Prognostic significance of BRCA1 and BRCA2 methylation status in circulating cell-free DNA of Pancreatic Cancer patients.

Introduction: Pancreatic cancer is the most fatal cancer type in the world. Its high mortality is mostly correlated to the absence of symptoms and the difficulty in early diagnosis, which in the majority of the cases occurs when the disease has already spread metastasis. Nowadays, tests that could predict early diagnosis are not available yet and the number of prognostic tests is limited. Hence, there is an urgent need for biomarkers capable of detecting early development or the rapid progression of the disease. Patients and Methods: DNA methylation represents the most frequent epigenetic event among tumor suppressor genes that are involved in various carcinogenic pathways. In the recent study we have tried to evaluate, for the first time, the prognostic value of BRCA1 and BRCA2 methylation in the cell-free DNA of pancreatic cancer patients. Using methylation-specific real-time PCR we examined the methylation status of BRCA1 and BRCA2 in 55 patients with operable and 50 patients with metastatic pancreatic cancer. In the operable disease setting, BRCA1 was found to be methylated in 33/55 (63.5%) patients examined while BRCA2 was also highly methylated in 31/55 (56.3%). In the metastatic disease, BRCA1 was found to be methylated in 26/50 (52%) while BRCA2 was found methylated in 23/50 (46%). Results: All control samples were negative for BRCA1 orBRCA2 promoter methylation. Patients with operable pancreatic cancer and a methylated BRCA1 and BRCA2 promoter status had a statistically significant poorer outcome as compared with patients with a non-methylated one (p=0.012 and p=0.001, respectively). Conclusion: In this study plasma methylation of BRCA1 and BRCA2 represents a frequent event in both the operable as well as in the metastatic setting. BRCA1 and BRCA2 methylation was significant and correlated with decreased survival in patients with operable pancreatic cancer. A larger cohort of patients is required to further explore the potential of these findings as well as to investigate whether BRCA1/2 methylation in plasma could serve as a potential prognostic biomarker in pancreatic cancer.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.

Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

Expression of Fas (CD95/APO-1) and Fas ligand (FasL) in experimentally-induced acute pancreatitis.

INTRODUCTION: Acinar cell death is a crucial event in acute pancreatitis (AP) and may occur either by apoptosis or necrosis. The aim of this study was to investigate the expression of the apoptosis associated proteins Fas and FasL in experimentally induced severe AP. METHODS: AP was induced in 30 rats by injecting 0.2 ml of 4.5% sodium taurocholate solution into the biliopancreatic duct. Sham operated animals (n = 30) and 10 normal controls were used for comparisons. Animals were killed at 6, 12, 24, 48, 72 hr and 1 week after operation (five animals at each time point) and both serum and pancreatic tissue were obtained. The severity of AP was graded by morphological evaluation and by measuring serum amylase levels. Acinar cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Tissue expressions of Fas and FasL were evaluated by immunohistochemistry. RESULTS: Sodium taurocholate injection resulted in severe acute necrotizing pancreatitis as early as six hr after taurocholate infusion with gradually increasing severity and a peak at 72 hr, and a significant increase of serum amylase at 6 and 12 hr. Apoptotic acinar cells were observed between 48 and 72 hr. The expression of both Fas and FasL in pancreatic tissue was induced in comparison with normal controls. Fas expression in AP was higher and statistically significant at 24 hr whereas FasL expression was consistently lower with a statistical significance observed at 12 hr when compared to sham-operated animals suggesting Fas upregulation and FasL downregulation in this model of AP. CONCLUSIONS: Induction and sequential changes in the expressions of Fas and FasL occur during taurocholate induced severe AP in rats and their temporal modulation might associate with acinar cell death by apoptosis.

Primary squamous cell carcinoma of the ampulla of Vater.

CONTEXT: Squamous carcinoma of the ampulla of Vater is a very rare tumor with only three cases been reported so far. CASE REPORT: Here, we report the case of a 68-year-old man who presented with painless obstructive jaundice, general fatigue, loss of appetite and weight loss. Laboratory tests revealed hypochromic anemia. Total and direct bilirubin, alkaline phosphatase, liver enzymes, carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA) were all elevated. Abdominal ultrasonography and computed tomography showed a distended gallbladder, dilatation of the intra- and extra-hepatic bile ducts and enlargement of the pancreatic head. Endoscopic retrograde cholangiopancreatography revealed a bulging papilla with infiltrative growth at the ampulla of Vater but endoscopic biopsies were inconclusive. The patient was treated with classical Whipple's pancreaticoduodenectomy. Histopathological examination showed a moderately differentiated squamous cell carcinoma. Multiple serial sectioning of the tumor specimen failed to detect an adenomatous component. Regional lymph nodes and resection margins were free of tumor and the disease was classified as stage IIA (T3N0M0) according to the TNM system. Adjuvant treatment was not given. Despite curative resection, multiple liver metastases developed after four months and the patient succumbed to progressive hepatic failure 5 months after the operation. CONCLUSION: Primary pure squamous cell carcinoma of the ampulla of Vater is a very rare histological type of carcinoma. Clinical characteristics and optimal treatment are obscure. Primary surgical treatment with curative intent should be performed although this type of carcinoma associates with dismal prognosis.

Spontaneous colo-umbilical fistula complicating diverticulitis of the sigmoid colon.

