Clinical epidemiology of gallbladder cancer in North-Central India and association of immunological markers, NLR, MLR and PLR in the diagnostic/prognostic prediction of GBC.

INTRODUCTION: The incidence of Gallbladder Cancer (GBC) is found to be increasing in the rural populations of north-central India. Role of multiple demographic factors, including poor socio-economic conditions, illiteracy and miserable primary healthcare services appear to be significant factors for this increase. Here, we aim to assess the present status of GBC in north-central India and evaluate the role of immunological markers in its management. METHODS: A total of 1845 cases of different Gallbladder diseases, including GBC, from rural and urban areas both, registered at CHRI, Gwalior during 2009-2014 and 2018 were included in this study. The demographic and clinical information of the patients were analysed using various statistical tests. RESULTS: Of all the cases (1845) included in this study, 1125 (60.97%) were diagnosed with GBC, of which, 707 (62.84%) were from rural background and 418 (37.15%) from urban settings. Mean age for GBC cases for both male and female was about 53.49 years. Females were more affected, being 70.37%, while male patients were only 29.63%. The pathological investigations showed elevated levels of total bilirubin and liver function enzymes both. The NLR, PLR and MLR were found to be significantly associated with different clinical parameters as well as OS. CONCLUSION: We infer that the growing trend of GBC, particularly in rural areas, in north-central India is primarily associated with the lack of awareness, inadequate medical support and poor socio-economic conditions. Evaluation of haematological markers may help in the predictive diagnosis/ prognosis and or management of GBC cases in the studied population.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

Antibacterial and catalytic activity of biogenic gold nanoparticles synthesised by Trichoderma harzianum.

This study reveals the antibacterial and catalytic activity of biogenic gold nanoparicles (AuNPs) synthesised by biomass of Trichoderma harzianum. The antibacterial activity of AuNPs was analysed by the means of growth curve, well diffusion and colony forming unit (CFU) count methods. The minimum inhibitory concentration of AuNPs was 20 µg/ml. AuNPs at 60 µg/ml show effective antibacterial activity as optical absorption was insignificant. The well diffusion and CFU methods were also applied to analyse the effect of various concentration of AuNPs. Further, the catalytic activity of AuNPs was analysed against methylene blue (MB) as a model pollutant in water. MB was degraded 39% in 30 min in the presence of AuNPs and sodium borohydrate and the rate constant (k) was found to be 0.2 × 10-3 s-1. This shows that the biogenic AuNP is an effective candidate for antibacterial and catalytic degradation of toxic pollutants.

Telomere length variation and expression analysis of shelterin complex genes during gallbladder carcinogenesis.

BACKGROUND: Telomeres, which are bound with shelterin protein complex, play an important role in maintaining genomic stability and its dysfunction may lead to carcinogenesis. Here, we aimed to analyze whether shelterin complex gene expression and telomere length variation, play any role in gallbladder carcinogenesis. METHODS: Telomere length analysis was carried out by monochrome multiplex qPCR, whereas expression analysis of shelterin genes was carried out using RT-qPCR. Statistical analysis was carried out using SigmaPlot 11 software. RESULTS: We found significantly reduced telomere length in tumor tissues, and this reduction was seen in both, tumors with or without gallstones in comparison to adjacent non tumor and gallstone (chronic calculous cholecystitis: Inflamed) tissues. Inflamed tissues showed increased telomere length as compared to both adjacent non tumor and tumor tissues. Expression analysis of five shelterin genes showed significant downregulation of TERF1, POT1, and TINF2 genes in inflamed tissues as compared to non tumor and tumor tissues. POT1 was also found to be significantly upregulated in tumor tissues and specifically in tumor tissues with gallstones compared to inflamed tissues. CONCLUSION: This study, thus, suggests that, gallstone does not affect telomere length and even after having increased telomere length, decreased expression of some shelterin genes in inflamed tissue might cause telomeres to cap improperly, possibly leading to telomere dysfunction and further, gallbladder carcinogenesis. Also, increased expression of POT1 in tumor tissues with gallstones could act as a diagnostic marker in patients with gallstones.

Epigenetic Downregulation of PTEN in Gallbladder Cancer.

