B-cell non-Hodgkin’s lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

This study provides convincing evidence that CD10 expression is related to a distinct GC signature in MCL cases, but without clinical or biological implications.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level, but other less common subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 755 B-NHL tumors at high depth; primarily including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated across major subtypes, such as the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations that are pathognomonic. The cumulative burden of mutations within a single cluster were more significantly discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic epistasis that contribute to disease etiology. We therefore provide a framework of co-occurring mutations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis of B-NHL subtypes.KEY POINTSGenetic perturbation of the ubiquitin proteasome system is an emerging hallmark of B-cell non-Hodgkin lymphoma (B-NHL).Co-occurring sets of genetic alterations define B-NHL subtypes and likely represent epistatic interactions.

The trophoblast cells at the maternal-fetal interface express an unusual combination of human leukocyte antigen (HLA)-C, HLA-E and HLA-G. Altered expression of HLA-G on the extravillous cytotrophoblast has been implicated in the etiology of recurrent miscarriages (RMs). We have assessed HLA-G expression in extravillous cytotrophoblast in cell cultures prepared from RM patients and compared with those of first-trimester voluntarily terminated normal pregnancies (control). Glucocorticoids, dexamethasone and hydrocortisone were examined for their role in modulation of the HLA-G expression. HLA-G promoter and 3'UTR variants were investigated for their effect on the transcription of HLA-G. Cultured cytotrophoblast cells from the first-trimester RM patients were treated with dexamethasone and hydrocortisone (dose concentration 0-1000 ng/ml). HLA-G gene transcription was determined by semiquantitative and quantitative real-time polymerase chain reaction (RT-PCR), while protein expression was determined by a specific enzyme-linked immunosorbent assay (ELISA), flow cytometry and western blot analyses. HLA-G polymorphisms were detected by PCR and/or sequence-based typing. Low level of HLA-G was observed in untreated trophoblast cells obtained from RM patients as compared with controls. Upon treatment with glucocorticoids, the expression of HLA-G in these cells was up-regulated in a dose-dependent manner (P < 0.05), with no change in cellular proliferation and viability. There was no significant association between HLA-G polymorphism in RM patients and controls. HLA-G is minimally expressed in cultured trophoblast cells of RM patients. It can be up-regulated upon exposure with both dexamethasone and hydrocortisone. Glucocorticoids have the potential to modulate HLA-G expression in vitro, and can be further examined for their therapeutic applicability in RM.

The objective of this study was to create a bioclinical model, based on clinical and molecular predictors of event-free and overall survival for relapsed/refractory diffuse large B-cell lymphoma patients treated on the Canadian Cancer Trials Group (CCTG) LY12 prospective study. In 91 cases, sufficient histologic material was available to create tissue microarrays and perform immunohistochemistry staining for CD10, BCL6, MUM1/IRF4, FOXP1, LMO2, BCL2, MYC, P53 and phosphoSTAT3 (pySTAT3) expression. Sixty-seven cases had material sufficient for fluorescent in situ hybridization (FISH) for MYC and BCL2. In addition, 97 formalin-fixed, paraffin-embedded tissue samples underwent digital gene expression profiling (GEP) to evaluate BCL2, MYC, P53, and STAT3 expression, and to determine cell-of-origin (COO) using the Lymph2Cx assay. No method of determining COO predicted event-free survival (EFS) or overall survival (OS). Factors independently associated with survival outcomes in multivariate analysis included primary refractory disease, elevated serum lactate dehydrogenase (LDH) at relapse, and MYC or BCL2 protein or gene expression. A bioclinical score using these four factors predicted outcome with 3-year EFS for cases with 0–1 vs. 2–4 factors of 55% vs. 16% (P<0.0001), respectively, assessing MYC and BCL2 by immunohistochemistry, 46% vs. 5% (P<0.0001) assessing MYC and BCL2 messenger ribonucleic acid (mRNA) by digital gene expression, and 42% vs. 21% (P=0.079) assessing MYC and BCL2 by FISH. This proposed bioclinical model should be further studied and validated in other datasets, but may discriminate relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients who could benefit from conventional salvage therapy from others who require novel approaches. The LY12 study; clinicaltrials.gov Identifier: 00078949.

Mantle cell lymphoma (MCL) is an aggressive disease with frequent relapse. Targeted therapies against B-cell receptor (BCR) molecules have demonstrated improved outcomes in relapsed cases. However, clinical responses are slow and selective, with failure to attain complete remission in a significant subset of patients. Complex interaction of BCR signal transduction with toll-like receptor (TLR) and other pathways in MCL remains unknown, thus averting progress in development of targeted therapies. We have performed detailed digital quantification of BCR/TLR signalling molecules and their effector pathways in a cohort (n = 81) of MCL patients and correlated these data with overall survival. Hierarchical clustering model based on BCR/TLR genes revealed two distinct (BCR high and BCR low ) subsets of patients (n = 32; 40%) with significant differences in expression (>1.5-fold change; p < 0.05). Higher levels of BTK/SYK/BLNK/CARD11/PLCG signalosome and lower expression of MALT1/BCL10 genes suggested tonic pattern of BCR activation. Amplified expression of TLR6/TLR7/TLR9 was noted in concert with hyper-responsiveness of BCR machinery. MYD88, a key TLR adaptor molecule, was not upregulated in any of these clusters, which may suggest a 'cross-talk' between BCR and TLR pathways. In sync with BCR/TLR signalling, we recorded significantly enhanced expression of genes associated with NF-kB pathway in BCR high subset of MCL patients. On univariate analysis, the BCR high patients showed a trend towards inferior clinical response to a standardized treatment protocol, compared with the BCR low group (log rank, p = 0.043). In conclusion, we have identified hyperactive BCR/TLR signalling pathways and their effector downstream targets in a subset of MCL patients and associated it with poor clinical outcomes. Our study provides quantitative evidence at RNA expression level of possible concomitant collaboration between TLR and BCR signalling molecules in MCL. These data will provide further insights for future functional studies and, hence, development of targeted therapies for MCL patients.

AimsThe cell of origin (COO) based molecular characterisation into germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) subtypes are central to the pathogenesis and clinical course in diffuse large B-cell lymphoma (DLBCL). Globally, clinical laboratories employ pragmatic but less than ideal immunohistochemical (IHC) assay for COO classification. Novel RNA-based platforms using routine pathology samples are emerging as new gold standard and offer unique opportunities for assay standardisation for laboratories across the world. We evaluated our IHC protocols against RNA-based technologies to determine concordance; additionally, we gauged the impact of preanalytical variation on the performance of Lymph2Cx assay.MethodsDiagnostic biopsies (n=104) were examined for COO classification, employing automated RNA digital quantification assay (Lymph2Cx). Results were equated against IHC-based COO categorisation. Assay performance was assessed through its impact on overall survival (OS).Results96 (92%) informative samples were labelled as GCB (38/96; 40%) and non-GCB (58/96; 60%) by IHC evaluation. Lymph2Cx catalogued 36/96 (37%) samples as GCB, 45/96 (47%) as ABC and 15/96 (16%) as unclassified. Lymph2Cx being reference, IHC protocol revealed sensitivity of 81% for ABC and 75% for GCB categorisation and positive predictive value of 81% versus 82%, respectively. Lymph2Cx-based COO classification performed superior to Hans algorithm in predicting OS (log rank test, p=0.017 vs p=0.212).ConclusionsOur report show that current IHC-based protocols for COO classification of DLBCL at UKM Malaysia are in line with previously reported results and marked variation in preanalytical factors do not critically impact Lymph2Cx assay quality.

Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd.