Comprehensive genetic screening for vascular Ehlers–Danlos syndrome through an amplification-based next-generation sequencing system

Vascular Ehlers–Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys–Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.     

Evaluation of esophageal bile reflux after total gastrectomy by gastrointestinal and hepatobiliary dual scintigraphy

Conducting the qualitative evaluation of reconstruction methods is difficult because of their complexity. The aim of the present study was to compare esophageal bile and food reflux by performing gastrointestinal and hepatobiliary dual scintigraphy (GHDS) after various methods of reconstruction following total gastrectomy. Of 17 patients studied, 4 had undergone Roux-en-Y anastomoses (R-Y); 6, jejunal pouch-Y anastomoses (P-Y); and 7, jejunal pouch interposition (P-I). GHDS was performed 1 year after surgery using¹¹¹In-diethylene triamine pentaacetic acid administered orally, and^99mTc-pyridoxyl-5-methyl tryptophan administered intravenously. Imaging data from a gamma camera were stored in and processed by a data analyzer. Three patients who had undergone R-Y and one who had undergone P-I complained of heartburn, while one who had undergone R-Y, two who had undergone P-Y, and three who had undergone P-I complained of a feeling of fullness. Esophageal bile reflux was confirmed by GHDS in four of the patients who had undergone R-Y, one who had undergone P-Y, and four who had undergone P-I. Moreover, GHDS demonstrated food retention in two patients who had undergone R-Y, five who had undergone P-Y, and four who had undergone P-I. Weight loss was closely related to the esophageal reflux of bile or food which can be accurately detected by GHDS. Despite the absence of heartburn, patients diagnosed as having bile reflux by GHDS showed poor recovery of body weight.     

Matrix metalloproteinase-2 status in stromal fibroblasts, not in tumor cells, is a significant prognostic factor in non-small-cell lung cancer.

PURPOSE: The purpose is to assess clinical significance of matrix metalloproteinase (MMP)-2 and MMP-9 status, especially MMP-2 status, in stromal cells in non-small-cell lung cancer (NSCLC) because experimental studies have revealed that stromal MMP-2 plays important roles in progression of malignant tumors, but most clinical studies focused on tumoral MMP-2 expression, not stromal MMP-2 expression. EXPERIMENTAL DESIGN: We conducted a retrospective study on MMP-2 and MMP-9 expression as evaluated immunohistochemically in a total of 218 consecutive patients with completely resected pathological stage I-IIIA, NSCLC. RESULTS: Strong MMP-2 expression in tumor cells and stromal fibroblasts were documented in 54 (24.8%) and 132 (60.6%) patients, respectively. Strong MMP-2 expression in stromal fibroblasts was more frequently seen in squamous cell carcinoma (72.7%) than in adenocarcinoma (54.9%; P = 0.016). Tumors showing strong MMP-2 expression in stromal fibroblasts showed a significantly higher intratumoral microvessel density (IMVD) than weak stromal MMP-2 tumors (mean intratumoral microvessel density, 50.9 versus 32.4, P = 0.003). In addition, postoperative prognosis of strong stromal MMP-2 patients was significantly poorer than that of weak stromal MMP-2 patients (5-year survival rate, 77.5 versus 60.2%, P = 0.032), and the prognostic significance was enhanced in squamous cell carcinoma patients but disappeared in adenocarcinoma patients. Multivariate analyses confirmed that strong stromal MMP-2 expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients. In contrast, MMP-2 or MMP-9 status in tumor cells was not a significant prognostic factor. CONCLUSIONS: MMP-2 status in stromal fibroblasts, not in tumor cells, was a significant prognostic factor associated with angiogenesis in NSCLC.     

Expression of Adhesion Molecules and Cytokines on Saphenous Veins in Chronic Venous Insufficiency

The objective of this study was to assess the relationship of signaling molecules to monocyte/ macrophages as a precursor to venous valve and venous wall dysfunction in patients with varicose veins. One of the hallmarks of venous dysfunction is destruction of venous valves with subsequent reflux and elevation of distal venous pressure. We recently observed that monocytes/macrophages migrate into the venous walls and valves of patients with venous insufficiency. There, they may play a role in the pathogenesis of primary venous insufficiency. If so, an important element in their performance would be the interaction between the monocytes and the endothelium as a precursor of damage to venous valves and the venous wall. To explore this interaction, immunohistochemistry was carried out to detect adhesion molecules and cytokines in surgical specimens removed during surgical therapy. Twenty-four surgical specimens consisting of proximal saphenous vein and subterminal valve were obtained using minimally traumatic technique in 6 males and 18 females who ranged in age from 31 to 79 years. Reflux was confirmed preoperatively by duplex technique, and severity was classified by the CEAP classification of the American Venous Forum. Ten patient limbs were class 2, eight were class 3, four were class 4, and two were class 6. The venous specimens were labeled using monoclonal antibody against ICAM-1, E-selectin, IL-1alpha, and TNF-alpha. CD68 was used for detection of monocytes/macrophages. Our results indicate that not only luminal venous endothelium but also endothelium in the vasa vasora of refluxing saphenous veins is activated, as indicated by the up-regulation of ICAM-1. However, IL-1alpha and TNF-alpha were increased in only selected specimens and are mainly detected in the vein wall. The factors that serve as trigger mechanisms to activate cells in the pathogenesis of primary venous dysfunction remain to be explored.     

