Shubhada Chiplunkar

IL17 producing γδT cells induce angiogenesis and are associated with poor survival in gallbladder cancer patients

Despite conventional treatment modalities, gallbladder cancer (GBC) remains a highly lethal malignancy. Prognostic biomarkers and effective adjuvant immunotherapy for GBC are not available. In the recent past, immunotherapeutic approaches targeting tumor associated inflammation have gained importance but the mediators of inflammatory circuit remain unexplored in GBC patients. In the current prospective study, we investigated the role of IL17 producing TCRγδ+ (Tγδ17), CD4+ (Th17), CD8+ (Tc17) and regulatory T cells (Tregs) in pathogenesis of GBC. Analysis by multi‐color flow cytometry revealed that compared to healthy individuals (HI), Tγδ17, Th17 and Tc17 cells were increased in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TIL) of GBC patients. Tregs were decreased in PBMCs but increased in TILs of GBC patients. The suppressive potential of Tregs from GBC patients and HI were comparable. Serum cytokines profile of GBC patients showed elevated levels of cytokines (IL6, IL23 and IL1β) required for polarization and/or stabilization of IL17 producing cells. We demonstrated that Tγδ17 cells migrate toward tumor bed using CXCL9‐CXCR3 axis. IL17 secreted by Tγδ17 induced productions of vascular endothelial growth factor and other angiogenesis related factors in GBC cells. Tγδ17 cells promote vasculogenesis as studied by chick chorioallantoic membrane assay. Survival analysis showed that Tγδ17, Th17 and Treg cells in peripheral blood were associated with poor survival of GBC patients. Our findings suggest that Tγδ17 is a protumorigenic subtype of γδT cells which induces angiogenesis. Tγδ17 may be considered as a predictive biomarker in GBC thus opening avenues for targeted therapies.

Common genetic variation and risk of gallbladder cancer in India: a case-control genome-wide association study

BACKGROUNDnGallbladder cancer is highly lethal, with notable differences in incidence by geography and ethnic background. The aim of this study was to identify common genetic susceptibility alleles for gallbladder cancer.nnnMETHODSnIn this case-control genome-wide association study (GWAS), we did a genome-wide scan of gallbladder cancer cases and hospital visitor controls, both of Indian descent, followed by imputation across the genome. Cases were patients aged 20-80 years with microscopically confirmed primary gallbladder cancer diagnosed or treated at Tata Memorial Hospital, Mumbai, India, and enrolled in the study between Sept 12, 2010, and June 8, 2015. We only included patients who had been diagnosed less than 1 year before the date of enrolment and excluded patients with any other malignancies. We recruited visitor controls aged 20-80 years with no history of cancer visiting all departments or units of Tata Memorial Hospital during the same time period and frequency matched them to cases on the basis of age, sex, and current region of residence. We estimated association using logistic regression, adjusting for age, sex, and five eigenvectors. We recruited samples for a replication cohort from patients visiting Tata Memorial Hospital between Aug 4, 2015, and May 17, 2016, and patients visiting the Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India, between July, 2010, and May, 2015. We used the same inclusion and exclusion criteria for the replication set. We examined three of the most significant single-nucleotide polymorphisms (SNPs) in the replication cohort and did a meta-analysis of the GWAS discovery and replication sets to get combined estimates of association.nnnFINDINGSnThe discovery cohort comprised 1042 gallbladder cancer cases and 1709 controls and the replication cohort contained 428 gallbladder cancer cases and 420 controls. We observed genome-wide significant associations for several markers in the chromosomal region 7q21.12 harbouring both the ABCB1 and ABCB4 genes, with the most notable SNPs after replication and meta-analysis being rs1558375 (GWAS p=3·8u2008×u200810-9; replication p=0·01; combined p=2·3u2008×u200810-10); rs17209837 (GWAS p=2·0u2008×u200810-8; replication p=0·02; combined p=2·3u2008×u200810-9), and rs4148808 (GWAS p=2·4u2008×u200810-8; replication p=0·008; combined p=2·7u2008×u200810-9). Combined estimates of per-allele trend odds ratios were 1·47 (95% CI 1·30-1·66; p=2·31u2008×u200810-10) for rs1558375, 1·61 (1·38-1·89; p=2·26u2008×u200810-9) for rs17209837, and 1·57 (1·35-1·82; p=2·71u2008×u200810-9) for rs4148808. GWAS heritability analysis suggested that common variants are associated with substantial variation in risk of gallbladder cancer (sibling relative risk 3·15 [95% CI 1·80-5·49]).nnnINTERPRETATIONnTo our knowledge, this study is the first report of common genetic variation conferring gallbladder cancer risk at genome-wide significance. This finding, along with in-silico and biological evidence indicating the potential functional significance of ABCB1 and ABCB4, underlines the likely importance of these hepatobiliary phospholipid transporter genes in the pathology of gallbladder cancer.nnnFUNDINGnThe Tata Memorial Centre and Department of Biotechnology.

