松下 一之

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

The present study was planned to identify novel serum antibody markers for digestive organ cancers. We have used screening by phage expression cloning and identified novel fourteen antigens in this experiment. The presence of auto-antibodies against these antigens in serum specimens was confirmed by western blotting. As for auto-antibodies against fourteen antigens, AlphaLISA (amplified luminescence proximity homogeneous assay) assay was performed in the sera of gastrointestinal cancers patients to confirm the results. Serum antibody levels against these fourteen recombinant proteins as antigens between healthy donors (HD) and esophageal squamous cell carcinoma (ESCC) patients, gastric cancer (GC), or colon cancer (CC) were compared. The serum levels of all fourteen auto-antibodies were significantly higher in ESCC and GC than those of HD. Among those auto-antibodies, except ECSA2 and CCNL2, were also detected significantly higher levels in CC than those of HD. Receiver operating curve (ROC) revealed similar results except CCNL2 in CC. AUC values calculated by ROC were higher than 0.7 in auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, HS3ST1, TUBA1B, TACSTD2, AKR1C3, BAMBI, DCAF15 in ESCC, auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, TACSTD2, AKR1C3, BAMBI, DCAF15 in GC, and auto-antibodies against TPI1, HOOK2, PUF60 in CC. AUC of the combination of HOOK2 and anti-p53 antibodies in ESCC was observed to be as high as 0.8228. Higher serum antibody levels against ten antigens could be potential diagnostic tool for ESCC. Higher serum antibody levels against eight antigens could be potential diagnostic tool for GC, and serum antibody levels against three antigens could be potential diagnostic tool for CC.

近年、臨床検査の国際的な検査値の標準化の中で、各種酵素活性測定法のJSCC勧告法を、IFCC勧告法へ変更する必要性の有無について議論されている。LD活性測定は、本邦ではほとんどの施設で、JSCC勧告法に準拠した試薬を常用酵素標準物質で校正するJSCC標準化対応法が使用されており、国内の施設間差の是正に大きな効果を上げてきた。一方、国際的にはほとんどの施設で、IFCC法に準拠した方法が採用されていると考えられる。JSCC勧告法とIFCC勧告法ではLDアイソザイムの反応性に差があることから、症例によっては両法間で解離する可能性が指摘されている。今回我々は、IFCC勧告法の処方に準拠し、かつ日常検査における汎用性を高めた試薬を使用する機会を得た。本法の性能および有用性を評価することを目的として検討を行った。その結果、本法の基礎的性能は良好であり、IFCC勧告法試薬とも高い相関性を示し、解離例も認められなかった。したがって本法は、今後JSCC勧告法からIFCC勧告法への変更の必要性を議論するにあたり、基本データの再確認に有用な試薬と考えられた。(著者抄録)

With the international standardization of reference values for clinical tests in recent years, there has been discussions on the necessity of changes in various enzyme assays from the JSCC-recommended method to the IFCC-recommended method. In Japan, for the measurement of LD activity, JSCC transferable method (calibrated with JCCLS certified reference material for enzyme of JSCC method) has been used in most facilities and is markedly effective for correcting differences among institutions. However, the IFCC-recommended method is more widely adopted by institutions in other countries. Since the reactivity of LD isozyme differs between the JSCC- and IFCC-recommended methods, disagreement between the two methods in some cases has been reported. We evaluated a reagent that is based on the IFCC-recommended method (IFCC-recommended evaluation reagent) which is more versatile for routine tests. We verified the performance and usefulness of this reagent. The result of this study shows that the basic performance of this method is good, and that it is highly correlated with the IFCC-recommended method. There was no case showing any difference between the two methods. Therefore, this reagent may be useful for the confirmation of basic data in discussing the necessity of future changes from the JSCC-recommended method to the IFCC-recommended method.

