Naohiro Tomita

Extraction and Analysis of Diagnostically Useful Proteins from Formalin-fixed, Paraffin-embedded Tissue Sections

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-μm-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as β-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

A risk model for esophagectomy using data of 5354 patients included in a Japanese nationwide web-based database

Objective:This study aimed to create a risk model of mortality associated with esophagectomy using a Japanese nationwide database. Methods:A total of 5354 patients who underwent esophagectomy in 713 hospitals in 2011 were evaluated. Variables and definitions were virtually identical to those adopted by the American College of Surgeons National Surgical Quality Improvement Program. Results:The mean patient age was 65.9 years, and 84.3% patients were male. The overall morbidity rate was 41.9%. Thirty-day and operative mortality rates after esophagectomy were 1.2% and 3.4%, respectively. Overall morbidity was significantly higher in the minimally invasive esophagectomy group than in the open esophagectomy group (44.3% vs 40.8%, P = 0.016). The odds ratios for 30-day mortality in patients who required preoperative assistance in activities of daily living (ADL), those with a history of smoking within 1 year before surgery, and those with weight loss more than 10% within 6 months before surgery were 4.2, 2.6, and 2.4, respectively. The odds ratios for operative mortality in patients who required preoperative assistance in ADL, those with metastasis/relapse, male patients, and those with chronic obstructive pulmonary disease were 4.7, 4.5, 2.3, and 2.1, respectively. Conclusions:This study was the first, as per our knowledge, to perform risk stratification for esophagectomy using a Japanese nationwide database. The 30-day and operative mortality rates were relatively lower than those in previous reports. The risk models developed in this study may contribute toward improvements in quality control of procedures and creation of a novel scoring system.

A pancreaticoduodenectomy risk model derived from 8575 cases from a national single-race population (japanese) using a web-based data entry system: The 30-day and in-hospital mortality rates for pancreaticoduodenectomy

Objective:To create a mortality risk model after pancreaticoduodenectomy (PD) using a Web-based national database system. Background:PD is a major gastroenterological surgery with relatively high mortality. Many studies have reported factors to analyze short-term outcomes. Subjects and Methods:After initiation of National Clinical Database, approximately 1.2 million surgical cases from more than 3500 Japanese hospitals were collected through a Web-based data entry system. After data cleanup, 8575 PD patients (mean age, 68.2 years) recorded in 2011 from 1167 hospitals were analyzed using variables and definitions almost identical to those of American College of Surgeons–National Surgical Quality Improvement Program. Results:The 30-day postoperative and in-hospital mortality rates were 1.2% and 2.8% (103 and 239 patients), respectively. Thirteen significant risk factors for in-hospital mortality were identified: age, respiratory distress, activities of daily living within 30 days before surgery, angina, weight loss of more than 10%, American Society of Anesthesiologists class of greater than 3, Brinkman index of more than 400, body mass index of more than 25 kg/m2, white blood cell count of more than 11,000 cells per microliter, platelet count of less than 120,000 per microliter, prothrombin time/international normalized ratio of more than 1.1, activated partial thromboplastin time of more than 40 seconds, and serum creatinine levels of more than 3.0 mg/dL. Five variables, including male sex, emergency surgery, chronic obstructive pulmonary disease, bleeding disorders, and serum urea nitrogen levels of less than 8.0 mg/dL, were independent variables in the 30-day mortality group. The overall PD complication rate was 40.0%. Grade B and C pancreatic fistulas in the International Study Group on Pancreatic Fistula occurred in 13.2% cases. The 30-day and in-hospital mortality rates for pancreatic cancer were significantly lower than those for nonpancreatic cancer. Conclusions:We conducted the reported risk stratification study for PD using a nationwide surgical database. PD outcomes in the national population were satisfactory, and the risk model could help improve surgical practice quality.

Microsatellite instability and mutated type II transforming growth factor-β receptor gene in gliomas

Microsatellite instability has been reported in familial cancer syndrome and in various kinds of human sporadic tumors. We investigated the replication error (RER) and mutation rate of the transforming growth factor-beta type II receptor (TGF-beta RII) gene to determine the frequency of the RER+ phenotype and elucidate the relation between the mutation of the TGF-beta RII gene and RER in the tumorigenesis of glioma. We screened genomic DNA from 40 gliomas, comprised from 24 glioblastomas (GB), 11 anaplastic astrocytomas (AA) and five astrocytomas (AS) and compared the results with DNA from corresponding leukocytes. Seven of the 40 (18%) gliomas had the RER+ phenotype: five (21%) of 24 GB and two (18%) of 11 AA. In six gliomas we detected mutation of the TGF-beta RII gene. Five (71%) of seven RER+ and one (3%) of 33 RER-tumors had one A deletion in the (A)10 repeat of the TGF-beta RII gene. No mutations were detected in the (GT)3 repeat area of the TGF-beta RII gene. As the normal cells of these glioma patients had no mutations, we concluded that the mutations were somatic. We posit that the observed mutations inactivate the receptor through a frameshift mutation resulting in protein truncation. Our data suggest that the TGF-beta RII (A)10 repeat may be one area of genomic instability in the early stages of malignant glioma tumorigenesis.