Mechanisms involved in the down-regulation of TCR ζ chain in tumor versus peripheral blood of oral cancer patients

Immune dysfunction is the hallmark of patients with oral cancer. Down‐regulation of T cell receptor (TCR) ζ chain expression was observed in T cells from patients with oral squamous cell carcinoma. In peripheral blood, the decrease in TCR ζ chain showed an inverse correlation with the tumor stage as demonstrated by western blotting, confocal microscopy and flow cytometry. The mechanism of TCR ζ chain degradation in the peripheral blood involves ubiquitination and subsequent targeting of TCR ζ for degradation in the lysosome. Decreased expression of PKC θ and the subsequent decrease of TCR ζ chain transcription factor Elf‐1 and its binding to DNA may contribute to the decreased/or absent TCR ζ chain transcripts in the tumor infiltrating lymphocytes. Oral cancer patients exhibiting TCR ζ chain defect also showed impaired lymphocyte proliferation, cytokine profile and intracellular calcium release upon stimulation with anti CD3 mAb. Our data shows that posttranslational degradation is primarily responsible for decreased TCR ζ chain expression in the peripheral blood, while a transcriptional defect is observed in the tumor compartment. The down‐regulation of TCR ζ chain culminates into impaired lymphocyte responses in these patients.

IL-10 production in non-small cell lung carcinoma patients is regulated by ERK, P38 and COX-2.

Immune dysfunction is hallmark of patients with non–small cell lung carcinoma (NSCLC). The molecular mechanism involved in COX‐2– and PGE2‐mediated production of immunosuppressive cytokine IL‐10 is not well‐understood. Our study addresses the involvement of T cell downstream signalling intermediates, cytokines (IL‐10 and IFN‐γ) and their transcription factors (T‐bet and GATA‐3) in COX‐2–mediated regulation of lymphocyte functions in NSCLC patients. In comparison to healthy individual, a marked decrease in lymphocyte proliferation to anti‐CD3 MAb was observed in NSCLC patients by thymidine incorporation assay. Using flow cytometry, decrease in intracellular calcium release with increase in reactive oxygen species was observed in lymphocytes of NSCLC patients. These patients showed increased IL‐10 and PGE2 with reduced IFN‐γ production by ELISA. Results demonstrated defect in regulation of transcription factors T‐bet and GATA‐3 as analysed by Western blotting (WB), immunoprecipitation and EMSA. Overexpression of p‐p38, p‐ERK and COX‐2 were observed with diminished p‐JNK by WB. IL‐10/IFN‐γ levels were found to be differentially regulated via p38 and ERK mitogen‐activated protein kinase (MAPK) pathways in cooperation with COX‐2. Inhibition of these pathways using selective inhibitors lead to increased lymphocyte proliferative response to anti‐CD3 MAb and IFN‐γ production with decrease in IL‐10 production. Studies showed involvement of ERK, p38 and COX‐2 pathways in high IL‐10 production, driven by lung tumour derived PGE2. The selective COX‐2 inhibitor rofecoxib showed ability to alter the cytokine balance by affecting regulation of T‐bet and GATA‐3 transcription factors.

