Circulating microRNAs as biomarkers for early detection of diffuse-type gastric cancer using a mouse model.

BACKGROUND: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and a poor prognosis. There are no effective serum biomarkers for early detection of DGC. We have established an E-cadherin/p53 double conditional knockout (DCKO) mouse line that recapitulates human DGC morphologically and molecularly. In this study we tried to identify circulating microRNAs (miRNAs) as non-invasive biomarkers for DGC diagnosis using DCKO mice. METHODS: We performed miRNA microarray and quantitative reverse transcription-PCR analyses of tissue and serum samples from DCKO mice with DGC and age-matched littermate controls. RESULTS: Comparative analyses showed that mouse and human primary gastric cancers have similar miRNA expression patterns. Next, we selected some candidate miRNAs highly expressed in sera and cancer tissues of DCKO mice for further evaluation. TaqMan quantitative RT-PCR analyses indicated that four of them, miR-103, miR-107, miR-194 and miR-210, were significantly upregulated in sera of both early and advanced-stage DGC-bearing mice compared with in corresponding controls. Receiver-operating characteristic curve analyses demonstrated that these four miRNAs can discriminate DGC-positive cases from normal ones with high sensitivity and specificity. CONCLUSION: These observations suggest that this mouse model of DGC is useful for identifying serum biomarkers, and we found circulating miRNAs that can accurately detect DGC at an early stage.

Phase II study of trifluridine/tipiracil plus bevacizumab by RAS mutation status in patients with metastatic colorectal cancer refractory to standard therapies: JFMC51-1702-C7.

BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status. PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort. RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals. CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.

Normal cells of patients with high cancer risk syndromes lack transforming activity in the NIH/3T3 transfection assay.

Oncogenes capable of transforming NIH/3T3 cells are often present in human tumors and tumor cell lines. Such oncogenes were not detected in normal fibroblast lines derived from patients with several clinical syndromes associated with greatly increased cancer risk. Thus, germ-line transmission of these oncogenes does not appear to be the predisposing factor responsible for these high cancer risk syndromes.

Frequent epigenetic silencing of the bone morphogenetic protein 2 gene through methylation in gastric carcinomas.

Recently, it was reported that exogenous bone morphogenetic protein (BMP)-2 acted as an antiproliferative agent in a variety of cell lines, including normal and cancerous gastric cell lines, indicating that BMP-2 plays an important role during cell growth. However, despite the loss of BMP-2 expression in several cancers, the underlying mechanism remains unknown. Epigenetic silencing through DNA methylation is one of the key steps during carcinogenesis. In this study, we found, through analysis by the methylation-specific polymerase chain reaction technique, CpG island methylation of the BMP-2 promoter region in gastric and colon cancer cell lines. BMP-2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. Moreover, 24 of the 56 (42.9%) gastric cancer tissues exhibited promoter methylation. Immunohistochemical staining revealed that 18 of the 24 (75%) gastric cancer tissues without methylation signals exhibited BMP-2 expression, whereas among 20 cancer tissues with strong methylation signals only four (20%) expressed BMP-2 (P = 0.0003). These findings indicate that BMP-2 methylation is strongly associated with the loss of BMP-2 protein expression in the primary gastric carcinomas. BMP-2 methylation was more often observed in diffuse type (60.7%) than in intestinal type (25%) gastric carcinomas (P = 0.007). Thus, aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be related to gastric carcinogenesis, particularly in the diffuse type.

Functional domains of rep2, a transcriptional activator subunit for Res2-Cdc10, controlling the cell cycle "start".

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2(+) gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2-Cdc10 complex. To identify the rep2(+) function and molecularly define its domain organization, we reconstituted the Res2-Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2-Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

Effect of protease inhibitors on focus formation by murine sarcoma virus.

The effect of six protease inhibitors, isolated from various species of actinomycetes, on focus formation by murine sarcoma virus was examined. Pepstatin was the only inhibitor. The treatment of cells with pepstatin at various times possibly retards the early stage of infection with murine sarcoma virus.

Development of a 3T3-like line from an embryo culture of an inbred strain of Syrian golden hamster.

