Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity.

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.

High incidence of synchronous or metachronous breast cancer in patients with malignant and benign thyroid tumor or tumor-like disorders.

BACKGROUND: Although many reports indicated an association between thyroid diseases and breast cancer, such an association still remains controversial. The present study was aimed to clarify the association of thyroid diseases with the breast cancer incidence. In the patients with benign and malignant thyroid tumor or tumor-like disorders, the incidence of other malignancies was surveyed, and the frequency of thyroid cancer in patients with breast cancer was also surveyed. PATIENTS AND METHODS: Between 1982 and 2005, a total of 201 female patients received surgery for tumor or tumor-like disorders, including 65 carcinoma, 68 adenoma, 61 adenomatous goiter and 7 chronic thyroiditis cases. Their outcomes were surveyed in December 2006. Furthermore, during the same periods, 340 female patients underwent breast cancer surgery and their outcomes were also surveyed in December 2006. RESULTS: The overall incidence rate of breast cancer was 16.4% (33/201) in the patients, who received thyroid surgeries and was much higher than other malignancies: 2.0% gastric cancer, 1.0% uterine and colorectal cancer. The incidence rate of breast cancer in each disease was 13.8% for thyroid cancer, 16.2% for adenoma and 21.3% for adenomatous goiter, but no incidence for chronic thyroiditis. On the other hand, in the patients with breast cancer during the same period in our department, the frequency of thyroid cancer was only 2.1% (7/340). CONCLUSION: It appears that thyroid cancer, adenoma and adenomatous goiter were associated with the risk of breast cancer, but chronic thyroiditis was not related.

Expression of orotate phosphoribosyl transferase in human pancreatic cancer: implication for the efficacy of uracil and tegafur-based adjuvant chemotherapy.

The enzyme orotate phosphoribosyl transferase (OPRT) is involved in the metabolism of the anticancer drug 5-fluorouracil (5-FU), and is a key enzyme for conversion of 5-FU to its active form in tumor tissue. Little is known regarding the significance of OPRT in human pancreatic cancer. The present study was designed to assess the association between the activity of OPRT in the tumor, and the clinicopathological status and prognosis of human resectable pancreatic cancer, especially regarding its relevance to the efficacy of adjuvant chemotherapy with uracil and tegafur (UFT), cyclophosphamide (CPA) and/or gemcitabine (GEM). The present study included 99 resectable pancreatic cancers, which were all invasive ductal tubular carcinomas. OPRT was immunostained with a rabbit anti-human OPRT polyclonal antibody. OPRT was positively stained in 54 (54.5%) of 99 pancreatic cancers. The post-surgical survival rate of the OPRT (+) pancreatic cancers was significantly higher than that of the OPRT (-) ones. In the OPRT (+) group, the survival rate of the patients, who received adjuvant chemotherapy (ACT) with UFT, CPA or GEM, was significantly higher than that of the patients without ACT; however, in the OPRT (-) group, there was no difference in the survival between the ACT (+) and (-) groups. Multivariate analyses demonstrated that for all patients, primary tumor, status of nodal involvement (pN), residual tumor, level of dissection and CPA were significant variables for the prognosis: in OPRT (+) groups, primary tumor, nodal involvement, GEM and CPA were significant variables. In contrast, in the OPRT (-) group, pN was the only significant variable. The present study is the first report on the significance of OPRT in human pancreatic cancer, and the results indicate that the expression of OPRT may be useful to predict the response to adjuvant chemotherapy in human pancreatic cancer.

Cyclophosphamide augments the anti-tumor efficacy of uracil and tegafur by inhibiting dihydropyrimidine dehydrogenase.

The present study assesses the effects of neo-adjuvant chemotherapy (NAC) with uracil and tegafur (UFT) alone vs UFT plus cyclophosphamide (CPA), on the activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in breast cancer tissues. Breast cancer patients were randomly assigned to 3 groups; the control (no-treatment) group (n=13), the UFT (5-8 mg/kg/day) alone group (n=10) and the UFT plus CPA (1 mg/kg/one day interval) (UC) group (n=9), and they received NAC for 2-4 weeks. A total of 32 invasive ductal breast carcinomas were used to assay for TS and DPD activity. There were no statistically significant differences in tumor size or stage classification between the 3 groups. The DPD activity was inversely and significantly correlated with the tumor size and pT, but the TS activity was not correlated with these clinicopathological factors. The TS activity was decreased by NAC with UFT, and the addition of CPA resulted in an increased inhibition of TS activity. In contrast, DPD activity was increased by NAC with UFT administration, but its increased activity was significantly inhibited by the addition of CPA. Multiple regression analyses demonstrated that the total dose of UFT was a significant variable for inhibiting TS activity, and that CPA was a significant variable for inhibiting DPD activity. The DPD activity increased by UFT can be inhibited by CPA, and this may represent one of the possible mechanisms responsible for the anti-tumor activity of 5-FU or its derivatives as enhanced by CPA.

