HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-Related HCC with Distinct Features.

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely, the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate, and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

Cell-Free Virus-Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer.

BACKGROUND AND AIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. APPROACH AND RESULTS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CONCLUSIONS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection.

Alteration of colonic epithelial cell differentiation in mice deficient for glucosaminyl N-deacetylase/N-sulfotransferase 4.

Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) are the first enzymes that mediate the initiation of heparan sulfate sulfation. We previously identified NDST4 as a putative tumor suppressor in human colorectal cancer. In the study, we generated an Ndst4 knockout (Ndst4-/-) mouse strain and explored its phenotypic characteristics, particularly in the development of colonic epithelial homeostasis. The Ndst4-deficient mice were viable and fertile, and their life spans were similar to those of wild-type littermates. No gross behavioral or morphological differences were observed between the Ndst4-/- and wild-type mice, and no significant changes were determined in the hematological or serum biochemical parameters of the Ndst4-/- mice. Ndst4 RNA transcripts were expressed in the brain, lung, gastrointestinal tract, pancreas, and ovary. However, Ndst4-null mice exhibited no gross or histological abnormalities in the studied organs, except for the colon. Although no alterations were observed in the crypt length or number of proliferating cells, the Ndst4-/- mice exhibited an increased number of goblet cells and a decreased number of colonocytes in the proximal colon compared with the wild-type mice. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic epithelium. Taken together, we established, for the first time, an Ndst4-/- mouse strain and revealed the involvement of Ndst4 in the development and homeostasis of colonic epithelium. Accordingly, NDST4 in human colon might direct the biosynthesis of specific heparan sulfate proteoglycans that are essential for the maintenance of colonic epithelial homeostasis. Thus, the loss of its function may result in the tumorigenesis and progression of colorectal cancer.

DNA Hypermethylation of SHISA3 in Colorectal Cancer: An Independent Predictor of Poor Prognosis.

BACKGROUND: Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown. METHODS: SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing. RESULTS: Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients. CONCLUSIONS: SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.

Protocadherin 10 suppresses tumorigenesis and metastasis in colorectal cancer and its genetic loss predicts adverse prognosis.

Protocadherin 10 (PCDH10), a novel tumor suppressor gene in human cancers, is located in a common deleted region at chromosome 4q28 in colorectal cancer (CRC). This study aimed to ascertain the genetic loss of PCDH10 and its clinical relevance in CRC and to explore the tumor suppressor function of PCDH10. The genetic deletion of PCDH10 was determined in 171 pairs of primary tumors and corresponding normal mucosae by loss of heterozygosity study. In total, 53 carcinomas were positive for allelic loss of PCDH10. The genetic aberration was significantly associated with tumor progression and distant metastasis (p = 0.021 and p = 0.018, respectively) and was an independent predictor of poor survival for CRC patients (p = 0.005). Expression of PCDH10 gene was silenced or markedly down-regulated in all of 12 CRC cell lines tested and in 41 of 53 colorectal carcinomas compared with their matched normal mucosae. Ectopic expression of PCDH10 suppressed cancer cell proliferation, anchorage-independent growth, migration and invasion in vitro. Subcutaneous injection of PCDH10-expressing CRC cells into SCID mice revealed the reduction of tumor growth compared with that observed in mock-inoculated mice. Furthermore, through intrasplenic implantation, the re-expression of PCDH10 in silenced cells restrained liver metastasis and improved survival in SCID mice. In conclusion, PCDH10 is a pivotal tumor suppressor in CRC, and the loss of its function promotes not only tumor progression but also liver metastasis. In addition, the genetic deletion of PCDH10 represents an adverse prognostic marker for the survival of patients with CRC.

NDST4 is a novel candidate tumor suppressor gene at chromosome 4q26 and its genetic loss predicts adverse prognosis in colorectal cancer.

BACKGROUND: Genomic deletion at tumor suppressor loci is a common genetic aberration in human cancers. The study aimed to explore candidate tumor suppressor genes at chromosome 4q25-q28.2 and to delineate novel prognostic biomarkers associated with colorectal cancer (CRC). METHODS: Deletion mapping of chromosome 4q25-q28.2 was conducted in 114 sporadic CRC by loss of heterozygosity study with 11 microsatellite markers. A novel candidate tumor suppressor gene, namely NDST4, was identified at 4q26. Gene expression of NDST4 was investigated in 52 pairs of primary CRC tissues by quantitative reverse transcription-polymerase chain reaction. Allelic loss of NDST4 gene was further determined in 174 colorectal carcinomas by loss of heterozygosity analysis, and then was assessed for clinical relevance. RESULTS: One minimal deletion region was delineated between D4S2297 and D4S2303 loci at 4q26, where NDST4 was the only gene that had markedly been downregulated in CRC tumors. By laser capture microdissection, NDST4 RNA expression was demonstrated in colonic epithelial cells, but was undetectable in tumor cells. In total, 30 (57.7%) of 52 colorectal carcinomas showed a dramatic reduction in NDST4 gene expression compared with matched normal mucosae. The genetic loss of NDST4 was significantly associated with advanced pathological stage (P = 0.039) and poorer overall survival of patients (P = 0.036). CONCLUSIONS: NDST4 gene is a novel candidate tumor suppressor gene in human cancer, and the loss of its function might be involved in CRC progression. In addition, the loss of heterozygosity assay, which was established to determine the allelic loss of NDST4 gene, could be a cost-effective tool for providing a useful biomarker of adverse prognosis in CRC.

Mapping of genetic deletions on chromosome 3 in colorectal cancer: loss of 3p25-pter is associated with distant metastasis and poor survival.

PURPOSE: There is no detailed analysis of loss of heterozygosity (LOH) on chromosome 3 in colorectal cancer (CRC). Our aim was to define frequently deleted loci on chromosome 3 and to explore novel prognostic markers and the locations of candidate tumor suppressor genes associated with CRC. METHODS: LOH at 23 microsatellite markers spanning on chromosome 3 was determined in 112 sporadic CRC by automated fluorescence-based polymerase chain reaction. Genetic loss was assessed for the clinicopathological significance by univariate and multivariate analyses. RESULTS: Fifty-eight (51.8%) of 112 carcinomas exhibited LOH at one or more loci tested. Among seven loci with high LOH rates, allelic losses at D3S1297 and D3S1266 occurred more frequently in younger patients. A marked gender distortion for genetic deletion was observed at six loci, where LOH was identified more frequently in male cases. For clinical outcome, LOH solely at D3S1297 (3p26.3) was significantly associated with distant metastasis (P = 0.001) and was indicative of a shorter overall survival (P = 0.014). In addition, loss of one common deletion region at 3p25-pter was significantly correlated to distant metastasis (P = 0.009) and had an adverse effect on patients' overall survival in univariate and multivariate tests (P = 0.009 and 0.001, respectively). CONCLUSIONS: Loss of chromosome 3p25-pter could act as an independent predicator of poor prognosis in CRC, suggesting that microsatellite analysis is a useful means to stratify patients into different risk groups. In addition, inactivation of candidate tumor suppressor genes in this region might involve in CRC progression.