL2+ follicles, careful examination with molecular studies should be carried out.

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

N-acetyltransferase polymorphisms are associated with risk of lymphoma subtypes.

Genes encoding for arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) have been investigated with alternate findings in relation to risk of non-Hodgkin lymphoma (NHL). We tested functional haplotype-based NAT1 and NAT2 gene polymorphisms in relation to risk of lymphoma overall and its major B cell subtypes, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). We used allele specific primers and multiplex PCR to detect NAT1 and NAT2 haplotypes in 248 patients with incident lymphoma and 208 population controls. We inferred the NAT1 rapid and slow acetylator and the NAT2 rapid, intermediate or slow acetylator phenotype, based on published functional data on the respective genotypes. Odds ratios and 95% confidence intervals (95% CIs) for lymphoma, B-NHL, DLBCL, FL, CLL, and other B-NHL combined associated with the inferred rapid NAT1 acetylator and with the intermediate and slow NAT2 acetylator phenotypes were estimated with unconditional and polytomous logistic regression analysis, adjusting for age, gender and education. NAT1 rapid acetylators showed a 2.8-fold excess risk (95% CI 1.5-5.2) for lymphoma (all subtypes combined). Risk was highest for CLL and FL, with significant heterogeneity detected across subtypes. Risk also increased with decreasing NAT2 acetylating capacity with no heterogeneity detected across B cell lymphoma subtypes. Risks did not vary by gender. Although poor statistical power was a major limitation in our study, larger studies and pooled analyses are warranted to test whether NAT1 and NAT2 gene polymorphisms might modulate risk of specific lymphoma subtypes through the varying metabolic activity of their products. Copyright © 2015 John Wiley & Sons, Ltd.

Idelalisib-associated Enterocolitis: Clinicopathologic Features and Distinction From Other Enterocolitides.

Idelalisib is a highly specific small-molecule phosphoinositide-3-kinase δ inhibitor that was recently approved by the Food and Drug Administration for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma and follicular lymphoma. The known side effects of idelalisib include severe diarrhea and colitis. Here we report the histologic findings in idelalisib-associated enterocolitis in 11 patients with chronic lymphocytic leukemia or follicular lymphoma receiving idelalisib over a 5-year period (2011 to 2015) at our institution. All 11 patients were receiving idelalisib and underwent colonoscopy for the evaluation of diarrhea. None of the patients had previously received a stem cell transplant. Histologically, the colon biopsies in all 11 cases showed some degree of apoptosis within crypts, with 5 cases showing moderate to severe apoptosis involving the majority of the crypts with loss of goblet cells. No viral inclusions were seen in any case and immunohistochemical stains for cytomegalovirus performed in 9/11 cases were negative. All cases showed at least focal acute cryptitis, and 8 of these cases showed mild architectural distortion. Increased inflammation within the lamina propria was seen in 7 cases, and increased intraepithelial lymphocytes within crypts was seen in 8 cases; the lymphocytes were mostly T cells with a predominance of CD8 T cells, with the majority expressing the α/ß T-cell receptor. Diagnoses of graft-versus-host disease, autoimmune enteropathy, infectious enterocolitis, and although thought to be less likely, inflammatory bowel disease were considered in each case. The presence of numerous intraepithelial lymphocytes in addition to severe villous blunting and apoptosis in the small intestinal biopsies from a subset of these patients additionally raised the possibility of autoimmune enteropathy, common variable immunodeficiency, or less likely, celiac disease. Awareness of the histologic features of idelalisib-associated enterocolitis is important to distinguish it from potential mimics, particularly graft-versus-host disease, autoimmune enteropathy, and cytomegalovirus/infectious enterocolitis.

A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXß, and ATXγ) and Novel Autotaxins (ATXδ and ATXε).

BACKGROUND: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXß, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated. METHODS: Anti-ATXß and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXß and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of "classical ATX" (ATXα, ATXß, and ATXγ) and "novel ATX" (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples. RESULTS: The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups. CONCLUSIONS: We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

Protein kinase CK2 is widely expressed in follicular, Burkitt and diffuse large B-cell lymphomas and propels malignant B-cell growth.