Primary structure of human pancreatic secretory trypsin inhibitor (PSTI) gene

The human pancreatic secretory trypsin inhibitor (PSTI) gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is approximately 7.5 kb long and is separated into four exons by three introns. The gene has multiple transcription start points and examination with a single-laser cell-sorter showed that it is located on chromosome 5.

Total gastrectomy risk model: Data from 20,011 Japanese patients in a nationwide internet-based database

Objective:To construct a risk model for total gastrectomy outcomes using a nationwide Internet-based database. Background:Total gastrectomy is a very common procedure in Japan. This procedure is among the most invasive gastrointestinal procedures and is known to carry substantial surgical risks. Methods:The National Clinical Database was used to retrieve records on more than 1,200,000 surgical cases from 3500 hospitals in 2011. After data cleanup, 20,011 records from 1623 hospitals were analyzed for procedures performed between January 1, 2011, and December 31, 2011. Results:The average patient age was 68.9 years; 73.7% were male. The overall morbidity was 26.2%, with a 30-day mortality rate of 0.9%, in-hospital mortality rate of 2.2%, and overall operative mortality rate of 2.3%. The odds ratios for 30-day mortality were as follows: ASA (American Society of Anesthesiologists) grade 4 or 5, 9.4; preoperative dialysis requirement, 3.9; and platelet count less than 50,000 per microliter, 3.1. The odds ratios for operative mortality were as follows: ASA grade 4 or 5, 5.2; disseminated cancer, 3.5; and alkaline phosphatase level of more than 600 IU/L, 3.1. The C-index of 30-day mortality and operative mortality was 0.811 (95% confidence interval [CI], 0.744–0.879) and 0.824 (95% CI, 0.781–0.866), respectively. Conclusions:We have performed the first reported risk stratification study for total gastrectomy, using a nationwide Internet-based database. The total gastrectomy outcomes in the nationwide population were satisfactory. The risk models that we have created will help improve the quality of surgical practice.

Risk stratification of 7,732 hepatectomy cases in 2011 from the National Clinical Database for Japan

BACKGROUND There has been no report on risk stratification for hepatectomy using a nationwide surgical database in Japan. The objective of this study was to evaluate mortality and variables associated with surgical outcomes of hepatectomy at a national level. STUDY DESIGN We analyzed records of 7,732 patients who underwent hepatectomy for more than 1 segment (MOS) during 2011 in 987 different hospitals, as identified in the National Clinical Database (NCD) of Japan. The NCD captured 30-day morbidity and mortality as well as 90-day in-hospital mortality outcomes, which were submitted through a web-based data entry system. Based on 80% of the population, independent predictors for 30-day mortality and 90-day in-hospital mortality were calculated using a logistic regression model. The risk factors were validated with the remaining 20% of the cohort. RESULTS The median postoperative length of hospitalization was 16.0 days. The overall patient morbidity rate was 32.1%. Thirty-day mortality and 90-day in-hospital mortality rates were 2.0% and 4.0%, respectively. Totals of 14 and 23 risk factors were respectively identified for 30-day mortality and 90-day in-hospital mortality. Factors associated with risk for 90-day in-hospital mortality were preoperative condition and comorbidity, operative indication (emergency surgery, intrahepatic/perihilar cholangiocarcinoma, or gallbladder cancer), preoperative laboratory data, and extent and location of resected segments (segment 1, 7, or 8). As a performance metric, c-indices of 30-day mortality and 90-day in-hospital mortality were 0.714 and 0.761, respectively. CONCLUSIONS Here we report the first risk stratification analysis of hepatectomy using a Japanese nationwide surgical database. This system would predict surgical outcomes of hepatectomy and be useful to evaluate and benchmark performance.

Expression of Tumor Suppressor Gene p16INK4 Products in Primary Gastric Cancer

Recent studies have shown that the cyclin-dependent kinase (CDK) inhibitor p27Kip1 represents an indicator for patients’ outcome in several human malignancies including gastric cancer. However, the clinicopathologic value of another class of CDK inhibitor, p16INK4, has not been determined. In a retrospective study, we examined the expression of p16INK4 by immunohistochemical assay of 80 samples of primary gastric cancers and their adjacent nonneoplastic mucosas. Less than 10% of non-tumor gastric mucosal cells were p16INK4 positive, whereas the expression of p16INK4 in gastric cancer cells varied widely from 0 to 100% (mean, 24.5%). The expression of p16INK4 was not seen in 11.3% (9/80) of the cancer cases, but in 65% (52/80) this protein was even overexpressed when compared with the nonneoplastic mucosa. A clinicopathologic survey indicated that a low or no expression of p16INK4 was associated with poorly differentiated carcinoma (p = 0.0133), but the level of expression did not correlate with other parameters including patients’ prognosis or with the expression of the pRb protein. In an effort to explore the underlying mechanism for the p16INK4-negative cases, a prospective study was also performed on 20 cases of gastric cancer to compare the level of the p16INK4 protein with the methylation status of the p16INK4 promoter. Gastric cancer tissues with methylation expressed significantly lower levels of the p16INK4 protein (p = 0.0013) and two of them lacked p16INK4 expression altogether, whereas all the cancer tissues without methylation expressed it. These findings suggest that the p16INK4 protein may be associated with differentiation of gastric cancer tissues and that methylation of the p16INK4 promoter may, in part, account for the loss of p16INK4 expression.