There is significant debate within the diagnostics community regarding the accuracy of variant identification by next-generation sequencing and the necessity of confirmatory testing of detected variants. Because the quality threshold to discriminate false positives depends on the workflow, no regulatory standard regarding this matter has yet been published. The goal of this study was to empirically determine the threshold to perform additional Sanger sequencing and to reduce the experimental cost to a practical level. Using 278 model genes, a hybridization capture-based protocol was examined to meet the clinical requirements of low cost, high efficiency, and high-quality data. To reduce excessive false-positive detection, filtering processes were introduced to remove mismapped reads and strand-biased detection to a published best-practices pipeline. With seven samples from the 1000 Genomes Project, 2750 single-nucleotide polymorphisms and 142 insertions/deletions were identified by our designed workflow. Compared with variants registered in the single nucleotide polymorphism database (dbSNP), a zero false-positive threshold value was determined (quality score > 1000). The variants satisfying these criteria accounted for 95.6% of single-nucleotide polymorphisms and 50.7% of insertions/deletions. Except for deletions located within the highly repeated sequences, the workflow achieved 100% sensitivity. The established threshold allowed us to discriminate between convincing variants and those requiring validation, a design that reconciles the competing objectives of cost minimization and quality maximization of clinical gene panel testing.

Familial adenomatous polyposis (FAP) is an inherited disorder characterized by numerous colorectal adenomatous polyps with predisposition to the development of colorectal cancer (CRC). Here, we conducted genome-wide DNA methylation analysis of FAP neoplasms, including seven cancer samples and 16 adenoma samples, using an Infinium 450K BeadArray. As controls for sporadic colorectal neoplasms and mucosae, we used Infinium 450k data from 297 CRC samples, 45 colorectal adenoma samples, and 37 normal mucosa samples with reference to The Cancer Genome Atlas and other databases. Unsupervised two-way hierarchical clustering analysis of FAP and sporadic CRC/adenoma revealed that CRC was classified into four DNA methylation epigenotypes (MEs): high-ME (HME), intermediate-ME (IME), low-ME (LME), and normal-like ME (NME). Five FAP neoplasms (two cancer and three adenoma) were clustered with IME, whereas 18 FAP neoplasms (five cancer and 13 adenoma) were clustered into NME. IME FAP neoplasms significantly correlated with KRAS mutations, similar to sporadic CRC. Within IME cases, however, aberrant DNA methylation was significantly less frequent in FAP neoplasms than sporadic neoplasms, and these unmethylated genes included WNT family genes and several types of oncogenes. In summary, FAP neoplasms were classified into at least two molecular subtypes, i.e., NME in the majority of cases showing mostly no aberrant methylation and IME in some cases accompanied by KRAS mutations but less frequent aberrant DNA methylation than sporadic neoplasms, suggesting that FAP may follow a tumorigenesis pathway different from that of sporadic CRC.

遺伝子関連検査と医療ビッグデータについては、とくにがん医療の分野で国内外に大きな動きがある。とりわけ、がんゲノム医療はこれまでの科学的な因果律に基づく研究成果から、確率・統計情報に基づいたデータ処理による医療に大きくパラダイムシフトしている。平成30年(2018)にはがん遺伝子パネル検査は先進医療Bから保険収載への移行が予定されており、同時に統合データベースとしての医療ビッグデータの構築の動きが進んでいる。人工知能を用いた補助診断ツールが実際のがんゲノム医療に役立つことがすでに臨床現場で行われつつある。このように胚細胞や体細胞変異の情報を最初に明らかにして医療に役立てる考え方は"genotype first"とよばれており、がん医療にとどまらない。一方、その実現には多くの課題があるため、本稿では「ゲノム解析技術の進歩と臨床検査への応用」と題して、近未来のわが国の遺伝子関連検査と医療ビッグデータの発展に必要な"倫理・法律(改正個人情報保護法)とゲノム解析"、"保存臨床検体や遺伝関連検査の精度保証"、"重要な人材育成"、それらの基盤(社会インフラ)としての"バイオバンク"などについて現況をまとめた。さらに遺伝子一括(パネル)検査とそれに伴う分子標的治療薬の選択、およびゲノム医療をわが国で実現するために解決するべき諸課題について簡単に概説を加えた。(著者抄録)

