TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication.

OBJECTIVE: We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. DESIGN: We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. RESULTS: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. CONCLUSIONS: Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.

Primary hepatolithiasis, recurrent pyogenic cholangitis, and oriental cholangiohepatitis: a tale of 3 countries.

Primary hepatolithiasis (HL), recurrent pyogenic cholangitis, and oriental cholangiohepatitis are terms commonly used in Japan, Hong Kong, and Korea respectively, and describing the different aspects of the same disease, with "HL" indicating the pathologic changes, "recurrent pyogenic cholangitis" emphasizing the clinical presentation and suppurative inflammation, and "oriental cholangiohepatitis" highlighting its ethnic preference and mysterious nature. HL is predominantly a disease of the far east and shows great regional differences in the incidence and the type of intrahepatic stones. Pathologically, it is characterized by pigmented calcium bilirubinate stones within dilated intrahepatic bile ducts featuring chronic inflammation, mural fibrosis, and proliferation of peribiliary glands, without extrahepatic biliary obstruction. Episodes of suppurative inflammation cumulate in sclerosing cholangitis in peripheral ducts and parenchymal fibrosis from scarring and collapse. Mass-forming inflammatory pseudotumor and neoplasms-like intraductal papillary neoplasms and cholangiocarcinoma are increasingly recognized complications. Bacterial infection and dietary factors are believed to be important in the formation of pigment stones within intrahepatic bile ducts, whereas parasitic infestation is likely coincidental. With improvement of environmental conditions and westernization of diet, the incidence of pigment stones has decreased. At the same time, cholesterol stones with milder clinical manifestations and pathologic changes are increasingly recognized, and for which stone dissolution therapy can be considered. Understanding the underlying pathology avoids confusion with other diseases more prevalent in the western world, and allows correct selection of the appropriate treatment.

Germline, somatic and epigenetic events underlying mismatch repair deficiency in colorectal and HNPCC-related cancers.

High-frequency microsatellite instability (MSI-H) results from deficiency in nucleotide mismatch repair. It contributes significantly to carcinogenesis in the human colorectal mucosa. Here we study 41 colorectal and three other HNPCC-related cancers with MSI-H to provide comprehensive information on the mechanisms of inactivation of the two major proteins involved, hMLH1 and hMSH2. Seventeen of the patients had family histories meeting the criteria for Bethesda grades 1, 2 or 3. Of these familial cases, 14 (83%) had early-onset disease, defined on the basis of diagnosis prior to the age of 50, but in three the disease was of late onset (>50 years). A second subset of 20 patients had early onset disease without family history. The remaining seven patients were selected to allow comparisons with sporadic, late-onset disease, the molecular basis of which has been extensively reported elsewhere. We stratified the tumours initially on the basis of hMLH1 or hMSH2 protein deficiency, detected by immunohistochemistry, and then by analysis of germline and somatic mutation, mRNA transcription, loss of heterozygosity (LOH) at the hMLH1 and hMSH2 loci, and methylation status in two regions of the hMLH1 promoter. The functional significance of several of these changes in the MSI-H tumours was confirmed by comparisons with 16 tumours with low-frequency microsatellite instability and 56 tumours with stable microsatellites. As anticipated, patients with family histories usually showed germline mutation of hMSH2 or hMLH1. In many cases the residual normal allele was silenced in their tumours by loss of heterozygosity (LOH). The small subset of late-onset, sporadic cases confirmed the preponderance in this group of biallelic hMLH1 promoter methylation. In the early-onset, apparently sporadic subset there were 11 tumours with hMLH1 deficiency, five with hMSH2 deficiency and four with no detectable abnormality in expression of either protein. These showed a complex mixture of lesions, including germline and somatic mutations, promoter methylation, LOH, suppression of wild-type RNA by as yet undiscovered mechanisms, or no detectable abnormality in any of these parameters. Evidence is presented to indicate that methylation in proximal region of the hMLH1 promoter is a more reliable correlate of transcriptional silencing in colorectal cancers than methylation in upstream region. These observations have significant implications for management of patients with MSI-H tumours.

Elucidating the prognostic significance of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese patients with metastatic colorectal cancer.

AIM: The prognostic significance of KRAS, NRAS, PIK3CA and BRAF mutations was evaluated in Chinese patients with metastatic colorectal cancer (CRC). METHOD: Tumor samples from 183 patients were retrospectively tested for KRAS, NRAS, PIK3CA and BRAF mutations. Multivariate analysis was performed to determine the relationship between mutational status, drug response and survival. RESULT: Over 70% of patients received two or more lines of chemotherapy, 50% had cetuximab and 18% had bevacizumab. The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations was 45, 3.2, 5 and 20%, respectively. For the entire cohort, the median overall survival was 24 months (95% confidence interval [CI] = 20.4-26.4 months). Of the genes tested, only KRAS mutation was an independent prognostic factor with a multivariate hazard ratio of 1.5 (95% CI = 1.05-2.16, P = 0.03). In the subgroup of patients who received cetuximab-based therapy in the first-line setting, KRAS mutation was associated with a lack of response to chemotherapy (28% vs 66%, chi-square, P = 0.01). Patients with KRAS mutant tumors (or KRAS wild-type tumors that harbored BRAF and/or PIK3CA mutations) tended to have lower response rates to chemotherapy and/or cetuximab (P = not significant). The number of NRAS mutant cases was too small to allow any statistical analysis. CONCLUSION: The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations in this cohort is consistent with reports from non-Asian populations, and KRAS mutation has both prognostic and predictive significance in Chinese patients with metastatic CRC.

