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.

Epigenetic downregulation of desmin in gall bladder cancer reveals its potential role in disease progression.

Background & objectives: Gall bladder cancer (GBC) is a fatal neoplasm, with a globally variable incidence rates. To improve the survival rate of patients, a newer set of biomarkers needs to be discovered for its early detection and better prognosis. Our earlier studies on GBC proteomics and whole-genome methylome data revealed expression of desmin to be significantly downregulated with correlated promoter hypermethylation during gall bladder carcinogenesis. Thus, to evaluate desmin as a potential biomarker for GBC, we carried out a detailed follow up study. Methods: Methylation-specific polymerase chain reaction (MS-PCR) (n=17, GBC and n=23, non-tumour control), real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) [n=14, GBC and n=14, adjacent non-tumour (ANT)], immunohistochemistry (n=27, GBC and n=14, non-tumour) and immunoblotting (n=13, GBC and n=13, ANT) were performed in surgically removed gall bladder tissue samples. Results: MS-PCR analysis showed methylation of desmin in 88.23 per cent (15/17) gall bladder tumour samples as compared to non-tumour tissues (39.13%, 9/23). Real-time qRT-PCR analysis revealed a significant downregulation of desmin expression in GBC as compared to ANT tissue. This was further confirmed by western blot, showing reduced expression of desmin protein in GBC, as compared to non-tumour tissue. Immunohistochemical analysis also showed a decreased level of desmin i.e., more than 95 per cent (26/27) in tumour cells compared to non-tumours (35.71%, 5/14). Interpretation & conclusions: The increased frequency of desmin promoter methylation which could be responsible for its significant downregulation, indicates its potential as a candidate biomarker for GBC. This requires further validation in a large group of patients to evaluate its clinical utility.

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.

Fine needle aspiration cytology of lesions of liver and gallbladder: An analysis of 400 consecutive aspirations.

BACKGROUND: Patients presenting with mass lesions of liver and gallbladder are a common occurrence in a cancer hospital in north central part of India. Fine-needle aspiration cytology (FNAC) serves as first line of pathological investigations, but there are pros and cons involved. AIM: The main objective of the present study was to establish adequacy of the procedure and to find out diagnostic pitfalls. An attempt was made to analyze inconclusive and inadequate aspirations. MATERIALS AND METHODS: A total of 400 consecutive fine-needle aspirates of liver, belonging to 328 cases over a period of 2 years, were analyzed. Hematoxylin and eosin and May-Grόnwald-Giemsa stains were used. Chi-square test was carried out to compare significant degree of difference in different kind of diagnosis. RESULTS: Out of 400 aspirations, 289 (72.2%) were adequate, 75 (18.7%), inconclusive and 36 (9%), inadequate. Among positive aspirations the most common was metastatic adenocarcinoma, 128 (44.2%). The positive diagnosis and adequate aspirations were significantly high (P < 0.0001). Major differential diagnostic problems were: Distinguishing the poorly differentiated hepatocellular carcinoma from the metastatic adenocarcinoma; and leukemia/lymphoma from other malignant round cell tumors. Common diagnostic pitfalls were repeated aspirations from the necrotic area and aspiration of atypical, disorganized and reactive hepatocytes, adjacent to a metastasis. No complications were observed. CONCLUSION: FNAC can be used successfully for the diagnosis of liver and gallbladder lesions, thus avoiding open biopsy. Study indicates the potential of using FNAC in clinical intervention where the incidence of gall-bladder and liver cancer is very high and open biopsy and surgery are not an option.

Identification of prosaposin and transgelin as potential biomarkers for gallbladder cancer using quantitative proteomics.

Gallbladder cancer is an uncommon but lethal malignancy with particularly high incidence in Chile, India, Japan and China. There is a paucity of unbiased large-scale studies investigating molecular basis of gallbladder cancer. To systematically identify differentially regulated proteins in gallbladder cancer, iTRAQ-based quantitative proteomics of gallbladder cancer was carried out using Fourier transform high resolution mass spectrometry. Of the 2575 proteins identified, proteins upregulated in gallbladder cancer included several lysosomal proteins such as prosaposin, cathepsin Z and cathepsin H. Downregulated proteins included serine protease HTRA1 and transgelin, which have been reported to be downregulated in several other cancers. Novel biomarker candidates including prosaposin and transgelin were validated to be upregulated and downregulated, respectively, in gallbladder cancer using tissue microarrays. Our study provides the first large scale proteomic characterization of gallbladder cancer which will serve as a resource for future discovery of biomarkers for gallbladder cancer.