Subjective and functional results after total gastrectomy: prospective study for longterm comparison of reconstruction procedures

Background: Many reconstruction procedures have been developed in efforts to resolve patients' complaints after total gastrectomy. However, there have been few reports of longterm comparisons between reconstruction procedures, especially with regard to the prevention of duodenal food passage. This study was undertaken to compare the longterm subjective and functional results among Roux-en-Y esophagojejunostomy (R-Y), R-Y with pouch (P-Y), and jejunal interposition with pouch (P-I) after total gastrectomy. Methods: Consecutive patients requiring curative total gastrectomy were enrolled in this prospective study by the envelope method. Results: Hospital stay was longer following a P-I than an R-Y or a P-Y. Over 50% of R-Y patients complained of heartburn, and 20% of R-Y patients showed dumping syndrome throughout the postoperative period, with this rate being significantly different from rates in the other two groups. P-Y patients complained of early satiety in the late postoperative period, while P-I patients complained of early satiety in the early postoperative period. The nutritional index in P-I patients was higher than those in patients with the other two procedures. Gastrointestinal and hepatobiliary dual scintigraphy (GHDS) showed that the rate of bile reflux with an R-Y was relatively high after surgery. Food reflux with a P-Y was increased (9.4% to 11.1%), but with a P-I food reflux was decreased at 3 years after surgery (13.3% to 9.9%). Patients with a P-Y had a faster recovery of body, weight in the early postoperative period; however, at 5 years after operation, body weight recovery with a P-I was greatest. Conclusion: Reconstruction should be performed with pouch formation after total gastrectomy with curative intent. Received: March 7, 2002 / Accepted: September 26, 2002 Acknowledgments This study was partly supported by the University of Tsukuba Research Project. Offprint requests to: S. Adachi     

Prognostic significance of loss of heterozygosity on chromosome 9p21–22 in human esophageal cancer

Background Deletions involving chromosome 9p21, on which the tumor suppressor genep16/MTS1 is located, have been noted in esophageal cancer. We investigated the relationship between the deletion of chromosome 9p21–22 and the clinical features of esophageal cancer. Methods We examined the loss of heterozygosity (LOH) on chromosome 9p21–22 in 56 esophageal cancers using polymerase chain reaction (PCR) analysis and 2 microsatellite markers (RPS6 and IFNA). Results In 18 out of 50 informative cases (36%), LOH had occurred at 1 or 2 loci on chromosome 9p21–22. We found no relationship between LOH on chromosome 9p21–22 and patient sex, age tumor length, location, histologic differentiation, depth of tumor invasion, the extent of lymph node metastasis, histologic stage, or curability. Among 35 patients without an absolute noncurative resection, the mean survival of 11 patients with LOH on chromosome 9p21–22 was 19.3 months, compared with 42.3 months for 24 patients with a normal allele; thus, the survival rate of those with LOH was significantly lower than that of patients without LOH on chromosome 9p21–22 (log-rank test;P=0.03). Conclusion These data suggest that LOH on chromosome 9p21–22, on which the cell-cycle regulatorp16/MTS1 gene is located, may be related to cancer development, and probably can serve as a clinical marker for evaluating a patient's prognosis.     

Tumor-specific Activation of Mitogen-activated Protein Kinase in Human Colorectal and Gastric Carcinoma Tissues

To search for the signaling events in colorectal carcinoma relevant to its tumorigenesis, we investigated the activity of mitogen-activated protein kinase (MAPK) in human colorectal carcinoma tissues and paired normal tissues. Of 64 cases examined, approximately 75% (48 cases) showed tumor-specific activation of MAPK by in situ kinase renaturation assay, as well as in vitro kinase assay with immunoprecipitated MAPK. In addition, tumor-specific activation of MAPK was associated with the activation of MAPK kinase in the cases we examined. However, no clear correlation of MAPK activation with lymph node involvement, metastatic rate, stage, histological classification, age or sex was observed. These results suggest that the MAPK pathway is involved in colorectal tumor development, but its activation alone is not sufficient for malignant conversion. In contrast to colorectal carcinoma, gastric carcinoma tissues showed a lower rate of MAPK activation, suggesting that the signaling pathway activated in colorectal carcinoma tissues may differ in part from that of gastric carcinoma.     

