Hidenori Kamiyama

Promoter hypermethylation of tumor-related genes in peritoneal lavage and the prognosis of patients with colorectal cancer

The predictive value of free cancer cells in the peritoneal fluid of patients with colorectal cancer (CRC) remain to be elucidated. The aim of this study was to determine the prognostic relevance of the methylation of tumor‐related genes detected in the peritoneal lavage fluid (PLF) of patients undergoing a resection for CRC.

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability

BackgroundRecent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.MethodsWith an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.ResultsUnsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3′ end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).ConclusionsSomatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Pancreas head carcinoma with total fat replacement of the dorsal exocrine pancreas

We report a case of pancreas head carcinoma associated with fat replacement of the body and tail. A 68-year-old man presented with obstructive jaundice and was admitted to our hospital. Ultrasonography and computed tomography showed pancreas head tumor with a neighboring cystic lesion and fatty replacement of parenchyma of the pancreas body and tail. By endoscopic retrograde pancreatography, abruption of the main pancreatic duct and the presence of an accessory duct were detected. After percutaneous transhepatic biliary drainage, pancreatoduodenectomy was successfully performed. At laparotomy, the pancreas head was easily dissected from the replaced fatty tissue of the body and tail without continuity of the ductal system or parenchyma. Microscopic examination revealed the existence of an infiltrating ductal adenocarcinoma and a neighboring. cyst in the pancreas head. The dorsal exocrine pancreas was completely replaced by the fat tissues, in which viable Langerhans’ islets were scattered. The patient’s postoperative course was uneventful, and exogenous insulin administration was unnecessary for the maintenance of normal blood sugar level. Acquired fat replacement of the body and tail of the pancreas is an uncommon disorder, mimicking congenital agenesis of the dorsal pancreas. Though the mechanism is controversial, obstruction of the main pancreatic duct by a cystic lesion or carcinoma in the pancreas head is a possible cause of fatty degeneration of the pancreatic parenchyma.

Risk factors for intra-abdominal infection after pancreaticoduodenectomy - a retrospective analysis to evaluate the significance of preoperative biliary drainage and postoperative pancreatic fistula.

BACKGROUND/AIMS Intra-abdominal infection (IAI) after pancreaticoduodenectomy (PD) is a common cause of prolongation of postoperative hospital stay and readmission to the hospital following discharge. METHODOLOGY Two hundred and six patients undergoing PD were reviewed to investigate the risk factors for IAI after PD. Patients were separated into two groups: those who developed IAI after PD (Group A; n=44), and those who had not developed IAI after PD (Group B; n=162), the two groups were then compared to identify the risk factors for IAI after PD. A hundred and six patients (51.5%) underwent preoperative biliary drainage (PBD). RESULTS Multivariate analysis revealed that pancreatic fistula (PF) was an independent risk factor for IAI after PD (p<0.001; odds ratio=9.58; 95% confidence interval=4.37-21.0), but PBD was not a significant risk factor. CONCLUSIONS We demonstrated that the adequate PBD might not affect IAI after PD. On the other hand, PF was an independent risk factor for IAI after PD. A large randomized controlled trial, which would prove the effect of early removal of a prophylactic placed drain to prevent IAI, should be planned.

The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals

Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers.

Molecular biomarkers for the detection of metastatic colorectal cancer cells

Approximately half of all patients with colorectal cancer develop local recurrence or distant metastasis during the course of their illness. Recently, the molecular detection of metastatic cancer cells in various types of clinical samples, such as lymph nodes, bone marrow, peripheral blood, and peritoneal lavage fluid, has been investigated as a potential prognostic marker. The prognostic value of molecular tumor cell detection was independent of the type of detection method used. As assays become more sensitive and quantitative, a more thorough assessment of the cancer status of patients will be based on molecular markers alone. At present, it is difficult to conclude that one specific molecular marker is superior to others. Comparative analyses are recommended to assess the prognostic impact of molecular analyses in the same patient and determine the biomarkers that provide the most accurate prognostic information.

Aberrant methylation of PSD disturbs Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis in ulcerative colitis-associated carcinogenesis