Role of nitric oxide in heat shock protein induced apoptosis of γδT cells

Activation induced cell death (AICD) has been proposed to serve as a mechanism to limit T lymphocyte proliferation induced by antigenic stimulation. Heat shock proteins (hsp60 and hsp70) expressed on oral tumor cells serve as ligands for peripheral blood γδT lymphocytes. Tumor cell lysis by γδT lymphocytes is mediated via recognition of hsp expressed on tumor cells. In the present study, we report that upon stimulation with hsp, γδT lymphocytes isolated from oral cancer patients undergo AICD as confirmed by DNA ploidy, annexin V staining and confocal microscopy. In cocultures of γδT lymphocytes and tumor cells, addition of antihsp60 and antihsp70 MAb, but not anti‐Fas MAb (ZB4), inhibited DNA fragmentation of γδT lymphocytes. Flow cytometric analysis revealed a down regulation of Fas expression on γδT lymphocytes upon incubation with hsp60 and hsp70. Increased expression of iNOS was observed in hsp‐stimulated γδT lymphocytes. Addition of monomethyl L‐arginine monoacetate, competitive inhibitor of NOS, inhibited nitric oxide (NO) production and apoptosis of γδT lymphocytes induced by hsp60 and hsp70. The NO‐induced apoptosis of γδT lymphocytes involves activation of caspase‐9 and loss of mitochondrial membrane potential. The present study explains a novel strategy adopted by tumor cells to evade immune recognition by γδT lymphocytes.

T-cell Receptor γ and δ Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in Indian Patients

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal lymphoid malignancy and junctional sequences of rearranged T-cell receptor (TCR) represent the best suitable marker to study clonality in these patients. A sensitive, non-radioactive, and rapid approach of PCR coupled with heteroduplex analysis was used to analyse clonality of TCR γ and 8 gene rearrangements in 26 Indian T-ALL patients. Amongst TCR γ gene family, Vγ1-Jγ1.3/2.3 sequences were most utilized (53.9%) while from TCRδ repertoire Vδ1-Jδ1 sequences were preferentially rearranged (23.1%) in these patients. 19.2% of Indian T-ALL patients demonstrated both clonal TCR γ and δ gene rearrangements along with surface expression of TCRγδ. Although the majority of T-ALL patients showed surface expression of TCRα β, the small fraction (19.2%) of TCRγδ+ T-ALL represent a distinct subgroup which needs further evaluation.

Extracellular 2′5′-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer

Decreased expression of CD3‐ζ chain, an adaptor protein associated with T‐cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3‐ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour‐derived factors promote immune suppression by down‐regulating CD3‐ζ chain expression. 2′5′‐Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3‐ζ chain down‐regulation and OAS2 stimulation. The surrogate situation was established by over‐expressing OAS2 in a HEK293 cell line and cell‐free supernatant was collected. These supernatants when incubated with T cells resulted in down‐regulation of CD3‐ζ chain, which shows that the secreted OAS2 is capable of regulating CD3‐ζ chain expression. Incubation of T cells with cell‐free supernatants of oral tumours or recombinant human OAS2 (rh‐OAS2) induced caspase‐3 activation, which resulted in CD3‐ζ chain down‐regulation. Caspase‐3 inhibition/down‐regulation using pharmacological inhibitor or small interfering RNA restored down‐regulated CD3‐ζ chain expression in T cells induced by cell‐free tumour supernatant or rh‐OAS2. Collectively these results show that OAS2 leads to impairment in CD3‐ζ chain expression, so offering an explanation that might be applicable to the CD3‐ζ chain deficiency observed in cancer and diverse disease conditions.

Next generation metronomic chemotherapy—report from the Fifth Biennial International Metronomic and Anti-angiogenic Therapy Meeting, 6–8 May 2016, Mumbai

The 5th Biennial Metronomic and Anti-angiogenic Therapy Meeting was held on 6th – 8th May in the Indian city of Mumbai. The meeting brought together a wide range of clinicians and researchers interested in metronomic chemotherapy, anti-angiogenics, drug repurposing and combinations thereof. Clinical experiences, including many from India, were reported and discussed in three symposia covering breast cancer, head and neck cancers and paediatrics. On the pre-clinical side research into putative mechanisms of action, and the interactions between low dose metronomic chemotherapy and angiogenesis and immune responses, were discussed in a number of presentations. Drug repurposing was discussed both in terms of clinical results, particularly with respect to angiosarcoma and high-risk neuroblastoma, and in pre-clinical settings, particularly the potential for peri-operative interventions. However, it was clear that there remain a number of key areas of challenge, particularly in terms of definitions, perceptions in the wider oncological community, mechanisms of action and predictive biomarkers. While the potential for metronomics and drug repurposing in low and middle income countries remains a key theme, it is clear that there is also considerable potential for clinically relevant improvements in patient outcomes even in high income economies.