Longer-term diabetic patients have a more frequent incidence of nosocomial infections after elective gastrectomy

Diabetes mellitus (DM) is one of the risk factors for the development of postoperative nosocomial infections in surgical patients. We conducted this retrospective study to elucidate the perioperative risk factors for postoperative nosocomial infections in diabetic patients undergoing elective gastrectomy. Chart review was performed on diabetic and nondiabetic patients undergoing elective gastrectomy for gastric malignancy from January 1992 through April 1999. Fourteen of the 83 diabetic patients, and 23 of the 284 nondiabetic patients developed postoperative nosocomial infections. Statistical comparisons of multiple variables were made between patients with and without postoperative nosocomial infections. In diabetic patients, univariate analysis showed that longer-term DM (especially longer than 10 yr) was associated with a significantly increased risk for postoperative nosocomial infections. Multiple logistic regression analysis showed that DM lasting longer than 10 yr was an independent risk factor for postoperative nosocomial infections (odds ratio, 6.8; 95% confidence interval, 1.7 to 27.1). In nondiabetic patients, similar analysis showed that age was an independent risk factor for postoperative nosocomial infections. We conclude that patients with longer-term DM had a significantly greater incidence of postoperative nosocomial infections after elective gastrectomy. Implications: Postoperative nosocomial infection is one of the major problems in diabetic patients. This study demonstrated that postoperative nosocomial infections were more common in patients undergoing elective gastrectomy if they had diabetes mellitus longer than 10 yr.     

Effects of portal-systemic shunt following 90% partial hepatectomy in rats

We studied the effects of portal-systemic shunt after massive hepatectomy. Male Wistar rats were divided into two groups: one group underwent laparotomy alone (C group) and in the other group a portal-systemic shunt was placed through laparotomy (S group). After 90% hepatectomy was performed, 3-day and 1-week survival rates and histopathology were examined, and hepatic hemodynamics during the early stage after hepatectomy were measured using dye-containing microspheres. The 3-day survival rate in the S group was significantly higher, and the 1-week survival rate was slightly higher, than those in C group. Sinusoidal dilation 7 days after hepatectomy in the S group was significantly milder than that in the C group. Fatty degeneration of hepatocytes in the S group was significantly milder than that in the C group. With respect to hepatic hemodynamics during the early stage after hepatectomy, the rate of shunt (26.3%) in the S group was significantly higher than that (9.5%) in the C group. Portal pressure, total hepatic blood flow, and total hepatic blood flow per gram of liver in the S group were significantly lower than those in the C group. These results suggest that approximately 26% shunt after 90% hepatectomy in rats increases the early survival rate and improves histological changes in surviving rats 7 days after resection.     

Formation of tamoxifen-DNA adducts via O-sulfonation, not O-acetylation, of alpha-hydroxytamoxifen in rat and human livers.

Tamoxifen (TAM) is used as the standard endocrine therapy for breast cancer patients and as a chemopreventive agent for women at high risk for this disease. Unfortunately, treatment of TAM increases the incidence of endometrial cancer; this may be due to the genotoxic damage induced by TAM metabolites. Formation of TAM-DNA adducts in rat liver correlates with the development of hepatocarcinoma. TAM-DNA adducts are proposed to be formed through O-sulfonation and/or O-acetylation of alpha-hydroxylated TAM and its metabolites. However, the role of O-sulfonation and O-acetylation in the formation of TAM-DNA adducts has not been extensively investigated. Rat or human hydroxysteroid sulfotransferases (HST), acetyltransferases, and liver cytosol were incubated with calf thymus DNA, alpha-OHTAM, and either 3'-phosphoadenosine 5'-phosphosulfate (PAPS) or acetyl coenzyme A (acetyl-CoA) as a cofactor and analyzed for TAM-DNA adduct formation, using 32P postlableling/polyacrylamide gel electrophoresis analysis. TAM-DNA adduct was formed when PAPS, not acetyl-CoA, was used. No TAM-DNA adducts were produced using human N-acetyltransferase I and II. HST antibody inhibited approximately 90% of TAM-DNA adduct formation generated by the cytosol or HST, suggesting that HST is primarily involved in the formation of TAM-DNA adducts. The formation of TAM-DNA adducts with rat liver cytosol and HST was much higher than that of human liver cytosol and HST. Our results indicate that TAM-DNA adducts are formed via O-sulfonation, not O-acetylation, of alpha-hydroxylated TAM and its metabolites.