We previously reported that the Pleckstrin and Sec7 domain-containing (PSD) gene is preferentially methylated in patients with ulcerative colitis (UC) who developed colorectal cancer (CRC), and is implicated in UC-associated carcinogenesis through its inhibition of apoptosis. This study aimed to determine the potential effect of PSD methylation on its downstream molecule, Ras-related C3 botulinum toxin substrate 1 (Rac1), which governs neutrophil chemotaxis and apoptosis signaling. PSD was knocked down in a normal human fibroblast cell line (HNDF) and a neutrophil-like cell line (HL-60). Both NHDF and HL-60 cells exhibited numerous filamentous-actin (F-actin) rich membrane extensions, resulting in the activation of Rac1; this activation was hampered by PSD silencing. Lipopolysaccharide, a reactive oxygen species (ROS) inducer, stimulated NHDF cells to release ROS and activated caspase-3/7 in the presence of neutrophils, which was inhibited by PSD knockdown. Migration assays demonstrated that chemotaxis of HL-60 cells was affected by PSD silencing in NHDF cells. Tissue sections from 6 UC patients with CRC and 15 UC patients without CRC were examined. To verify Rac1-mediated chemotaxis in tissue sections, we evaluated the grade of neutrophil infiltration by histological assessment and assessed F-actin and PSD expression by immunohistochemistry. Neutrophil infiltration, F-actin and PSD expression were significantly decreased in specimens from UC patients with PSD methylation compared with those without. Decreased levels of F-actin expression were observed in colorectal mucosa, as well as in infiltrating cells with PSD methylation. PSD expression was preferentially inhibited in colorectal mucosa by PSD methylation, whereas PSD expression was rarely observed in infiltrating cells, regardless of PSD methylation status. These data indicate that aberrant methylation of PSD occurs in UC-associated colorectal mucosa, enabling circumvention of Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis, and thus plays a pivotal role in the mechanisms underlying UC-associated carcinogenesis.

Aberrant methylation of the Pleckstrin and Sec7 domain-containing gene is implicated in ulcerative colitis-associated carcinogenesis through its inhibitory effect on apoptosis.

The Pleckstrin and Sec7 domain-containing (PSD) gene, which regulates skeletal rearrangements, has been found to be more frequently methylated both in ulcerative colitis (UC)-associated colorectal cancer tissues (5 of 7; 71.4%) and matched normal epithelia (4 of 7; 57.1%) compared to non-neoplastic UC epithelia (6 of 22; 27.3%) and sporadic colorectal cancer tissues (6 of 32; 18.8%). The levels of PSD mRNA were positively correlated with the methylation status of PSD, as shown by both MSP and bisulfite sequencing. To determine the potential role of PSD silencing in the mechanisms underlying UC-associated carcinogenesis, the levels of senescence, proliferation and apoptosis were evaluated in a normal human fibroblast cell line (NHDF) in which 93% of PSD expression was knocked down by a small-interfering RNA (si-RNA). Although there were no significant differences in the levels of senescence and proliferation caused by PSD knockdown, the level of apoptosis was significantly decreased by PSD knockdown (5.3% in siControl-treated cells vs. 0.67% in siPSD-treated cells, p=0.0001). In addition, reactive oxygen species inducers accelerated apoptosis in NHDF and a neutrophil-like cell line, which was significantly reduced by PSD knockdown. To verify the effect of PSD methylation in tissue sections including 21 samples from UC patients with or without tumors, we elucidated PSD promoting accumulation of filamentous-actin (F-actin) and apoptosis by immunohistochemistry and TUNEL assay, respectively. Both levels of accumulation of F-actin and apoptosis were significantly decreased in specimens from UC patients with PSD methylation compared to those without PSD methylation (F-actin: 0.69±0.86 with vs. 1.57±0.51 without, p=0.0031, apoptotic index: 0.31±0.63 with vs. 1.0±0.88 without, p=0.0277). In conclusion, our results indicate that PSD methylation plays a significant role in the mechanisms underlying UC-associated carcinogenesis through its inhibitory effect on apoptosis in the interaction between colorectal mucosa and neutrophils.

Giant fibrovascular polyp of the esophagus successfully treated by endoscope-assisted resection

Fibrovascular polyps of the esophagus are extremely rare. A 52-year-old woman had the sensation of food sticking in her throat for 3 weeks. Examination revealed a polypoid lesion within the esophagus that extended from the pyriform sinus to the distal esophagus above the gastric cardia. With the patient under general anesthesia, the stalk of the lesion was cut through the rigid laryngoscope; however, the tumor could not be retrieved through the mouth. Large biopsies of the lesion were taken. The remaining lesion was later passed in the fecal matter. Pathological findings were consistent with the features of fibrovascular polyps.

Risk factor for pancreatic fistula after pancreaticoduodenectomy performed by a surgeon during a learning curve: analysis of a single surgeon's experiences of 100 consecutive patients.

BACKGROUND/AIMS Among several kinds of morbidities, pancreatic fistula (PF) is the most common complication of pancreaticoduodenectomy (PD). However, it has not been clarified what kind of perioperative factors are risk factors of PF after PD is performed by a training surgeon. METHODOLOGY We evaluated the risk factors of PF after PD in which all procedures for 100 consecutive patients were performed by a single training surgeon, retrospectively. The 100 cases were divided into two groups and the first 50 cases were named Group A and the latter 50 cases were named Group B. RESULTS Multivariate analysis demonstrated that the absence of main pancreatic duct dilatation was an independent risk factor for grade B and grade C PF (p=0.0080; OR=5.311; 95% CI=1.116-7.025). There was no significant difference of the frequencies of grade B and grade C PF between Group A and Group B (p=0.13361). CONCLUSIONS We demonstrated that the absence of main pancreatic duct dilatation was an independent risk factor for grade B and grade C PF after PD was performed by a training surgeon; for those without pancreatic duct dilatation, PD can be performed by a surgeon in the earlier training period with an acceptable rate of PF.