Immunohistochemical expression of HER-1 and HER-2 in extrahepatic biliary carcinoma.

The clinicopathological significance of HER-1- and HER-2-overexpressions (OE) (HercepTest score 2+ or 3+) in biliary cancer and their relationship to the efficacy of adjuvant chemotherapy (ACT) were assessed. In 72 biliary cancer (28 gallbladder and 44 bile duct cancer), HER-1 and HER-2 were stained immunohistochemically in formalin-fixed, paraffin-embedded specimens. The ACT included uracil and tegafur (UFT)-based chemotherapies. Out of the 72 cancer, OE was observed in 31 specimens (43%) for HER-1 and 47 (65%) for HER-2. However, their OEs were not correlated with each other. HER-2-OE was inversely correlated with the clinical stage (p=0.0482). HER-1-OE was correlated with distant metastasis (p=0.0263), but not with the clinical stage. Neither the OE of HER-1 or HER-2, nor their co-expression, showed any significant effect in term of patient survival. In the HER-1-OE (-) patients, the survival rate of the ACT group was significantly higher than that of the surgery-alone (SA) group (p=0.0423), but in the HER-1-OE (+) patients, there was no statistical difference in survival rate between the ACT and the SA group. In contrast, HER-2-OE had no significant effect on the efficacy of ACT. Multivariate analysis also demonstrated that the histological grade and ACT were significant variables, but T, N, M and HER-1 and HER-2 were not significant variables. In conclusion, neither HER-1-OE or HER-2-OE were prognostic factors of the biliary cancer. However, HER-1-OE may be a useful marker for the indication of ACT.

Immunohistochemical expression of receptor-tyrosine kinase c-kit protein and TGF-beta1 in invasive ductal carcinoma of the pancreas.

BACKGROUND: The receptor tyrosine kinase c-kit is known to play an important role in the progression of gastrointestinal stromal tumors, but its biological significance in other solid malignancies is unclear. Recent publications have suggested a regulatory role for TGF-beta1 in c-kit-mediated cell growth. The present study assessed the clinicopathological significance of c-kit protein (KIT) and TGF-beta1 expression in resectable invasive ductal carcinomas (IDCs) of the pancreas. PATIENTS AND METHODS: This study included 91 pancreatic IDC patients who received a pancreatectomy between 1982 and 2003. The expression of KIT and TGF-beta1 was analyzed by immunohistochemistry. RESULTS: KIT and TGF-beta1 were expressed in 77% (70/91) and 59% (54/91) of the IDC, respectively. The expression of KIT was not correlated with that of TGF-beta1. TGF-beta1 expression correlated inversely with nodal involvement, but KIT expression did not correlate with any clinicopathological factors. KIT expression had no significant influence on the survival of the patients, whereas the survival rate of TGF-beta1 (+) IDC patients was significantly higher than that of TGF-beta1 (-) IDC patients. Co-expression analysis of KIT and TGF-beta1 indicated that, in patients with KIT (+) IDC, the TGF-beta1 (+) group showed a significantly better survival rate than the TGF-beta1 (-) group. Neither KIT expression nor TGF-beta1 expression had a significant effect on the efficacy of adjuvant chemotherapy (ACT). In multivariate analysis, TGF-beta1 expression was one of the significant variables for survival in IDC patients overall, but KIT expression was not. CONCLUSION: TGF-beta1 expression is suggested to have a significant influence on c-kit-mediated cell proliferation in human pancreatic IDCs.

The dissociated expression of protein and messenger RNA of DPC4 in human invasive ductal carcinoma of the pancreas and their implication for patient outcome.

