Boris H. Ruebner

MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer

Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker α-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.

Developmental Context Determines Latency of MYC-Induced Tumorigenesis

One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYCs ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.

Immunohistochemical staining of cancer stem cell markers in Hepatocellular Carcinoma

BACKGROUND Cancer stem cells (CSCs) are thought to be a critical subpopulation in tumor development, progression, metastasis and recurrence, and the identification of these cells is an initial step in understanding their role in oncogenesis and in seeking valuable markers for diagnosis or development of targeting therapeutics. AIMS To identify CSCs in hepatocellular carcinoma (HCC) specimens and define their tissue specificity. METHODS Immunohistochemical staining of CSC markers: CD44, CD90, CD133 and aldehyde dehydrogenase (ALDH) was performed in 25 HCC specimens, 4 hepatoblastomas, 8 peri-malignant tissues, and 19 cases of viral hepatitis. RESULTS The positivity of CD44 staining in HCC specimens was significantly lower than in viral hepatitis specimens. The positive rate of CD133 in HCC was similar to viral hepatitis specimens. CD133(+) cells were largely localized to ALDH-positive cells in HCC as revealed by confocal microscopy. In contrast, the co-expression of both markers was visualized within vessels or in the portal areas in viral hepatitis. Moreover, among 7 liver specimens adjacent to HCC tissue, 3-6 samples were positive for CD44, CD90, CD133 and ALDH, especially in dysplastic cells. One of 4 hepatoblastoma cases was positive for all these markers; whereas, the other three specimens were negative for all these CSC markers. CONCLUSIONS In HCC and dysplastic tissues, clusters of CD133(+)/ALDH(high) cells were identified. The use of cancer stem cell markers to screen tissues with chronic liver diseases provides limited guidance in the identification of malignant cells.

Abnormal expression of PDC‐E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody‐negative primary biliary cirrhosis

The presence of antimitochondrial antibodies (AMA) is a major criterion for the diagnosis of primary biliary cirrhosis (PBC). Although it is not clear that AMA are involved in the pathogenesis of the disease, the study of these autoantibodies has enabled much information to be accumulated about the specificity of this response. The autoantigens have been identified as components of a functionally related enzyme family, the 2‐oxo‐acid‐dehydrogenase complex. Within this complex, pyruvate dehydrogenase E2 subunit (PDC‐E2) has been determined to be the immunodominant autoantigen. Using a panel of mouse monoclonal antibodies and human combinatorial autoantibodies, it has been demonstrated that patients with PBC, but not controls, have an abnormal expression of either PDC‐E2 or a cross‐reacting molecule in the apical region of biliary epithelium. Others have shown a similar reaction using rabbit sera directed to PDC‐E2. Our previous studies have concentrated on AMA‐positive patients. In this study, the presence of PDC‐E2, class II, immunoglobulin (Ig) A, and B7/BB1 in the bile duct epithelial cells of AMA‐positive as well as AMA‐negative patients is addressed. Most patients with AMA‐negative PBC (seven of nine) react in a fashion similar to AMA‐positive patients with intense staining of the apical region of the bile duct epithelial cells for “PDC‐E2,” increased IgA expression, and little major histocompatibility complex (MHC) class II staining in the early‐stage patients. Interestingly, the two AMA‐negative patients that did not express PDC‐E2 on the apical side of their biliary epithelium had anticentromere antibodies and Sjögrens syndrome. Based on these data, it can be concluded that the disease process for both AMA‐positive and AMA‐negative patients with PBC has a similar pathogenic mechanism, which is likely to involve the abnormal expression of PDC‐E2 or a molecular mimic of PDC‐E2, and does not require MHC class II expression. (Hepatology 1995; 22:1440–1446).

Role of bacterial infection in Diet-Induced acute pancreatitis in mice

SummaryThis study was performed to elucidate the role of bacterial infection in acute pancreatitis in young female mice fed a choline-deficient diet supplemented with 0.5% DL-ethionine (CDE diet). Mice were randomly classified into two groups: one had been fed CDE diet alone (nonantibiotic group), the other was fed a CDE diet with oral administration of antibiotics (antibiotic group). Survival rates at 96 and 144 h after introduction of the CDE diet were significantly improved in the antibiotic group, 25.0 and 19.4%, respectively, as compared with 3.6 and 0% in the nonantibiotic group (p, 0.05). Aerobic and anaerobic bacterial cultures of blood, ascites, spleen, and pancreas were taken from living mice 72 h after introduction of the CDE diet. Positive bacterial growth from one or more of the specimens occurred in 29.4% of the nonantibiotic group, and in 10.0% of the antibiotic group. Mice with pancreatic necrosis had a higher positive culture rate, 62.5% in the nonantibiotic group vs 20.0% in the antibiotic group. These results suggest that reduction of intestinal flora in mice inhibits secondary infection caused by bacterial transloca-tion and improves survival in diet-induced hemorrhagic pancreatitis. We believe the development of bacterial infection of gut origin may be a factor influencing mortality in severe pancreatitis.