An embryo culture of an inbred strain of Syrian hamster developed into a permanent cell line under the "3T3" 3 X 10-5 cells/60-mm dish. The resulting cell line had properties very similar to those of the mouse 3T3 series and was named HAMS 3T3. The cell showed density-dependent inhibition of division with a saturation density of 1.0 to 1.2 x 10-6 cells/60-mm dish or 4.5 to 5.5 x 10-4 cells/sq cm. Addition of fresh medium containing 5 or 10% fetal calf serum to a confluent culture induced DNA synthesis in 18 hr with subsequent cell division. Cells were hyperdiploid with a mode of 45 chromosomes (80% of the cells). When cells at the 60th passage were injected into the skin or cheek pouch of an inbred hamster of the same strain as that from which they were derived, they produced a benign tumor that regressed after 3 weeks. Morphological transformation was obtained by infection with the Moloney strain of murine sarcoma virus.

Genomic structure of the transforming growth factor beta type II receptor gene and its mutations in hereditary nonpolyposis colorectal cancers.

To characterize the tumorigenetic role of the transforming growth factor beta type II receptor (RII) gene, we defined its genomic structure, which consists of seven exons. The sequences of exon-intron junctions were determined to facilitate mutation analysis of each exon. Twenty-five carcinomas and five adenomas from hereditary nonpolyposis colorectal cancer patients were analyzed for mutations in the entire coding region. Four missense mutations (two in adenomas and two in carcinomas) were found in the 10 cases carrying the polyadenine deletions in one allele. These results indicate that RII shares the two-hit inactivation mechanism with tumor suppressor genes and that mutations of it may occur in the early stage of tumorigenesis.

Point mutations and allelic deletion of tumor suppressor gene DCC in human esophageal squamous cell carcinomas and their relation to metastasis.

Since tumor suppressor gene DCC exhibits amino acid sequence homology to the neural cell adhesion molecule, there is a possibility that DCC might be related to tumor metastasis. In the present study, we examined 51 cases of primary esophageal carcinomas with regard to point mutations and loss of the DCC gene. We detected point mutations in two cases by screening using polymerase chain reaction-single strand conformation polymorphism analysis. When we determined the sequences, one case with lymph node metastasis showed an ATG (Met) to ACG (Thr) missense mutation in codon 168. Another case showed a CGA (Arg) to GGA (Gly) mutation in codon 201, which might be a polymorphic change, and two other mutations resulting in no amino acid change. We also examined loss of heterozygosity of the DCC gene. Forty-four of the 51 cases (86%) were informative, and among them 10 cases (23%) showed allelic deletion. The further away the lymph node metastasis was from the primary tumor, the higher the frequency of allelic deletions became. We also found allelic deletions in moderately and poorly differentiated squamous cell carcinomas but not in well differentiated ones. These results indicate that alterations of the DCC gene are related to the degree of lymph node metastasis and the degree of differentiation.

Germ-line mutation of the hMSH6/GTBP gene in an atypical hereditary nonpolyposis colorectal cancer kindred.

A germ-line mutation of hMSH6 (also called GTBP) was found in a hereditary nonpolyposis colorectal cancer (HNPCC)-like patient in whom germ-line mutations of hMSH2, hMSH3, or hMLH1 had not been detected. The patient had rectal cancer and two colon adenomas at 62 years of age and a weak family history of gastrointestinal tumors, indicating atypical HNPCC. Somatic mutations of hMSH6 were observed in three colorectal tumors from the patient, indicating two-hit inactivation. Microsatellite instabilities at mononucleotide repeats were detected in all three tumors. These data suggest that hMSH6 is responsible for tumorigenesis in atypical HNPCC.

Transforming genes from familial adenomatous polyposis patient cells detected by a tumorigenicity assay.