BACKGROUND: The loss of expression of Dpc4 protein (pDpc4) has been demonstrated in about half of invasive ductal carcinoma (IDC) of the pancreas, but the expression of DPC4-mRNA remains to be evaluated. The present study assessed the comparative expression of pDpc4 and DPC4-mRNA in pancreatic IDC and their implication for patient outcome. MATERIALS AND METHODS: In the freshly separated specimens of 21 IDCs and the paraffin-embedded specimens of 88 resectable IDCs, the expression of mRNA was assessed by in situ hybridization and the expression of pDpc4 was assessed by immunohistochemistry. RESULTS: In the freshly separated specimens, DPC4-mRNA was expressed in 71% of the IDC, but pDpc4 expression was lost in 76% of the IDC. In 88 resectable IDCs, pDpc4 expression was lost in 75 (85.2%) and loss of pDpc4 expression was significantly correlated with the grade of nodal involvement (p =0.0265). Furthermore, the survival rate of the pDpc4 (-) group was significantly lower than that of the pDpc4 (+) group (p=0.0391). Adjuvant chemotherapy (ACT) significantly improved the survival rate and, in the ACT group, pDpc4 (-) patients had a significantly lower survival rate than the pDpc4 (+) patients. CONCLUSION: In human pancreatic IDC, although DPC4-mRNA was usually expressed, the expression of pDpc4 was lost. The expression of pDpc-4 is an indicator of better prognosis and response to ACT.

Implication of HER-2/neu overexpression for the efficacy of oral fluoropyrimidine-based adjuvant chemotherapy in the patients with estrogen receptor negative breast cancer after surgery.

BACKGROUND: The implications of HER-2/neu overexpression for the efficacy of adjuvant chemotherapy (ACT) and endocrine therapy (AET) have been controversial. The present study retrospectively assessed the effects of HER-2/neu overexpression on the efficacy of oral fluoropyrimidine-based ACT and AET after breast cancer surgery. PATIENTS AND METHODS: The expression of HER-2/neu protein and estrogen receptor (ER) in 217 primary breast cancers was assessed immunohistochemically using the HercepTest and an anti-ER monoclonal antibody. The overexpression of HER-2/neu was classified into 4 categories (0-3+) according to standard criteria, and 3+ was categorized as HER-2/neu-overexpression. Of the 217 patients, 26 received surgery alone (SA), 32 received ACT, 20 received AET alone and 139 received both AET and ACT. The regimen of the ACT included oral fluoropyrimidines in all patients. RESULTS: HER-2/neu (3+) overexpression and ER expression were seen in 31.8% (69 out of 217) and 47.5% (103 out of 217) of the patients, respectively. The survival of the HER-2/neu (3+) group was significantly lower than other groups (p = 0.0134), especially in the ER (+) patients (p = 0.0229). However, in the ER (+) patients, HER-2/neu overexpression had no significant effect. The effects of HER-2/neu overexpression and ER expression on the efficacy of the ACT and AET were analyzed by patient survival. In the ER (-) patients, the ACT (+) group had a significantly higher survival rate than the ACT (-) group (p = 0.0459), and this was noted especially in the HER-2/neu-overexpressing cases (p = 0.0485). In the ER (+) patients, ACT and AET did not have any significant influence on the survival regardless of the HER-2/neu status. CONCLUSION: Fluoropyrimidines-based ACT improves the long-term outcome of patients with HER-2/neu-overexpression (3+) and ER (-) breast cancer after surgery.

A fractal dimension analysis: a new method for evaluating the response of anticancer therapy.

BACKGROUND: The therapeutic effects of various anticancer therapies on malignant tumors are evaluated as objective response (OR) by comparing the tumor size before and after the therapy However, it is difficult to evaluate the OR because malignant tumors frequently have very irregular or stellate shapes. In the present study we investigated a new method for evaluating the response of esophageal cancer to radio-chemotherapy using the fractal dimension (FD). PATIENTS AND METHODS: The changes in tumor size or shape during therapy were recorded in 6 patients with esophageal cancer by esophageal fluoroscopy. The OR was evaluated by WHO standard criteria by calculating the tumor regression rate (TRR), while the FD was calculated by a standard compass-counting method. RESULTS: All 6 patients complained of dysphagia before therapy, but the food passage improved after therapy in all patients. The TRR after therapy ranged between -90% and 43.4%, and the OR was evaluated as no change (NC) in 5 patients and progressive disease (PD) in one patient. On the other hand, the RD gradually decreased in all 6 patients during the therapy. Furthermore, 5 patients underwent esophagectomy and the histological effect was evaluated as grade-2 response in two patients and grade-1 response in three patients. The decreases in the FD were 0.069 and 0.079 in grade-2 responses and 0.022-0.034 in grade-1 responses. By contrast, 4 NCs included 2 grade-2 responses and 2 grade-1 responses, and PD was evaluated as grade-1 response. These results suggested that the standard criteria for an OR could not accurately evaluate the therapeutic effect in some cases, and the changes in FD correlated with the improvement in the symptoms and histological effect more precisely than the TRR or the OR. CONCLUSION: The FD represented the therapeutic effect of radio-chemotherapy well for esophageal cancer, and may be widely applied for evaluating the therapeutic effect of cancer therapy.