Colocutaneous fistula caused by diverticulitis is relatively uncommon with colo-umbilical fistulas being even rarer. We herein report a rare case of a spontaneous colo-umbilical fistula due to diverticulitis of the sigmoid colon. The fistula developed from a diverticulum of the sigmoid colon that discharged through the umbilicus after two episodes of acute diverticulitis. The condition was successfully treated by resectional surgery.

Common bile duct obstruction secondary to a periampullary diverticulum.

Periampullary duodenal diverticula are not uncommon and are usually asymptomatic although complications may occasionally occur. Here, we report the case of a 72-year-old woman who presented with painless obstructive jaundice. Laboratory tests showed abnormally elevated serum concentrations of total and direct bilirubin, of alkaline phosphatase, of γ-glutamyl transpeptidase, and of aspartate and alanine aminotransferases. Serum concentrations of the tumor markers carbohydrate antigen 19-9 and carcinoembryonic antigen were normal. Abdominal ultrasonography showed dilatation of the common bile duct (CBD), but no gallstones were found either in the gallbladder or in the CBD. The gallbladder wall was normal. Computed tomography failed to detect the cause of CBD obstruction. Magnetic resonance imaging and magnetic resonance cholangiopancreatography revealed a periampullary diverticulum measuring 2 cm in diameter and compressing the CBD. The pancreatic duct was normal. Hypotonic duodenography demonstrated a periampullary diverticulum with a filling defect corresponding to the papilla. CBD compression by the diverticulum was considered as the cause of jaundice. The patient was successfully treated by surgical excision of the diverticulum. In conclusion, the presence of a periampullary diverticulum should be considered in elderly patients presenting with obstructive jaundice in the absence of CBD gallstones or of a tumor mass. Non-interventional imaging studies should be preferred for diagnosis of this condition, and surgical or endoscopic interventions should be used judiciously for the effective and safe treatment of these patients.

Synchronous breast and rectal cancers in a man.

Breast cancer in men is relatively rare and its coexistence with other primary non-breast cancers exceptional. Here, we report the case of a 50-year-old man who presented with symptoms of rectal adenocarcinoma and in whom a synchronous, asymptomatic cancer of the left breast was found incidentally at physical examination.

Synchronous carcinoma of the ampulla of vater and colon cancer.

Carcinoma of the papilla of Vater is a relatively rare tumor and its coexistence with other primary sporadic cancers is very exceptional. Here we report the case of a 76-year-old man who presented with painless obstructive jaundice, pathologically elevated liver function tests and increased serum levels of carbohydrate antigen 19-9 and carcinoembryonic antigen. Endoscopic retrograde cholangiography revealed a large polypoid mass in the ampulla of Vater. A large tumor in the ascending colon was also incidentally detected by abdominal computed tomography. Endoscopic biopsies from both lesions showed adenocarcinomas. Metastases to the liver and to the hepatoduodenal ligament and hepatic artery lymph nodes were found during surgery. Right colectomy and a biliary bypass were performed. Histological analysis showed an ampullary adenocarcinoma with metastases to regional lymph nodes and the liver and a colonic adenocarcinoma with local invasion into the pericolic fat. Treatment with gemcitabine plus cisplatin was suggested postoperatively. The association of sporadic ampullary and colonic adenocarcinomas and the mutually increased risk of developing either a synchronous or a metachronous tumor following each other should be considered in patients with primary ampullary or colorectal cancer during the preoperative evaluation and postoperative follow-up of these patients.

Ischemic colitis of the left colon in a diabetic patient.

Diabetes mellitus may affect the gastrointestinal tract possibly as a result of autonomic neuropathy. Here we present a 68-year-old male with non-insulin-dependent diabetes mellitus who presented with prolonged watery diarrhea and in whom imaging studies demonstrated ischemic colitis of the left colon. Resection of the affected colon resulted in sustained disappearance of symptoms.

Cutaneous metastasis at a surgical drain site after gastric cancer resection.

Cutaneous metastasis from intra-abdominal malignant solid tumours such as gastric adenocarcinoma is very rare. Here, we report the case of a 76-year-old male patient with a T4N2M0, poorly differentiated, signet-ring cell gastric carcinoma, who underwent potentially curative resection of the tumour and developed cutaneous metastasis at the site of the surgical drain 4 months after the operation while he was on chemotherapy. The lesion involved the skin and the subcutaneous fat only. A CT scan revealed local recurrence at the resection bed but no distant metastases. The patient died 1 month later. It is concluded that the development of cutaneous metastasis after gastric carcinoma resection indicates tumour recurrence or disseminated disease and is associated with poor prognosis.

Serum vascular endothelial growth factor levels in pancreatic cancer patients correlate with advanced and metastatic disease and poor prognosis.

The serum concentrations of vascular endothelial growth factor (VEGF) were measured by an enzyme linked immunosorbent assay in 51 healthy controls and in 58 patients with pancreatic cancer before and 30 days after surgery. Pancreatic cancer patients had significantly higher serum VEGF levels compared with healthy controls with a significant association between serum VEGF levels, disease stage and the presence of both lymph node and distant metastases. Serum levels of VEGF decreased significantly after radical resection of the tumor. Elevated preoperative serum VEGF level was a significant prognostic factor, although not independent of stage, for patient survival. These findings suggest that serum VEGF concentrations may reflect pancreatic cancer progression and that their determination may be clinically useful.