Serine-threonine kinase CK2 is highly expressed and pivotal for survival and proliferation in multiple myeloma, chronic lymphocytic leukemia and mantle cell lymphoma. Here, we investigated the expression of α catalytic and ß regulatory CK2 subunits by immunohistochemistry in 57 follicular (FL), 18 Burkitt (BL), 52 diffuse large B-cell (DLBCL) non-Hodgkin lymphomas (NHL) and in normal reactive follicles. In silico evaluation of available Gene Expression Profile (GEP) data sets from patients and Western blot (WB) analysis in NHL cell-lines were also performed. Moreover, the novel, clinical-grade, ATP-competitive CK2-inhibitor CX-4945 (Silmitasertib) was assayed on lymphoma cells. CK2 was detected in 98.4% of cases with a trend towards a stronger CK2α immunostain in BL compared to FL and DLBCL. No significant differences were observed between Germinal Center B (GCB) and non-GCB DLBCL types. GEP data and WB confirmed elevated CK2 mRNA and protein levels as well as active phosphorylation of specific targets in NHL cells. CX-4945 caused a dose-dependent growth-arresting effect on GCB, non-GCB DLBCL and BL cell-lines and it efficiently shut off phosphorylation of NF-κB RelA and CDC37 on CK2 target sites. Thus, CK2 is highly expressed and could represent a suitable therapeutic target in BL, FL and DLBCL NHL.

Open-label, single-arm, phase II study of enzastaurin in patients with follicular lymphoma.

This open-label, phase II study investigated whether enzastaurin, a protein kinase C-beta (PKCß) inhibitor, had activity in patients with grade 1 or 2 follicular lymphoma (FL). Adults with grade 1 or 2 FL who had no more than one prior treatment received oral enzastaurin continuously for up to 3 years. Of the 66 patients who received enzastaurin, 53 were evaluable for response. Overall response rate (ORR, primary efficacy endpoint) was 26.4% (3.8% complete response). Median (95% confidence interval) progression-free survival, time to response, and duration of response were 18.1 (11.5-28.3), 4.9 (2.8-8.1), and 22.3 (8.8-not applicable) months, respectively. In patients with tumour tissue available for biomarker analysis, ORRs in low versus high PKCß2 expression groups were 41.7% and 8.3%, respectively (P = 0.041). The most common, mainly low-grade drug-related adverse events were fatigue (25.8%), diarrhoea (25.8%), nausea (18.2%), and chromaturia (18.2%). Four (6.1%) patients had Grade 3 toxicity and one (1.5%) patient had Grade 4 toxicity. Enzastaurin demonstrated limited clinical activity in grade 1 or 2 FL. Patients with low PKCß2 expression in tumours had higher ORR than those with high PKCß2 expression. Enzastaurin was well tolerated with mostly grade 1 or 2 toxicities. Further studies may be warranted in select patient populations.

Correlations between functional imaging markers derived from PET/CT and diffusion-weighted MRI in diffuse large B-cell lymphoma and follicular lymphoma.