PURPOSE: Gallbladder cancer (GBC) is becoming a global health problem. Phosphatase and tensin homolog (PTEN), also known as guardian gene of cancer, encodes a protein, which dephosphorylates phosphatidylinositol (3,4,5) triphosphate {PtdIns (3,4,5) P3 } into phosphatidylinositol (4,5) diphosphate {PtdIns (4,5) P2} that results in the inhibition of AKT/PKB signaling pathway. PTEN plays a key role in the pathogenesis of various cancers. However, its role in GBC is still obscure. The present study was aimed to identify the epigenetic role of PTEN in GBC and GSD. METHODS: PTEN promoter methylation was studied by methylation-specific PCR in 50 GBC and 30 GSD tissues. Transcript level expression of PTEN was analyzed by reverse transcriptase PCR and quantitative PCR in 20 GBC and 20 GSD tissue samples. Immunohistochemistry was performed on tissue microarrays of 136 GBC samples to correlate the methylation pattern with the pattern of in situ expression of PTEN protein. Student's t test was performed to test the significant level of difference between case and control samples. Values showing p ≤ 0.05 were considered significant. RESULTS: MS PCR showed PTEN promoter methylation in 30% (15/50) GBC (p = 0.0486) and 22.86% (8/30) GSD (p = 0.0530) cases. RT-PCR and qPCR revealed downregulation of PTEN in advanced GBC cases (p < 0.0001), but, not in GSD (p = 0.901). Immunohistochemistry of GBC tissue microarray scored 1+ in 20.29% (p = 0.0028) and zero or negative in 50% (p < 0.0001) GBC cores. CONCLUSIONS: Thus, PTEN may be considered as a useful biomarker for the management of GBC, however, needs larger sample size to validate it further.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

BACKGROUND & OBJECTIVES: Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD. METHODS: Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues. RESULTS: Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC ( P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression. INTERPRETATION & CONCLUSIONS: The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.

Global methylation profiling to identify epigenetic signature of gallbladder cancer and gallstone disease.

Promoter methylation in various tumor suppressor genes is reported to influence gallbladder carcinogenesis. Here, we aimed to identify methylation status in gallbladder cancer (GBC) by performing a comprehensive genome-wide DNA methylation profiling. The methylation status of 485,577 CpG sites were investigated using Illumina's Infinium Human Methylation 450 BeadChip array in 24 tissues (eight each of tumor, adjacent non-tumor, and gallstone). About 33,443 differentially methylated sites (DMRs) were obtained in the whole human genome, of which 24,188 (72 %) were hypermethylated and 9255 (28 %) were hypomethylated. The data also revealed that majority of the DMRs are localized on the proximal promoter region [Transcription start sites (TSS200, TSS1500) and 5' untranslated region (5'UTR)] and first exon. Exclusion of first exon detected a total of 10,123 (79 %) hypermethylated and 2703 (21 %) hypomethylated sites. Comparative analysis of the later with our differential proteomics data resulted in identification of 7 hypermethylated or down-regulated (e.g., FBN1, LPP, and SOD3) and 61 hypomethylated or up-regulated markers (e.g., HBE1, SNRPF, TPD52) for GBC. These genes could be further validated on the basis of their methylation/expression status in order to identify their utility to be used as biomarker/s for early diagnosis and management of GBC.

Dysfunction of subtelomeric methylation and telomere length in gallstone disease and gallbladder cancer patients of North Central India.

BACKGROUND: Telomeres play an important role in cancer progression. Recently it has been shown that subtelomeric methylation negatively regulates telomere length in various diseases, including cancers. Here, we evaluated the influence of subtelomeric methylation in telomere dysfunction in gallbladder cancer (GBC), and whether this dysfunction is affected by the presence of gallstones. METHODS: Relative telomere length and subtelomeric methylation levels were assessed using monochrome multiplex quantitative polymerase chain reaction and bisulfite sequencing, respectively, in different gallbladder tissue types including different grades of GBC, gallstones and adjacent non-tumor. RESULTS: We found telomere length to shorten significantly in overall GBC, but specifically in early grade cancer. We also found D4Z4 and DNF92 subtelomeric sequences to be hypermethylated and hypomethylated, respectively, in GBC; however, their methylation levels differed significantly, only in early grade cancer. We could not find any specific correlation between subtelomeric methylation and telomere length in GBC. Interestingly, telomere length and subtelomeric methylation differed significantly in GBC without gallstones but not in GBC with gallstones. CONCLUSIONS: This study, thus, suggests that telomere dysfunction and changes in methylation levels may occur earlier in the progression of GBC, while the presence of gallstones may have no influence on telomere length as well as on methylation levels.

Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes.

BACKGROUND: As on today, the global mortality rate of gallbladder cancer is still very high. Both genetic and epigenetic alterations play pivotal roles in the development of cancer. We selected seven tumour associated genes, implicated in other cancers, to assess their methylation status in gallbladder cancer and gallstone diseases. AIM OF STUDY: To study the promoter methylation of certain tumour associated genes in the molecular pathogenesis of gallbladder cancer and gall stone diseases. MATERIALS AND METHODS: Methylation specific PCR for seven tumour associated genes, viz., MASPIN, 14-3-3 sigma gene, THBS1, FLNC, HLTF, COX-2 and SOCS1, was performed in 50 gallbladder cancer (GBC), 30 gall stone diseases (GSD) and their respective adjacent control tissues. Semi-quantitative PCR and immunohistochemistry was carried out to check the expression level. Student's t-test was carried out to compare the differences in the methylation and expression patterns between cases and control tissues. RESULTS: We observed methylation of CpG islands in seven of the studied markers, but, the frequency of methylation was found varying among different samples. Of them, 14-33 sigma showed methylation in 45 GBC (90%; p=0.0001) and 25 GSD (86.66%; p=0.001), MASPIN in 35 GBC (70%; p=0.0008) and 18 GSD (51.43%; p=0.040), FLNC in 16 GBC (32%; p=0.0044) and 9 GSD (25.71%; p=ns), THBS1 in 26 GBC (52%; p=0.0009) and 10 GSD (28.57%; p=0.0505), HLTF in 8 GBC (16%; p=ns) and 2 GSD (5.71%; p=ns), COX2 in 10 GBC (20%; p=ns) and 6 GSD (17.14%; p=ns) and SOCS-1 in 3 GBC samples only (6%; p=ns), but not in GSD. Semi-quantitative PCR revealed down regulation in MASPIN, 14-3-3 sigma, THBS1, HLTF, COX2 and SOCS1 in advanced gallbladder cases. Immunohistochemistry further confirmed the down-regulation of SOCS1 in GBC. CONCLUSION: The present study infers that accumulation of epigenetic alterations increases poor prognosis of GBC patients. Out of seven genes, MASPIN and THBS1 play key epigenetic role in GBC, but not in GSD. The reason for downregulation of SOCS1 only in GBC, and unaltered expression of 14-3-3 sigma protein in all the GBC and GSD tissue samples is not clear. Further investigation on the expression pattern of these genes in GBC cell lines may elucidate their likely functional role in in association with gallbladder cancer.

Quantitative Assessment of Expression of Lactate Dehydrogenase and its Isoforms 3 and 4 may Serve as Useful Indicators of Progression of Gallbladder Cancer: A Pilot Study.

We have studied the expression of lactate dehydrogenase and its isoforms in gall bladder cancer, cholelithiasis and chronic cholecystitis. Quantitative and qualitative assays of lactate dehydrogenase and its various isoforms were carried out in the blood sera of patients and healthy controls along with parallel estimation of various liver function test enzymes. Statistical analysis was done using the software Graph Pad Prism. Significantly high expression of lactate dehydrogenase along with alkaline phosphatase and total bilirubin (P ≤ 0.05) was observed in all the three clinical conditions as compared to controls. LDH showed an increasing trend from stage I to stage IV of GBC indicating a significant positive association with the disease progression. The levels of LDH 3 and 4 isoforms appeared significantly more elevated in GBC than cholelithiasis or chronic cholecystitis. We suggest that a careful estimation of total LDH and its isoforms 3 and 4 alone or along with alkaline phosphatase and total bilirubin during different clinical stages, like chronic cholecystitis, cholelithiasis and GBC, may prove to be a potentially useful biomarker in the prognostic management of gall bladder diseases, specifically GBC.