Outcome of the 'waiting until spontaneous extrusion' strategy for long-term tympanostomy tube placement in children with cleft palate.

BACKGROUND: Otitis media with effusion (OME) in children with cleft palate (CP) is known to be refractory to treatment and most of these patients undergo surgery for ventilation tube (VT) placement. OBJECTIVES: To identify the outcomes of children with CP using long-term VT with a 'waiting until spontaneous extrusion' strategy. MATERIAL AND METHODS: We retrospectively reviewed the medical records of all children with CP who visited our department from December 2016 to November 2017 and who received long-term VT placement in our department. Risk factors related to residual perforation and recurrence of OME were analyzed. RESULTS: A total of 106 children were included in this study. Our statistical analysis of 94 ears followed for more than three months after VT loss revealed that longer VT placement was associated with residual perforation, and shorter VT placement was associated with OME recurrence. Although a longer duration of VT placement was associated with an increased rate, extremely long-term VT placement was not associated with residual perforation, as expected. Half of the VTs were spontaneously extruded at 40 months after insertion. CONCLUSIONS AND SIGNIFICANCE: Long-term VT insertion using a waiting until spontaneous extrusion strategy is a potential option for children with CP.

FRY site-specific methylation differentiates pancreatic ductal adenocarcinoma from other adenocarcinomas.

Adenocarcinoma is a type of cancer that occurs in the glandular cells throughout the body. There are several metastatic adenocarcinoma of unknown primary origin. Currently, there is no highly effective method to differentiate pancreatic ductal adenocarcinoma (PDAC) from other adenocarcinomas. Here, we identified pancreas tissue by site-specific methylation at FRY and found that it can also detect PDAC. The establishment of Combined Bisulphite Restriction Analysis (COBRA) and quantitative real-time PCR techniques of FRY revealed FRY hypermethylation in 21 out of 24 normal pancreatic tissue samples, whereas all other normal tissue samples from thirteen other organs (80 samples) remained totally unmethylated. Similarly in application to PDAC, this marker effectively indicated 25 PDAC among 151 other common adenocarcinomas with values of 100%, 98.7%, 92.6%, and 100% in sensitivity, specificity, positive predictive value and negative predictive value, respectively. In summary, we have demonstrated that this epigenetic site-specific marker has high potential for pancreatic tissue identification and can be applied in PDAC diagnosis.

Middle ear myoclonus cured by selective tenotomy of the tensor tympani: strategies for targeted intervention for middle ear muscles.

OBJECTIVE: To describe a case of middle ear myoclonus that was successfully cured by selective transection of the tensor tympani (TT) without sectioning the stapedius tendon (ST) and to review previously reported cases, elucidating precipitating factors for interventions targeting middle ear muscles. DATA SOURCES: One case we encountered and a recent systematic review published in 2012. STUDY SELECTIONS: In addition to our case, 23 cases identified by the previous systematic review regarding middle ear myoclonus in which surgical interventions were conducted. DATA SYNTHESIS: Outcomes for selective tenotomy of TT or ST were analyzed focusing on the following 6 preoperative factors: 1) history of facial palsy, 2) provoking factors for tinnitus, 3) auscultation of the ear, 4) movement of the ear drum, 5) complication with palatal myoclonus, and 6) confirmation of myoclonus during surgery. Among these, the first 2 factors represented significant factors for selective tenotomy of ST (p < 0.05 and p < 0.01, respectively). Furthermore, no auscultation of the ear was significant for selective tenotomy (p < 0.01), specifically for ST. Confirmation of muscle contraction during surgery contributed significantly (p < 0.01) to targeted intervention, but selective tenotomy of TT was successfully performed in 3 cases without such confirmation by confirming variations in compliance with tympanometry CONCLUSION: Assessment of the history of facial palsy, provoking factor of tinnitus, auscultation of the ear, and confirmation of myoclonus during surgery appear helpful in predicting which middle ear muscle is undergoing myoclonus. Furthermore, long-time-based tympanometry offers objective information for planning targeted intervention for middle ear muscles and clarifying clinical outcomes.

New treatment for invasive fungal sinusitis: three cases of chronic invasive fungal sinusitis treated with surgery and voriconazole.