Acinar cell cystadenoma of the pancreas: a new entity?

This report describes a newly observed cystic lesion of the pancreas showing acinar cell differentiation. The patients affected by this lesion included seven women and three men (age range 16-66 years). In six patients, all of whom were female and all but one of whom suffered from abdominal pain, the cystic lesions (diameters, 4-15 cm) were detected by imaging techniques and subsequently removed. In four patients the cystic lesions were incidental findings. Eight lesions occurred as unifocal, unilocular or multilocular cysts in the head (n = 6) or tail (n = 2) of the pancreas. One lesion was bifocal (head and tail) and another involved the entire pancreas. The cysts were only rarely connected with the pancreatic duct system, but with acinar structures. Their lining cells expressed pancreatic enzymes and lacked any cellular atypia or proliferative activity (Ki67 index <1%). For a follow-up period of 6-84 months all patients remained alive and well. Although a nonneoplastic nature cannot be fully excluded, we propose that this lesion, composed of well-differentiated acinar cells, may represent the benign counterpart of the well-recognized acinar cystadenocarcinoma. We therefore suggest the term acinar cell cystadenoma.

Inflammatory pseudotumor-like follicular dendritic cell tumor of liver and spleen: granulomatous and eosinophil-rich variants mimicking inflammatory or infective lesions.

Inflammatory pseudotumor-like follicular dendritic cell (FDC) tumor is an uncommon, Epstein-Barr virus-associated neoplasm of the liver or spleen, characterized by spindly tumor cells dispersed in a background of small lymphocytes and plasma cells. We report 6 diagnostically challenging cases in which the neoplastic component is further overshadowed by granulomas or eosinophils. The patients included 2 men and 4 women with a median age of 45.5 years, and 1 of them showed concurrent involvement of the liver and spleen. The presence of extensive coalescent epithelioid granulomas in 3 splenic tumors and 3 liver tumors raised the possibilities of an infective process or sarcoidosis. In another liver tumor, the massive infiltrate of eosinophils, accompanied by geographic eosinophilic abscesses, suggested parasitic infestation or so-called eosinophilic granuloma of the liver. However, scrutiny of the tissue between the granulomas or among the eosinophils revealed scattered atypical spindly cells with indistinct cell borders, large vesicular nuclei, and distinct nucleoli. The atypical cells were positive for FDC markers on immunostaining (CD21, CD35) and Epstein-Barr virus on in situ hybridization. Thus, a diagnosis of inflammatory pseudotumor-like FDC tumor could be confirmed. Awareness of the granulomatous and eosinophil-rich variants of this tumor type will facilitate the correct diagnosis to be made.

MSH2 c.1452-1455delAATG is a founder mutation and an important cause of hereditary nonpolyposis colorectal cancer in the southern Chinese population.

Hereditary nonpolyposis colorectal cancer (HNPCC) accounts for approximately 2% of all colorectal cancer (CRC) cases and is the most common hereditary CRC syndrome. We have previously reported a high incidence of microsatellite instability (MSI) and germline mismatch repair (MMR) gene mutations in young Hong Kong Chinese with CRC. Ongoing studies at the Hereditary Gastrointestinal Cancer Registry in Hong Kong have revealed a unique germline MSH2 c.1452-1455delAATG mutation that has not been reported in other ethnic groups. Detailed analysis showed that this specific MSH2 mutation constituted 21% of all germline MMR gene mutations and 36% of all MSH2 germline mutations identified. We designed a specific PCR-based diagnostic test on paraffin-embedded tissues and identified this germline mutation in 2 (1.5%) of 138 consecutive patients with early-onset CRC (<46 years of age at diagnosis). Haplotype analysis was performed using 11 microsatellite markers located between D2S391 and D2S123. All 10 families had the same disease haplotype, suggesting a founder effect. These 10 families all originated from the Chinese province of Guangdong, which historically included Hong Kong. It is the most populous of the Chinese provinces, with a population of >93 million. Further analysis suggested that this founder mutation may date back to between 22 and 103 generations ago. The identification of this MSH2 founder mutation has important implications for the design of mutation-detection strategies for the southern Chinese population. Since there were major emigrations from Hong Kong and Guangdong province during the 19th and 20th centuries, this finding is also significant for Chinese communities worldwide.