OBJECTIVES: To investigate the correlations between functional imaging markers derived from positron emission tomography/computed tomography (PET/CT) and diffusion-weighted magnetic resonance imaging (DWI) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Further to compare the usefulness of these tumor markers in differentiating diagnosis of the two common types of Non-Hodgkin's lymphoma (NHL). MATERIALS AND METHODS: Thirty-four consecutive pre-therapy adult patients with proven NHL (23 DLBCL and 11 FL) underwent PET/CT and MRI examinations and laboratory tests. The maximum standardized uptake value (SUV(max)), metabolic tumor volume (MTV), and metabolic tumor burden (MTB) were determined from the PET/CT images. DWI was performed in addition to conventional MRI sequences using two b values (0 and 800 s/mm(2)). The minimum and mean apparent diffusion coefficient (ADC(min) and ADC(mean)) were measured on the parametric ADC maps. RESULTS: The SUV(max) correlated inversely with the ADC(min) (r =  -0.35, p<0.05). The ADC(min), ADC(mean), serum thymidine kinase (TK), Beta 2-microglobulin (B2m), lactate dehydrogenase (LD), and C-reactive protein (CRP) correlated with both whole-body MTV and whole-body MTB (p<0.05 or 0.01). The SUV(max), TK, LD, and CRP were significantly higher in the DLBCL group than in the FL group. Receiver operating characteristic curve analysis showed that they were reasonable predictors in differentiating DLBCL from FL. CONCLUSIONS: The functional imaging markers determined from PET/CT and DWI are associated, and the SUV(max) is superior to the ADC(min) in differentiating DLBCL from FL. All the measured serum markers are associated with functional imaging markers. Serum LD, TK, and CRP are useful in differentiating DLBCL from FL.

Phase II study of alisertib, a selective Aurora A kinase inhibitor, in relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas.

PURPOSE: Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can correlate with more histologically aggressive forms of disease. We therefore designed a phase II study of alisertib, a selective AAK inhibitor, in patients with relapsed and refractory aggressive non-Hodgkin lymphomas. PATIENTS AND METHODS: Patients age ≥ 18 years were eligible if they had relapsed or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL), transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in 21-day cycles. RESULTS: We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n = 13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n = 5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%), stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths during the study were attributed to progressive non-Hodgkin lymphoma (n = 2), treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall response rate was 27%, including responses in three of 21 patients with DLBCL, three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with transformed follicular lymphoma, and four of eight with noncutaneous T-cell lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the expected pharmacokinetic variability, with a trend for higher incidence of adverse event-related dose reductions at higher trough concentrations. Analysis for AAK gene amplification and total AAK protein revealed no differences between histologies or correlation with clinical response. CONCLUSION: The novel AAK inhibitor alisertib seems clinically active in both B- and T-cell aggressive lymphomas. On the basis of these results, confirmatory single-agent and combination studies have been initiated.

The serine-threonine kinase p90RSK is a new target of enzastaurin in follicular lymphoma cells.

BACKGROUND AND PURPOSE: Follicular lymphoma is the second most common non-Hodgkin's lymphoma and, despite the introduction of rituximab for its treatment, this disease is still considered incurable. Besides genetic alterations involving Bcl-2, Bcl-6 or c-Myc, follicular lymphoma cells often display altered B-cell receptor signalling pathways including overactive PKC and PI3K/Akt systems. EXPERIMENTAL APPROACH: The effect of enzastaurin, an inhibitor of PKC, was evaluated both in vitro on follicular lymphoma cell lines and in vivo on a xenograft murine model. Using pharmacological inhibitors and siRNA transfection, we determined the different signalling pathways after enzastaurin treatment. KEY RESULTS: Enzastaurin inhibited the serine-threonine kinase p90RSK which has downstream effects on GSK3ß. Bad and p70S6K. These signalling proteins control follicular lymphoma cell survival and apoptosis; which accounted for the inhibition by enzastaurin of cell survival and its induction of apoptosis of follicular lymphoma cell lines in vitro. Importantly, these results were replicated in vivo where enzastaurin inhibited the growth of follicular lymphoma xenografts in mice. CONCLUSIONS AND IMPLICATIONS: The targeting of p90RSK by enzastaurin represents a new therapeutic option for the treatment of follicular lymphoma.

EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.

In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

Quantitative MRI establishes the efficacy of PI3K inhibitor (GDC-0941) multi-treatments in PTEN-deficient mice lymphoma.