Invasive fungal sinusitis is a relatively rare disease and can be divided into acute fulminant, chronic, and granulomatous invasive fungal sinusitis. The conventional treatment is radical surgery combined with systemic amphotericin B administration, but the poor prognosis and unestablished treatment options require a better therapeutic strategy. We report three cases of chronic invasive fungal sinusitis successfully treated with a combination of surgery and voriconazole, a new antifungal agent, with good responses in all patients. Voriconazole administration could form the basis for a new standard treatment for invasive fungal sinusitis.

Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene expression profiles of pancreatic ductal cells.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1-positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing >44 000 probe sets. Application of both Welch's analysis of variance and effect size-based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3-D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome-based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC.

Retroviral expression screening of oncogenes in natural killer cell leukemia.

Aggressive natural killer cell leukemia (ANKL) is an intractable malignancy that is characterized by the outgrowth of NK cells. To identify transforming genes in ANKL, we constructed a retroviral cDNA expression library from an ANKL cell line KHYG-1. Infection of 3T3 cells with recombinant retroviruses yielded 33 transformed foci. Nucleotide sequencing of the DNA inserts recovered from these foci revealed that 31 of them encoded KRAS2 with a glycine-to-alanine mutation at codon 12. Mutation-specific PCR analysis indicated that the KRAS mutation was present only in KHYG-1 cells, not in another ANKL cell line or in clinical specimens (n=8).

High-throughput screening of genome fragments bound to differentially acetylated histones.

Although acetylation-deacetylation of histones contributes to regulation of gene expression, few methods have been available to determine the whole-genome histone acetylation profile in specific cells or tissues. We have now developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genome fragments embedded in histones subject to differential acetylation. This DCS screening was applied to a human gastric cancer cell line incubated with or without an inhibitor of histone deacetylase (HDAC) activity, resulting in the rapid identification of more than 250 genome fragments. Interestingly, a number of cancer-related genes were revealed to be the targets of HDAC in the cancer cells, including those for tumour protein 73 and cell division cycle 34. Such differential acetylation of histone was also shown to be linked to the regulation of transcriptional activity of the corresponding genes. Among the isolated genome fragments, 94% (32/34) of them were confirmed to be bound to differentially acetylated histones, and the genes corresponding to 78% (7/9) of them exhibited differential transcriptional activity consistent with the level of histone acetylation. With its high fidelity, the DCS method should open a possibility to rapidly compare the genome-wide histone acetylation profiles and to provide novel insights into molecular carcinogenesis.

A phase II study of S-1 in patients with metastatic breast cancer--a Japanese trial by the S-1 Cooperative Study Group, Breast Cancer Working Group.

BACKGROUND: S-1 is a newly developed novel oral dihydrouracil dehydrogenase inhibiting fluoropyrimidine drug consisting of 1 M tegafur (FT), 0.4 M 5-chloro-2, 4-dihydroxypyrimidine (gimeracil), and 1 M potassium oxonate (oteracil), with efficient antitumor activity and low gastrointestinal toxicity which is widely used in Japan against advanced gastric, head and neck cancers. We investigated its clinical efficacy against metastatic breast cancer. METHODS: A non-blind phase II study was carried out to evaluate the efficacy and toxicity in metastatic breast cancer patients. Patients with measurable metastasis foci (n=111) were enrolled, and 108 patients were regarded as eligible. S-1 was administered orally at a standard dose of 80 mg/m2/day b.i.d. One course consisted of 28 consecutive days of administration followed by a 14-day rest, and courses were repeated up to six times. RESULTS: Among the eligible patients, 10 had a complete response and 35 had a partial response, with an overall response rate (CR+PR) of 41.7% (95% confidence interval: CI, 32.3-51.5%). The incidences of toxicity (> or =grade 3) were neutropenia 9.1%, anemia 0.9%, anorexia 3.6%, stomatitis 1.8%, nausea/vomiting 1.8%, diarrhea 0.9%, and fatigue 2.7%, however no treatment-related deaths were observed. The median survival time was 872 days (95% CI, 572-1,110 days). There was no difference in response rate or toxicity between the under 65-year-old group and the older group. CONCLUSION: S-1 was demonstrated to have high efficacy with low gastrointestinal toxicity even in older patients and will be a promising new chemotherapy drug for metastatic breast cancer.