Anastomotic failure of a gastric tube inserted for reconstruction following esophagectomy, which is relatively rare, causes pleural infection and persistent pleural irritation, leading to communication with the pulmonary parenchyma. Although several interventions have been reported to treat such broncho-gastric tube fistulas, refractory cases remain. We herein report the successful treatment by endo-scopic bronchial occlusion with an endobronchial Watanabe spigot in 2 patients who suffered from the above complication..

Ashizawa K, Murata S, Terada T, Ito D, Bunya M, Watanabe K, Teruuchi Y, Tsuchida S, Satoh M, Nishimura M, Matsushita K, Sugama Y, Nomura F, Journal of microbiological methods, 2017, vol. 139, pp. 54-60, 2017

Nishimura M, Ueda M, Ebata R, Utsuno E, Ishii T, Matsushita K, Ohara O, Shimojo N, Kobayashi Y, Nomura F, BMC medical genetics, 2017, vol. 18, no. 1, pp. 66, 2017

Genetic testing for breast cancer predisposing genes, BRCA1 and BRCA2, can take advantage for early identification of carriers with pathogenic germline mutations. However, conventional approaches based on Sanger sequencing are laborious and expensive. Next-generation sequencing technology has a great impact on investigation of medical genomics and now applied clinical genetics. We provide a protocol based on a pool and capture method followed by high-throughput sequencing, which realizes a rapid, high-quality, high-accuracy and low-cost testing for mutations in BRCA1 and BRCA2 by using small amounts of input DNA. Custom capture probes were designed for 195 kb regions encompassing the entire BRCA1 and BRCA2. DNA libraries of 96 samples with distinct indices were pooled before hybridizing to the capture probes, which largely reduced labor and cost. The captured library was run on the Illumina MiSeq sequencer. We applied the method to 384 Japanese individuals including 11 patients with breast cancer whose mutation statuses had been determined by standard clinical testing and 373 individuals from a general population. 99.99% of coding exons and their 20 bp flanking regions were covered with a minimum of 20 reads and the average depth was 179.5, supporting confident variant detection. The sequencing method rendered concordant results for 11 patients with breast cancer compared with the standard clinical testing including nine mutations in eight patients. Among 373 individuals from the general population, novel stop gain and frameshift deletion in BRCA2 were identified, which led to truncated protein and were most likely to be pathogenic. The result suggests the importance of a large-scale population-wide screening for carriers of mutations in these genes.

Gastric cancer is the second leading cause of cancer death in the world, and effective diagnosis is extremely important for good outcome. We assessed the diagnostic potential of an autoantibody panel that may provide a novel tool for the early detection of gastric cancer. We analyzed data from patients with gastric cancer and normal controls in test and validation cohorts. Autoantibody levels were measured against a panel of six tumor-associated antigens (TAAs) by ELISA: p53, heat shock protein 70, HCC-22-5, peroxiredoxin VI, KM-HN-1, and p90 TAA. We assessed serum autoantibodies in 100 participants in the test cohort. The validation cohort comprised 248 participants. Autoantibodies to at least one of the six antigens showed a sensitivity/specificity of 49.0% (95% confidence interval [CI], 39.2-58.8%)/92.4% (95% CI, 87.2-97.6%), and 52.0% (95% CI, 42.2-61.8%)/ 90.5% (95% CI, 84.8-96.3%) in the test and validation cohorts, respectively. In the validation cohort, no significant differences were seen when patients were subdivided based on age, sex, depth of tumor invasion, lymph node metastasis, distant metastasis, peritoneal dissemination, or TNM stage. Patients who were positive for more than two antibodies in the panel tended to have a worse prognosis than those who were positive for one or no antibody. Measurement of autoantibody response to multiple TAAs in an optimized panel assay to discriminate patients with early stage gastric cancer from normal controls may aid in the early detection of gastric cancer.