Adenosquamous carcinoma arising in a mucinous cystadenoma of the pancreas

Approximately 500 cystic neoplasms of the pancreas have been reported, and among these the mucinous pancreatic cystadenomas are known to have malignant potential. We report a rare case of a mucinous cystadenoma containing adenosquamous carcinoma.

Microsomal and cytosolic epoxide hydrolases in rhesus monkey liver, and in normal and neoplastic human liver

The cytosolic epoxide hydrolase (EH-LC) was observed in rhesus monkey liver cytosol, and in both normal and neoplastic human liver. Microsomal epoxide hydrolase (EH-LM) was detected not only in the microsomes of normal and neoplastic human liver and normal rhesus monkey liver, but also in the cytosol of these tissues. No apparent differences were observed between the EH-LM in liver cytosol and that in microsomes. No major differences were observed between the levels of EH-LM in the cytosol of normal and that in neoplastic human liver.

Role of fine needle aspiration cytology and endoscopic biopsy in the preoperative assessment of pancreatic and peripancreatic malignancies

SummarySixty-seven of 207 patients with pancreatic and peripancreatic malignancies underwent preoperative fine needle aspiration cytology (FNA), and 24 patients underwent intraluminal endoscopic biopsies. All patients had confirmation of the diagnosis of malignancy either at operation, autopsy, or by clinical follow-up. FNA of liver metastases was positive for malignancy in 12 of 12 patients. FNA of the pancreas was performed on 44 patients with pancreatic adenocarcinoma and 11 patients with other pancreatic or peripancreatic malignancies. The diagnosis of cancer was established by FNA in 32 of 44 (72.4%) patients with pancreatic adenocarcinoma and 1 of 11 patients (9.1%) with other pancreatic or peripancreatic malignancies. In the patients with pancreatic adenocarcinoma, 17 of 18 patients (94.4%) who had no operative intervention, 12 of 18 (66.7%) patients who had palliative bypass procedures, and 3 of 8 (37.5%) patients resected had positive FNA. Eighteen of 24 patients (75%) who underwent intraluminal endoscopic biopsies, and 11 of 15 (73.3%) with ampullary carcinoma were positive. We believe that FNA is of limited value in the diagnosis of small resectable tumors of the pancreas as it identified cancer in only 3 of 8 patients in whom it was employed. False negative FNA may delay the diagnosis and treatment of pancreatic malignancies. Patients in whom there is a high index of suspicion of pancreatic or peripancreatic malignancy based on clinical presentation, CT scan, or ERCP assessment do not require preoperative, histologic proof of malignancy prior to pancreaticoduodenectomy. Patients deemed to be unresectable or candidates for bypass on the basis of the size of the tumors or suspected liver metastases should be considered for FNA cytology, which is mainly successful under these circumstances.

Immunological similarities between primary sclerosing cholangitis and chronic sclerosing sialadenitis: report of the overlapping of these two autoimmune diseases.

Primary sclerosing cholangitis (PSC) is characterized by destructive inflammation and fibrosis affecting the bile ducts. The etiology of PSC is still unknown, although lymphocytic infiltration in the portal areas suggests an immune-mediated destruction of the bile ducts. Patients with one autoimmune disease often suffer from one or more other autoimmune diseases. It is well known that there is a close relationship between PSC and inflammatory bowel disease, particularly ulcerative colitis(UC). However, the pathological findings in UC and other overlap diseases do not resemble those of PSC. In the present study, we report a patient with chronic sclerosing sialadenitis (Kuttners tumor) and PSC. It is compared the sclerosing changes in both salivary glands and bile ducts histologically. In addition, the expression pattern of mast cell tryptase, b-FGF, and HLA-DR were examined in both tissues immunohistochemically. Histological features of sclerosing change in both salivary and bile ducts were quite similar. Marked mast cell infiltration and b-FGF expression were seen in the sclerosing areas in both tissues. In active inflammatory areas of the salivary glands, HLA-DR expression was also seen. We hypothesized that similar immune reactions occur in both the salivary gland and bile ducts and are responsible for the fibrosis that follows.

Sclerosing mesenteritis seen clinically as pancreatic pseudotumor: two cases and a review.

Sclerosing mesenteritis is an uncommon nonneoplastic inflammatory process in the mesentery that is seen as a pseudotumor, usually involving the small bowel mesentery, the mesenteric fat, and less commonly, the mesentery of the large bowel. We report two cases of sclerosing mesenteritis and review the literature on this rare disease. Both patients had pain, profound weight loss, and a mass on computed tomography (CT) scan of the abdomen. The provisional diagnosis was pancreatic neoplasm on the basis of clinical presentation and imaging studies. The diagnosis of sclerosing mesenteritis was established by histologic findings in biopsy material obtained at laparotomy in both cases. Interval histologic studies in one patient who had a high CA 19-9 level, progressive biliary ductal and partial duodenal compression, revealed a transitional histologic pattern from predominant inflammation and fat necrosis to predominant fibrosis. This may explain the varied descriptive terms used in the literature to describe this entity.