DNA microarray analysis of dysplastic morphology associated with acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) develops de novo or secondarily to either myelodysplastic syndrome (MDS) or anticancer treatment (therapy-related leukemia, TRL). Prominent dysplasia of blood cells is apparent in individuals with MDS-related AML as well as in some patients with TRL or even with de novo AML. The clinical entity of AML with multilineage dysplasia (AML-MLD) is likely to be an amalgamation of MDS-related AML and de novo AML-MLD. The aim of this study was to clarify, by the use of high-density oligonucleotide microarrays, whether these subcategories of AML are intrinsically distinct from each other. MATERIALS AND METHODS: The AC133+ hematopoietic stem cell-like fractions were purified from the bone marrow of individuals with de novo AML without dysplasia (n = 15), AML-MLD (n = 11), MDS-related AML (n = 11), or TRL (n = 2), and were subjected to the synthesis of cRNA which was subsequently hybridized to microarray harboring oligonucleotide corresponding to more than 12,000 probe sets. RESULTS: We could identify many genes whose expression was specific to these various subcategories of AML. Furthermore, with the correspondence analysis/three-dimensional projection strategy, we were able to visualize the independent, yet partially overlapping, nature of current AML subcategories on the basis of their transcriptomes. CONCLUSION: Our data indicate the possibility of subclassification of AML based on gene expression profiles of leukemic blasts.

Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas.

Activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas (CRCs), in which mutations of BRAF and KRAS are mutually exclusive. Previously, we reported that hypermethylation of hMLH1 might play an important role in the tumorigenesis of right-sided sporadic CRCs with MSI showing less frequency of KRAS/TP53 alteration. Therefore, we have assumed that BRAF mutations might be highly associated with hMLH1 methylation status rather than MSI status. In this study, mutations of BRAF and KRAS and their relationship with MSI and hMLH1 methylation status were examined in 140 resected specimens of CRC. The methylation status was classified into 3 types: full methylation (FM), partial methylation (PM) and nonmethylation (NM). Only FM closely linked to reduced expression of hMLH1 protein. BRAF mutations were found in 16 cases (11%), all leading to the production of BRAF(V599E). As for MSI status, BRAF mutations were found in 43% of MSI+ and 4% of MSI- cases (p < 0.0001). Among the MSI+ individuals, BRAF mutations were more frequent in cases with hMLH1 deficiency (58%) than those with hMSH2 deficiency (0%; p=0.02). Moreover, they were found in 69% of FM, 4% of PM and 4% of NM, revealing a striking difference between FM and the other 2 groups (FM vs. PM or NM; p < 0.0001). These findings suggest that BRAF activation may participate in the carcinogenesis of sporadic CRCs with hMLH1 hypermethylation in the proximal colon, independently of KRAS activation.

DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic stem cells. Despite the high incidence of MDS in the elderly, effective treatment of individuals in its advanced stages is problematic. DNA microarray analysis is a potentially informative approach to the development of new treatments for MDS. However, a simple comparison of 'transcriptomes' of bone marrow mononuclear cells among individuals at distinct stages of MDS would result in the identification of genes whose expression differences only reflect differences in the proportion of MDS blasts within bone marrow. Such a 'population shift' effect has now been avoided by purification of haematopoietic stem-like cells that are positive for the cell surface marker AC133 from the bone marrow of healthy volunteers and 30 patients at various stages of MDS. Microarray analysis with the AC133+ cells from these individuals resulted in the identification of sets of genes with expression that was specific to either indolent or advanced stages of MDS. The former group of genes included that for PIASy, which catalyses protein modification with the ubiquitin-like molecule SUMO. Induction of PIASy expression in a mouse myeloid cell line induced apoptosis. A loss of PIASy expression may therefore contribute directly to the growth of MDS blasts and stage progression.

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a "population-shift" effect, the pancreatic ductal epithelial cells were purified by MUC1-based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background-matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC-specific markers, including those for AC133 and carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real-time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting.