We tried to detect oncogenes associated with familial adenomatous polyposis by a tumorigenicity assay in nude mice. One polyp and two peripheral blood lymphocyte DNAs out of 12 samples from patients induced Alu-positive tumors. Lymphocyte DNAs from one of 5 healthy people also showed tumorigenic activity. The transforming genes of polyps from a patient and lymphocytes from a normal person were found to be the human N-ras gene. Since these N-ras genes were amplified in nude mouse tumors and did not show any alterations in the nucleotide sequences around codons 12 and 61, it is likely that the tumors were induced by the amplified normal N-ras genes. The transforming sequences from two patients' lymphocytes did not hybridize with 12 known oncogene probes, suggesting that these two genes are novel oncogenes or genes for which we have not yet examined the homology. One oncogene derived from a patient's lymphocytes was partially cloned and shown to be located on human chromosome 7. This gene did not hybridize with the met and erbB1 genes, which are potential oncogenes located on chromosome 7. These data indicate that this gene is a new oncogene.

Infrequent germ-line mutation of the E-cadherin gene in Japanese familial gastric cancer kindreds.

Germ-line mutation of the E-cadherin gene was reported in familial gastric cancer (FGC) kindreds from New Zealand. Therefore, we analyzed all of the exons of E-cadherin by PCR-single-strand conformational polymorphism analysis in 16 patients from 14 Japanese FGC kindreds. However, no germ-line mutation was detected, suggesting that a predisposition to FGCs by E-cadherin gene mutation is infrequent in Japanese cases.

Two short sequences have positive effects on the human p27Kip1 gene transcription.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 plays an important role in the progression from G1 to S phase in the cell cycle. To study the activities of its promoter and other regulatory elements, we have cloned and characterized the 5'-flanking region of the human p27Kip1 gene. This region, about 3kb in length, is GC-rich and shares homology with that of the mouse p27Kip1 gene. Transcription start points (tsp) determined by the oligo-capping method are mapped in two regions, the cluster I (-479 to -403) and cluster II (-280 to -273). The cluster I was the primary functional site in transcription initiation. The luciferase activities of serial deletion mutants indicated that two short sequences (-581 to -557 and -556 to -526) had positive effects on transcription. The gel shift assay showed that factors in HeLa nuclear extract bound to these sequences. Sp1 was the major binding factor to the sequence of -556 to -526, wheres yet unidentified positive factors bound to the sequence of -581 to -557.

Promoter analysis of the human mismatch repair gene hMSH2.

The human DNA mismatch repair gene homologue hMSH2 is involved in hereditary nonpolyposis colorectal cancer. We isolated and characterized the 5' upstream region, about 4.4kbp, of the hMSH2 gene. This region contains CpG islands and a number of elements involved in constitutive expression, but there is no TATA-box nearby the transcription start points. This is the typical structure for many promoters of housekeeping genes. Alu sequences and mononucleotide repeats are clustered in this region and there are two transcription start points. Deletion analysis revealed that less than 300bp was sufficient to initiate transcription. Although no mutation that influences promoter activity of this region was found, a polymorphism was detected by PCR-RFLP analysis. Because informative cases (C/T heterozygous) were relatively high ( approximately 30%), this polymorphism is suitable for a marker to examine allelic losses.

Pharmacologic effect of recombinant human IFN-alpha, continuously released from a matrix prepared from a polyglycerol ester of fatty acids, on 2',5'-oligoadenylate synthetase activity in murine liver.

The objective of this study was to assess the pharmacologic effect of continuously released recombinant human interferon-alpha (rHuIFN-alpha) in the liver, the target organ of chronic hepatitis B and C, using 2',5'-oligoadenylate synthetase (2',5'-OAS) activity as an indicator of an antiviral state. A cylindrical matrix prepared from tetraglycerol dipalmitate (TGDP), a polyglycerol ester of fatty acids (PGEF), released rHuIFN-alpha in a pseudo-zero-order manner for about 1 week after implantation into mice, without any major loss of rHuIFN-alpha biologic activity during the release period. To evaluate the pharmacologic effect of the rHuIFN-alpha continuously released from this type of matrix, we established a murine test system. Bolus injections of rHuIFN-alpha solution at three doses increased 2',5'-OAS activities in murine liver extract and serum in a dose-dependent manner, indicating that this system is suitable for evaluating rHuIFN-alpha activity. After subcutaneous insertion of TGDP-matrix implants containing 5.5x10(7) IU rHuIFN-alpha per animal, 2',5'-OAS activities in both liver extracts and serum increased rapidly and remained high for over 1 week. Subcutaneous injections of an equivalent total dose (5.0x10(7) IU/animal per week) of rHuIFN-alpha solution in three or seven fractions prolonged 2',5'-OAS activities compared with a single bolus injection. Comparing 2',5'-OAS activity on day 7 and the portion of the area under the 2',5'-OAS activity-time curve above the normal level (deltaAUC) between the TGDP-matrix implant and multiple injections of the solution revealed that continuously released rHuIFN-alpha has an effect almost equivalent to that of three or seven injections of the solution per week.