Distribution and haplotype analysis of all the non-synonymous and autoimmunity-related single nucleotide polymorphisms in the human deoxyribonuclease II gene using worldwide populations.

We have focused on the 14 SNPs including all the non-synonymous and autoimmunity-related ones in the DNase II gene (DNASE2). The distribution of each allele and haplotype in these SNPs was examined in eight Asian, three African, three Mexican and two Caucasian populations using the newly developed PCR-RFLP methods. Eight SNPs among nine non-synonymous ones were monomorphic, indicating that a specific allele generating the intact activity-harboring DNase II in these SNPs is well conserved in worldwide populations. On the other hand, five other SNPs (-1951G>A, -1066G>C, -390A>C, +2630T>C, and +6235G>C) related to autoimmunity exhibited polymorphism common in worldwide populations, and especially their distributions were ethnic-dependent in the same manner as those of haplotypes. Furthermore, a strong linkage between SNPs -1951G>A and -1066G>C was confirmed in most populations. This study was the first to report any worldwide population analysis regarding all the non-synonymous and autoimmunity-related SNPs in the DNASE2, providing genetic information on the DNASE2 as a genetic marker for personal identification and/or genetic factor for susceptibility to autoimmunity.

Determination of ABO genotypes by real-time PCR using allele-specific primers.

ABO grouping of biological specimens is informative for identifying victims and narrowing down suspects. In Japan and elsewhere, ABO grouping as well as DNA profiling plays an essential role in crime investigations. In the present study, we developed a new method for ABO genotyping using allele-specific primers and real-time PCR. The method allows for the detection of three single nucleotide polymorphisms (SNPs) at nucleotide positions 261, 796, and 803 in the ABO gene and the determination of six major ABO genotypes. This method required less than 2 h for accurate ABO genotyping using 2.0 ng of DNA. This method could be applicable for rapid and simple screening of forensic samples.

Simultaneous determination of seven informative Y chromosome SNPs to differentiate East Asian, European, and African populations.

Identification of the population origin of an individual is very useful for crime investigators who need to narrow down a suspect based on specimens left at a crime scene. Single nucleotide polymorphisms of the Y chromosome (Y-SNPs) are a class of markers of interest to forensic investigators because many of the markers indicate regional specificity, thus providing useful information about the geographic origin of a subject. We selected seven informative Y-SNPs (M168, M130, JST021355, M96, P126, P196, and P234) to differentiate the three major population groups (East Asian, European, and African) and used them to develop forensic application. SNP genotyping was carried out by multiplex PCR reaction and multiplex single base extension (MSBE) reaction followed by capillary electrophoresis of extension products. This method can be used to assign a haplogroup from both degraded male DNA samples and DNA samples containing a mixture of female and male DNA through PCR primers that generate small amplicons (less than about 150 bp) and are highly specific for targets on the Y chromosome. The allelic state of each marker was definitively determined from a total of 791 males from the three major population groups. As expected, samples from the three major population groups showed Y-haplogroups common in the region of provenance: Y haplogroups C, D, and O for East Asians; IJ and R1 for Europeans; and AB and E for Africans.

Hyperbilirubinemia-related behavioral and neuropathological changes in rats: a possible schizophrenia animal model.

