The Expression of the RUVBL1 Component of the R2TP Complex Correlates with Poor Prognosis in DLBCL.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin's lymphoma (NHL) accounting for 30% of adult NHL worldwide and 50% in developing countries like India. DNA damage and Myc-induced transformation are well-known contributing factors towards development of DLBCL. A recently identified HSP90 co-chaperone complex R2TP has been shown to contribute towards DNA damage and Myc-induced transformation. This study aimed to analyse the immunohistochemical (IHC) expression of R2TP complex components RUVBL1, PIH1D1, and RPAP3 in DLBCL patients and correlate with prognosis. METHODS: DLBCL (n = 54) histological slides were retrieved from archives, and detailed histomorphological and clinical features were noted. IHC staining of R2TP complex components RUVBL1, PIH1D1, and RPAP3 was performed on 54 cases (FFPE) of DLBCL. Expression data were correlated with survival and clinical features. RESULTS: Out of the 54 DLBCL cases, 59.26% (n = 32) stained positive for RUVBL1. The RUVBL1 expression was associated with poor prognosis in both progression-free survival (PFS) (p = 0.0146) and overall survival (OS) (p = 0.0328). The expression was positively correlated with bone marrow involvement (p = 0.0525). The expression of PIH1D1 was observed in 68.51% (n = 32) of DLBCL cases, and positive correlation was observed with international prognostic index score (p = 0.0246); however, no correlation was observed with PFS or OS. Finally, RPAP3 was found immunopositive in only 1 case of DLBCL. CONCLUSIONS: Immunopositivity for RUVBL1 is associated with poor prognosis along with a higher relapse rate amongst the DLBCL patients. PIH1D1 immunopositivity correlated with a higher IPI score.

Genetic Analysis of HIBM Myopathy-Specific GNE V727M Hotspot Mutation Identifies a Novel COL6A3 Allied Gene Signature That Is Also Deregulated in Multiple Neuromuscular Diseases and Myopathies.

The GNE-associated V727M mutation is one of the most prevalent ethnic founder mutations in the Asian HIBM cohort; however, its role in inducing disease phenotype remains largely elusive. In this study, the function of this hotspot mutation was profoundly investigated. For this, V727M mutation-specific altered expression profile and potential networks were explored. The relevant muscular disorder-specific in vivo studies and patient data were further analyzed, and the key altered molecular pathways were identified. Our study found that the GNEV727M mutation resulted in a deregulated lincRNA profile, the majority of which (91%) were associated with a down-regulation trend. Further, in silico analysis of associated targets showed their active role in regulating Wnt, TGF-ß, and apoptotic signaling. Interestingly, COL6a3 was found as a key target of these lincRNAs. Further, GSEA analysis showed HIBM patients with variable COL6A3 transcript levels have significant alteration in many critical pathways, including epithelial-mesenchymal-transition, myogenesis, and apoptotic signaling. Interestingly, 12 of the COL6A3 coexpressed genes also showed a similar altered expression profile in HIBM. A similar altered trend in COL6A3 and coexpressed genes were found in in vivo HIBM disease models as well as in multiple other skeletal disorders. Thus, the COL6A3-specific 13 gene signature seems to be altered in multiple muscular disorders. Such deregulation could play a pivotal role in regulating many critical processes such as extracellular matrix organization, cell adhesion, and skeletal muscle development. Thus, investigating this novel COL6A3-specific 13 gene signature provides valuable information for understanding the molecular cause of HIBM and may also pave the way for better diagnosis and effective therapeutic strategies for many muscular disorders.

Follicular Helper T-Cell-derived Nodal Lymphomas: Study of Histomorphologic, Immunophenotypic, Clinical, and RHOA G17V Mutational Profile.