S-1 as adjuvant chemotherapy for stage III colon cancer: a randomized phase III study (ACTS-CC trial)

BACKGROUND S-1 is an oral fluoropyrimidine whose antitumor effects have been demonstrated in treating various gastrointestinal cancers, including metastatic colon cancer, when administered as monotherapy or in combination chemotherapy. We conducted a randomized phase III study investigating the efficacy of S-1 as adjuvant chemotherapy for colon cancer by evaluating its noninferiority to tegafur-uracil plus leucovorin (UFT/LV). PATIENTS AND METHODS Patients aged 20-80 years with curatively resected stage III colon cancer were randomly assigned to receive S-1 (80-120mg/day on days 1-28 every 42 days; four courses) or UFT/LV (UFT: 300-600mg/day and LV: 75mg/day on days 1-28 every 35 days; five courses). The primary end point was disease-free survival (DFS) at 3 years. RESULTS A total of 1518 patients (758 and 760 in the S-1 and UFT/LV group, respectively) were included in the full analysis set. The 3-year DFS rate was 75.5% and 72.5% in the S-1 and UFT/LV group, respectively. The stratified hazard ratio for DFS in the S-1 group compared with the UFT/LV group was 0.85 (95% confidence interval: 0.70-1.03), demonstrating the noninferiority of S-1 (noninferiority stratified log-rank test, P < 0.001). In the subgroup analysis, no significant interactions were identified between the major baseline characteristics and the treatment groups. CONCLUSION Adjuvant chemotherapy using S-1 for stage III colon cancer was confirmed to be noninferior in DFS compared with UFT/LV. S-1 could be a new treatment option as adjuvant chemotherapy for colon cancer. CLINICALTRIALS.GOV: NCT00660894.This phase III study, ACTS-CC, is the first study in which demonstrated the efficacy of S-1, an oral fluoropyrimidine, as adjuvant chemotherapy for stage III colon cancer by confirming its noninferiority to UFT/LV in terms of disease-free survival. S-1 could be a new treatment option as adjuvant chemotherapy for colon cancer.

Randomised phase III trial of adjuvant chemotherapy with oral uracil and tegafur plus leucovorin versus intravenous fluorouracil and levofolinate in patients with stage III colorectal cancer who have undergone Japanese D2/D3 lymph node dissection: final results of JCOG0205.

BACKGROUND NSABP C-06 demonstrated the non-inferiority of oral adjuvant uracil and tegafur plus leucovorin (UFT/LV) to weekly fluorouracil and folinate (5-FU/LV) with respect to disease-free survival (DFS) for stage II/III colon cancer. This is the first report of JCOG0205, which compared UFT/LV to standard 5-FU/levofolinate (l-LV) for stage III colorectal cancer patients who have undergone Japanese D2/D3 lymph node dissection. METHODS Patients were randomised to three courses of 5-FU/l-LV (5-FU 500 mg/m(2), l-LV 250 mg/m(2) on days 1, 8, 15, 22, 29, 36 every 8 weeks) or five courses of UFT/LV (UFT 300 mg m(-2)day(-1), LV 75 mg/day on days 1-28 every 5 weeks). The primary end-point was DFS. The sample size was 1100 determined with one-sided alpha of 0.05, power of 0.78 and non-inferiority margin of hazard ratio of 1.27. This trial is registered with UMIN-CTR (C000000193). FINDINGS Between February 2003 and November 2006, 1,101 patients (1092 eligible patients) were randomised to 5-FU/l-LV (n=550) or UFT/LV (n=551). Median age: 61 years, colon/rectum: 67%/33%, number of positive nodes ⩽3/>3: 73%/27%, stage IIIa/IIIb: 75%/25%. The hazard ratio of DFS was 1.02 (91.3% confidence interval, 0.84-1.23), demonstrating the non-inferiority of UFT/LV (P=0.0236). Five-year overall survival (87.5%) was higher than that in NSABP C-06 (69.6%). Grade 3/4 toxicities were 8.4% neutropenia in 5-FU/l-LV and 8.7% alanine aminotransferase elevation in UFT/LV, respectively. The incidences of diarrhoea (9.6% versus 8.5%) and anorexia (4.0% versus 3.7%) were similar between the two arms. No treatment-related deaths were reported. INTERPRETATION Adjuvant UFT/LV is non-inferior to standard 5-FU/l-LV with respect to DFS. UFT/LV should be an oral treatment option for patients with stage III colon cancer who have undergone Japanese D2/D3 lymph node dissection.