BACKGROUND: Patients with schizophrenia show a significantly higher frequency of hyperbilirubinemia than patients suffering from other psychiatric disorders and the general healthy population. We examined the hyperbilirubinemia on behavioral and neuropathological changes in rats as a possible animal model of schizophrenia. METHODS: Gunn rats with severe hyperbilirubinemia (j/j), Gunn rats without severe hyperbilirubinemia (+/j), and Wistar rats were examined by open-field, social interaction, and prepulse inhibition tests. TUNEL, AgNOR and Ki-67 were also assayed on paraffin-embedded brain sections of these rats. RESULTS: Compared to Wistar rats, both Gunn j/j and +/j rats showed hyperlocomotion, high sniffing scores, and low defecation scores. They showed significantly more aggressive behaviors and impaired prepulse inhibition. The numbers of Ki-67-labeled cells and AgNOR were lower and the number of TUNEL-positive cells was higher than that of Wistar rats. CONCLUSIONS: These results might support the neurodevelopmental hypothesis of schizophrenia. Both Gunn j/j and +/j rats may be a useful animal model and provide clues to the role of hyperbilirubinemia in schizophrenia.

p53 alteration is not an independent prognostic indicator, but affects the efficacy of adjuvant chemotherapy in human pancreatic cancer.

BACKGROUND AND OBJECTIVES: Mutations in the p53 gene are found in more than 50% of human cancers and are observed in 60-80% of pancreatic cancers. The clinicopathologic implications of p53 abnormalities and their effects on the efficacy of the adjuvant chemotherapy for pancreatic cancer remain controversial. METHODS: We investigated the p53 status in core exon-4 to -9 (codon 33-331) by direct DNA sequencing in a series of 72 pancreatic cancers and analyzed the effects of p53 abnormalities on the patients' survival and the efficacy of adjuvant chemotherapy. RESULTS: p53 mutations were found in 62.5% (45/72) of cases, including 38 point mutations and 7 frameshift mutations. The subtypes of p53 mutations included 68.9% (31/45) transitions and 15.6% (7/45) transversions. 39.5% (15/38) of point mutations were CGT (Arg) to CAT (His) mutation at codon-273 of exon-8. 34.2% (13/38) of point mutations were CGG (Arg) to TGG (Trp) mutation at codon-248 of exon-7. Of seven frameshift mutations, four were seen at exon-4, two at exon-5, and one at exon-6. Of overall cases, p53 abnormalities were not associated with a poorly differentiated grade and an advanced stage. The relationship of adjuvant chemotherapy to survival is approaching statistical significance. Univariate analysis showed that in the p53 mutation group, the patients who received adjuvant chemotherapy had a better survival ratio than that of patients who did not do. Multivariate analysis indicated that in the group with p53 mutations, the significant factors for survival were adjuvant chemotherapy, histologic grade, and clinical stage. However, in the group with a wild-type p53 gene, only histologic grade was a significant factor. In addition, 34.7% (25/72) of the cases harbor p53 polymorphism mutation only at codon-72 of exon-4, which did not show any significant effect on the pathology, prognosis, and efficacy of adjuvant chemotherapy of the pancreatic cancers. CONCLUSIONS: A p53 abnormality was not an independent factor for evaluating the prognosis of patients with pancreatic cancer, but was a beneficial indicator for selecting a reasonable strategy of adjuvant chemotherapy against pancreatic cancer.

Immunohistochemical expression of receptor-tyrosine kinase c-kit protein in invasive ductal carcinoma of the pancreas.

The expression of receptor tyrosine kinase c-kit and its biologic significance in pancreatic cancer are unclear. We studied the expression of c-kit protein (c-KIT) in resectable invasive ductal carcinomas (IDCs) of the pancreas, in order to assess whether a selective c-kit inhibitor, STI571 (Glivec), may be applied for the treatment of pancreatic IDCs. This study included 72 pancreatic IDC patients who received a pancreatectomy between 1982 and 2002. The expression of c-KIT was analyzed retrospectively by immunohistochemistry. c-KIT was expressed in 78% (56/72) of the pancreatic IDCs. c-KIT expression did not correlate with any clinicopathological factor of pancreatic IDC and c-KIT expression had no significant influence on the survival of the patients. The survival rate of the adjuvant chemotherapy (ACT) (+) group was significantly higher than that of the ACT (-) group, but c-KIT expression had no significant effects on the efficacy of the ACT. Multivariate analysis indicated that the pTNM stage, grade and ACT were all significant variables for survival in IDCs overall. As c-KIT was expressed in 78% of the pancreatic IDCs, it suggests that STI571 may be a beneficial agent for chemotherapy against human pancreatic IDCs.