Clonal T-cell Receptor γ and δ Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia at Diagnosis: Predictor of Prognosis and Response to Chemotherapy

Risk-based treatment assignment requires the availability of prognostic factors that reliably predict clinical outcome. Junctional regions of T-cell receptor (TCR) genes provide the best tool to study clonality, lineage association and minimal residual disease (MRD) in T-ALL. In this study, we have analyzed the suitability of clonal TCR γ and δ junctional gene rearrangement status of T-ALL patients at diagnosis as a prognostic marker for T-ALL. We studied peripheral blood samples of 50 newly diagnosed patients with T-ALL in India for incidence of clonal TCR γ and δ junctional region gene rearrangements by PCR-coupled heteroduplex analysis. Of these, 17 T-ALL patients uniformly treated on MCP 841 protocol were followed for more than 40 months (range: 41.26u200a–u200a55.82 months; mean: 49.26) and their clonal TCRγδ genotype was correlated with clinical outcome with respect to duration of complete remission, disease-free survival (DFS) and event-free survival. We also compared the clinical and biological features of TCRγδu200a+ T-ALL and TCRαβu200a+ T-ALL for their relative order of significance. Thirty per cent (15 of 50) of Indian T-ALL patients exhibited clonal rearrangements of both TCR γ and δ genes. A significant proportion of these patients (73.3%, 11 of 15 Pu200a<u200a0.005) showed predominant usage of VγIu200a–u200aJγ1.3/2.3 with Vδ1u200a–u200aJδ1 genes. A statistically significant association of L2 and L1 FAB blast morphology with TCRγδu200a+ T-ALL and TCRαβu200a+ T-ALL, respectively was observed (Pu200a=u200a0.001 by Fishers Exact Test). The survival rate in DFS group was higher for TCRγδu200a+ T-ALL compared to TCRαβu200a+ T-ALL (Pu200a=u200a0.1378 by Log rank test). Thus we have identified clonal TCR γ and δ junctional gene rearrangement status of T-ALL patients at diagnosis as a prognostic marker and predictor of response to chemotherapy. In future, this may help in designing tailored and risk-adjusted (less aggressive and less toxic) therapies for subset of T-ALL patients.

Place of birth and risk of gallbladder cancer in India

CONTEXTnWithin India, the incidence of gallbladder cancer (GBC) is characterized by marked geographical variation; however, the reasons for these differences are unclear.nnnAIMSnTo evaluate the role of place of birth, length of residence, and effect of migration from high- to low-risk region on GBC development.nnnSETTINGS AND DESIGNnPopulation-based cancer registries (PBCRs); case-control study.nnnSUBJECTS AND METHODSnData of PBCRs were used to demonstrate geographical variation in GBC incidence rates. A case-control study data examined the role of birth place, residence length, and effect of migration in etiology of GBC.nnnSTATISTICAL ANALYSISnRate ratios for different PBCRs were estimated using Chennai Cancer Registry as the reference population. Odds ratios (ORs) for developing GBC in a high-risk region compared to a low-risk region and associated 95% confidence interval (CI) were estimated through unconditional logistic regression models using case-control study.nnnRESULTSnGBC shows marked variation in incidence with risk highest in Northeast regions and lowest in South India. OR of 4.82 (95% CI: 3.87-5.99) was observed for developing GBC for individuals born in a high-risk region compared to those born in a low-risk region after adjusting for confounders. A dose-response relationship with increased risk with increased length of residence in a high-risk region was observed (OR lifetime 5.58 [95% CI: 4.42-7.05]; Ptrend ≤ 0.001). The risk persisted even if study participant migrated from high- to low-risk region (OR = 1.36; 95% CI: 1.02-1.82).nnnCONCLUSIONSnThe present study signifies the importance of place of birth, length of stay, and effect of migration from high- to low-risk region in the development of GBC. The data indicate role of environmental and genetic factors in etiology of disease.