Randomized controlled trial comparing oral doxifluridine plus oral cyclophosphamide with doxifluridine alone in women with node-positive breast cancer after primary surgery.

PURPOSE: We compared the therapeutic usefulness of doxifluridine (5'-DFUR) alone and a combination of 5'-DFUR plus cyclophosphamide (CPM), both of which are considered effective against advanced and recurrent breast cancer, to determine which treatment is more beneficial as postoperative adjuvant chemotherapy. PATIENTS AND METHODS: A total of 1,131 women with node-positive primary breast cancer were randomly assigned after primary surgery to receive 5'-DFUR alone or 5'-DFUR plus CPM. All patients initially received 5'-DFUR in an oral dose of 1,200 mg/d for 4 weeks, starting 4 weeks after surgery. Chemotherapy was then not given for 2 weeks. Patients in the 5'-DFUR group subsequently received five 4-week cycles of treatment consisting of oral 5'-DFUR (1,200 mg/d) for the first 2 weeks and no chemotherapy for the next 2 weeks. Those assigned to the 5'-DFUR plus CPM group also received oral CPM 100 mg/d for the first 2 weeks and no chemotherapy for the next 2 weeks. Women 50 years or older concurrently received 20 mg/d of tamoxifen for 2 years in both groups. RESULTS: Of the 1,088 eligible women, 546 were assigned to receive 5'-DFUR alone and 542 were assigned to receive 5'-DFUR plus CPM. Overall disease-free survival was significantly better in women who received 5'-DFUR plus CPM than in those who received 5'-DFUR alone (log-rank test, P =.021). Toxic effects occurred in 20.0% of patients (109 of 546) in the 5'-DFUR group and 32.3% of patients (175 of 542) in the 5'-DFUR plus CPM group (chi(2) test, P <.001). CONCLUSION: Combination therapy with 5'-DFUR plus CPM is more effective in preventing recurrence than 5'-DFUR alone.

DNA microarray analysis of T cell-type lymphoproliferative disease of granular lymphocytes.

Lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by the clonal proliferation of large granular lymphocytes of either T- or natural killer cell origin. To better understand the nature of T cell-type LDGL, we purified the CD4-CD8+ proliferative fractions from LDGL patients (n=4) and the surface marker-matched T cells isolated from healthy volunteers (n=4), and compared the expression profiles of 3456 genes using DNA microarray. Through this analysis, we identified a total of six genes whose expression was active in the LDGL T cells, but silent in the normal ones. Interestingly, expression of the gene for interleukin (IL) 1beta was specific to LDGL T cells, which was further confirmed by the examination of the serum level of IL-1beta protein. Given its important role in inflammatory reactions, the disease-specific expression of IL-1beta may have a causative relationship with the LDGL-associated rheumatoid arthritis. Spectratyping analysis of the T-cell receptor repertoire also proved the monoclonal or oligoclonal nature of LDGL cells. These data have shown that microarray analysis with a purified T-cell subset is an efficient approach to investigate the pathological condition of Tcell-type LDGL.

The effect of adjuvant 5'-deoxy-5-fluorouridine in early stage breast cancer patients: results from a multicenter randomized controlled trial.

To assess the efficacy of 5'-DFUR, an intermediate of capecitabine, for adjuvant treatment of early breast cancer, we conducted an open-labeled multi-center randomized controlled trial to compare postoperative 5'-DFUR treatment with surgery alone. We enrolled 1217 primary breast cancer patients and randomly assigned them into two treatment groups; one received six-month postoperative 5'-DFUR treatment by consecutive or intermittent administration, and the other surgery alone. Follow-up surveys were conducted once a year for all subjects simultaneously and examined their outcome/presence or absence of the cancer recurrence. The central study committee reviewed all follow-up data and judged the recurrence data to be used for the analysis. Eight-year follow-up data showed no significant differences in relapse-free and overall survival between the two groups, and 5'-DFUR treatment regimen showed an extremely high tolerance. Possible explanations are discussed for the finding of no significant survival difference between adjuvant 6-month 5'-DFUR monotherapy and surgery alone in early breast cancer.