AIM: To assess the efficacy of multiple treatment of phosphatidylinositol-3-kinase (PI3K) inhibitor on autochthonous tumours in phosphatase and tensin homologue (Pten)-deficient genetically engineered mouse cancer models using a longitudinal magnetic resonance imaging (MRI) protocol. MATERIALS AND METHODS: Using 3D MRI, B-cell follicular lymphoma growth was quantified in a Pten(+/-)Lkb1(+/hypo) mouse line, before, during and after repeated treatments with a PI3K inhibitor GDC-0941 (75 mg/kg). RESULTS: Mean pre-treatment linear tumour growth rate was 16.5±12.8 mm(3)/week. Repeated 28-day GDC-0941 administration, with 21 days 'off-treatment', induced average tumour regression of 41±7%. Upon cessation of the second treatment (which was not permanently cytocidal), tumours re-grew with an average linear growth rate of 40.1±15.5 mm(3)/week. There was no evidence of chemoresistance. CONCLUSION: This protocol can accommodate complex dosing schedules, as well as combine different cancer therapies. It reduces biological variability problems and resulted in a 10-fold reduction in mouse numbers compared with terminal assessment methods. It is ideal for preclinical efficacy studies and for phenotyping molecularly characterized mouse models when investigating gene function.

Mechanism of fragility at BCL2 gene minor breakpoint cluster region during t(14;18) chromosomal translocation.

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg(2+) and Mn(2+) are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression.

Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patient's disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. The abundance of BCR-insensitive cells in each tumor negatively correlated with overall patient survival. These lymphoma negative prognostic (LNP) cells increased as tumors relapsed following chemotherapy. Loss of antigen receptor expression did not explain the absence of BCR signaling in LNP tumor cells, and other signaling responses were intact in these cells. Furthermore, BCR signaling responses could be reactivated in LNP cells, indicating that BCR signaling is not missing but rather specifically suppressed. LNP cells were also associated with changes to signaling interactions in the tumor microenvironment. Lower IL-7 signaling in tumor infiltrating T cells was observed in tumors with high LNP cell counts. The strength of signaling through T cell mediator of B cell function CD40 also stratified patient survival, particularly for those whose tumors contained few LNP cells. Thus, analysis of cell-cell interactions in heterogeneous primary tumors using signaling network profiles can identify and mechanistically define new populations of rare and clinically significant cells. Both the existence of these LNP cells and their aberrant signaling profiles provide targets for new therapies for follicular lymphoma.

Activation-induced cytidine deaminase expression in diffuse large B-cell lymphoma with a paracortical growth pattern: a lymphoma of possible interfollicular large B-cell origin.

CONTEXT: Activation-induced cytidine deaminase, necessary for immunoglobulin somatic hypermutation and class switch recombination, is usually expressed within the follicular dendritic network but is also expressed in a population of interfollicular large B cells outside the germinal center. OBJECTIVE: To report 7 cases of diffuse large B-cell lymphoma with a distinct paracortical distribution. Expression of activation-induced cytidine deaminase, previously described in interfollicular large B cells, was evaluated. DESIGN: A panel of immunohistochemical markers, including double staining for activation-induced cytidine deaminase and CD20, was used to illustrate the cases. Molecular studies were performed by polymerase chain reaction in the paraffin-embedded tissue for t(14;18) chromosomal translocation and immunoglobulin heavy chain and T-cell receptor rearrangements. RESULTS: Patients included 3 males and 4 females ranging in age from 11 to 59 years (mean, 39 years). All specimens were lymph nodes (4 from the groin, 2 from the neck, and 1 from the axilla). Malignant lymphocytes were positive for CD20 and negative for CD5 and CD10. Staining for CD30, CD43, and BCL-2 was variable. The malignant cells showed at least focal staining with activation-induced cytidine deaminase. All cases were found to be monoclonal by immunoglobulin heavy-chain gene rearrangement or showed light-chain restriction. None of the tested cases showed t(14;18). CONCLUSIONS: Diffuse large B-cell lymphoma with a paracortical distribution is unusual and may be a distinct morphologic variant. More study is necessary to determine the stage of B-cell development and the cell of origin of these tumors. However, activation-induced cytidine deaminase expression suggests they may arise from a putative interfollicular large B cell.