DNA amplifications and elevated expression of proto-oncogene in addition to altered DNA ploidy in metastatic brain tumors.

The histopathological characteristics, proto-oncogene amplification, immunohistopathology of the c-erbB-2 product distribution, and the DNA content of nuclei were examined in metastatic brain tumors, which consisted of seven adenocarcinomas, a large cell carcinoma, a squamous cell carcinoma, a renal cell carcinoma and a mucoepidermoid carcinoma. A very high incidence of DNA changes was seen in these tumors. Proto-oncogene amplification and abnormal DNA content in the nuclear portion were found in 64% (7/11) and 67% (6/9) of cases, respectively. We also found double oncogene alteration in three cases metastasizing from lung, esophagus and kidney, and triple oncogene alteration in one case metastasizing from breast. We could not identify the common alterations in the group of metastatic brain tumor cells. These data suggest that the proto-oncogene amplifications and the alteration of DNA ploidy pattern may contribute to the metastatic process.

Distribution of basal lysosomes in exocrine acinar cells.

We examined the distribution of trimetaphosphatase (TMPase)-positive basal lysosomes in pancreas, parotid, submandibular, sublingual, and exorbital lacrimal glands from rats, rabbits, and guinea pigs. The location of the basal lysosomes was compared to that of the acid phosphatase (AcPase)-positive lysosomes. In all of the tissues examined from rat and rabbit, AcPase activity was localized primarily to the Golgi region. Reaction product was localized in GERL, immature secretory granules, and lysosomes lying adjacent to the Golgi apparatus. TMPase activity was found in basal lysosomes and in occasional elongated lysosomes adjacent to the Golgi apparatus. In guinea pig, the distribution of TMPase activity was identical to that seen in the other two species, but a significant number of lysosomes in the basal region of the cells also contained AcPase activity. These results confirm and extend our previous finding (J Histochem Cytochem 31:1209, 1983) that exocrine acinar cells possess two distinct populations of lysosomes. The lysosomes in the Golgi region contain both AcPase and TMPase activity, whereas those in the basal portion of the cells are reactive predominantly for TMPase. The functional significance of the two populations of lysosomes is not understood at present.

Serologic, virologic, and histologic characteristics of chronic phase hepatitis C virus disease in children infected by transfusion.

OBJECTIVE: We studied the time course of hepatic dysfunction, seropositivity to hepatitis C virus (HCV) antibodies, viremia, and histologic evidence of hepatic injury to evaluate the course of HCV infection in children infected by blood transfusion. PATIENTS AND METHODS: Twenty-nine patients (ages 4 to 18 years) who underwent open-heart surgeries for congenital heart disease were grouped into three categories based on alterations in serum alanine aminotransferase (ALT) levels: Group A, acute infection; Group B, subacute infection; and Group C, chronic infection. RESULTS: In Group C, all 13 patients had detectable HCV RNA in serum. In contrast, all patients in Group A had no detectable HCV RNA: In Group B, one of nine patients had detectable HCV RNA and two of four patients examined had persistent chronic hepatitis by histologic criteria. Antibodies directed against C100-3 antigen or core-antigen were more useful than second-generation HCV antibody assays in determining the relationship between viremia and immunologic response. Infection with HCV genotype II and the presence of higher HCV RNA copy numbers were associated with histologic evidence of hepatic damage. CONCLUSION: An abnormal ALT value is frequently associated with viremia, and biochemically resolved acute infection reflects clearance of HCV. However, a normal ALT does not always reflect an absence of hepatocyte damage and HCV replication in patients with subacute disease. The measures outlined in this study are useful indicators of disease activity during the chronic