The study was designed to review the demographic, clinical, and pathologic characteristics of follicular helper T cells (TFH)-derived nodal PTCL in India including angioimmunoblastic T-cell lymphoma (AITL), peripheral T-cell lymphoma (PTCL) with follicular helper T cell phenotype (P-TFH), and follicular T-cell lymphoma with additional immunohistochemistry (IHC) and RHOAG17V mutational analysis, as well as their impact on survival. This retrospective study included 88 cases of PTCL that were reclassified using IHC for TFH markers (PD1, ICOS, BCL6, and CD10) and dendritic-meshwork markers (CD21, CD23). Cases of TFH cell origin were evaluated for RHOAG17V mutation using Sanger sequencing and amplification-refractory mutation system-polymerase chain reaction (PCR) (validated using cloning and quantitative PCR) with detailed clinicopathologic correlation. Extensive re-evaluation with added IHC panel resulted in a total of 19 cases being reclassified, and the final subtypes were AITL (37 cases, 42%), PTCL-not otherwise specified (44, 50%), P-TFH (6, 7%), and follicular T-cell lymphoma (1, 1%). The presence of at least 2 TFH markers (>20% immunopositivity) determined the TFH origin. AITL patients tended to be male and showed increased presence of B-symptoms and hepatosplenomegaly. Histomorphology revealed that 92% of AITL cases had pattern 3 involvement. Sanger sequencing with conventional PCR did not yield any mutation, while RHOAG17V was detected by amplification-refractory mutation system-PCR in AITL (51%, P =0.027) and P-TFH (17%), which was validated with cloning followed by sequencing. Cases of RHOAG17V-mutant AITL had a worse Eastern Cooperative Oncology Group performance status initially but fared better in terms of overall outcome ( P =0.029). Although not specific for AITL, RHOAG17V mutation shows an association with diagnosis and requires sensitive methods for detection due to low-tumor burden. The mutant status of AITL could have prognostic implications and translational relevance.

NFκB (RelA) mediates transactivation of hnRNPD in oral cancer cells.

Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5' flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (-1358/-1347) motif and 99% reduction after the deletion of second motif (-1052/-1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen's Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.

Evaluation of Heterogeneous Nuclear Ribonucleoprotein D Expression as a Diagnostic Marker for Oral Squamous Cell Carcinoma.

The heterogeneous nuclear ribonucleoprotein D (hnRNPD) serves as a prognostic marker for oral squamous cell carcinoma (OSCC). We evaluated the diagnostic potential of hnRNPD to differentiate between OSCC and normal mucosa. Immunohistochemistry for hnRNPD and a routinely used diagnostic marker deltaNp63 (p40) was performed in 32 normal mucosae and 46 OSCC specimens. Subsequently, receiver-operating characteristic analysis was performed to evaluate the diagnostic potential of hnRNPD in comparison to that of p40. Immunostaining for p40 and hnRNPD was observed in 39 (84.78%) and 38 (82.60%) cases, respectively, in OSCC specimens. The poorly differentiated squamous cell carcinoma displayed 100% (eight cases) immunoreactivity for hnRNPD as compared to 87.5% (seven cases) for p40. Nuclear staining of p40 and hnRNPD was observed in all OSCC specimens. p40 staining was restricted to basal cells, whereas both basal and para-basal cells displayed hnRNPD staining in OSCC specimens. Areas under the curve for p40 and hnRNPD were 0.86 and 0.87, respectively. p40 and hnRNPD showed equal sensitivities (80.95%). However, hnRNPD displayed marginally higher (88.23%) specificity for tumor cells as compared to that of p40 (85.29%). Conclusion: In addition to being a well-established prognostic marker, hnRNPD can serve as a diagnostic marker for OSCC.

Lymphoma subtypes in India: a tertiary care center review.

Lymphomas are a group of neoplasm arising from immune cells with varied clinical presentation, molecular profile, morphology and immunophenotype. The epidemiology and response to treatment varies among patients from different geographical locations. We analyze the demographic characteristics of lymphomas in a tertiary care center of India over a period of five years. This was a retrospective study including cases from 2015 to 2019 which were classified according to WHO classification 2017. A total of 4115 lymphoma cases were diagnosed. Hodgkin lymphomas (HL) comprised 30.35% (n = 1249), and non-Hodgkin lymphoma (NHL) was 69.65% (n = 2866). Site of presentation was nodal in 64.76% cases, and 35.23% were extranodal. There was an overall male predominance. Among the NHLs, B-cell type comprised of 84.08% and 15.38% was T- and NK cell lymphomas. Mature B cell lymphomas comprised 82.41% with predominant being diffuse large B cell lymphoma type (42.53%) followed by follicular lymphoma (10.81%) and small lymphocytic lymphoma (6.10%). Among the T-cell type, PTCL NOS (2.65%) was the predominant subtype followed by ALK positive anaplastic large cell lymphoma (ALCL-ALK+) (2.44%), extranodal NK-T cell lymphoma (2.02%) and others. Classical type was predominant type (97.91%) among HL, and 2.08% were nodular lymphocyte predominant type. Among the classical HL, nodular sclerosis (28.1%) and mixed cellularity (32.18%) co-dominated. Our study indicates that the Indian population differs in the prevalence, presentation and the subtyping among various lymphomas. Higher prevalence of Hodgkin lymphoma, DLBCL, ALK + ALCL and immature cell neoplasm was noted.