Hoshino I, Nagata M, Takiguchi N, Nabeya Y, Ikeda A, Yokoi S, Kuwajima A, Tagawa M, Matsushita K, Satoshi Y, Hideaki S, Cancer science, 2017, vol. 108, no. 3, pp. 308-315, 2017

Background: Bile duct stones after hepaticojejunostomy are considered a troublesome adverse event. Although percutaneous transhepatic procedures using a cholangioscopy is performed to treat these bile duct stones, a peroral endoscopic procedure using a short, double-balloon enteroscope (sDBE) is an alternative. This study aimed to compare the immediate and long-term outcomes of both treatments for bile duct stones in patients who underwent prior hepaticojejunostomy. Methods: Between October 2001 and May 2013, 40 consecutive patients were treated for bile duct stones after hepaticojejunostomy at a tertiary care hospital. Initial success with biliary access, biliary intervention-related technical success, clinical success, adverse events, hospitalization duration, and stone-free survival were retrospectively evaluated. Results: The initial success rates for biliary access were 100% (8/8) with percutaneous transhepatic cholangioscopy (PTCS) and 91% (29/32) with sDBE. In three patients in whom biliary access during initial sDBE failed, successful access with subsequent PTCS was achieved, and biliary intervention-related technical success and clinical success were eventually achieved in all 40 patients. The rate of adverse events was significantly lower with sDBE than with PTCS (10% versus 45%; p = 0.025). The median hospitalization duration for complete stone clearance was significantly shorter with sDBE than with PTCS (10 versus 35 days; p < 0.001). During the median 7.2 year or 3.1 year follow up, the probabilities of being stone-free at 1, 2, and 3 years were 100%, 73%, and 64% for PTCS and 85%, 65%, and 59% for sDBE, respectively (p = 0.919). Conclusions: sDBE was useful, with few adverse events and short hospitalization; therefore, experienced endoscopists can consider it as first-line treatment for bile duct stones in patients with prior hepaticojejunostomy.

Beppu M, Sawai S, Misawa S, Mori M, Ito S, Sogawa K, Nishimura M, Matsushita K, Nomura F, Kuwabara S, Journal of neuroimmunology, 2017, vol. 302, pp. 20-22, 2017

Takane K, Matsusaka K, Ota S, Fukuyo M, Yue Y, Nishimura M, Sakai E, Matsushita K, Miyauchi H, Aburatani H, Nakatani Y, Takayama T, Matsubara H, Akagi K, Kaneda A, Oncotarget, 2016, vol. 7, no. 51, pp. 84003-84016, 2016

Satoh M, Ishige T, Ogawa S, Nishimura M, Matsushita K, Higashi T, Nomura F, Analytical and bioanalytical chemistry, 2016, vol. 408, no. 27, pp. 7617-7627, 2016

Kobayashi S, Hoshino T, Hiwasa T, Satoh M, Rahmutulla B, Tsuchida S, Komukai Y, Tanaka T, Matsubara H, Shimada H, Nomura F, Matsushita K, Oncotarget, 2016, vol. 7, no. 50, pp. 82493-82503, 2016

Ishige T, Itoga S, Matsushita K, Nomura F, Clinica chimica acta; international journal of clinical chemistry, 2016, vol. 457, pp. 75-80, 2016

The Ras-like GTPases, RalA and RalB are members of the Ras superfamily of small GTPases. Aberrant activation of Ral is a major cause of human tumorigenesis induced by oncogenic Ras. Serum anti-RalA antibodies are induced in esophageal carcinoma patients. However, detailed comparisons of their pathological characteristics are unavailable, and conventional serum markers have not been well evaluated. Serum samples of 171 patients with esophageal squamous cell carcinoma and 73 healthy individuals were analyzed using specifically developed ELISA system for serum anti-RalA antibodies. A cut-off optical density value was fixed at 0.255 (the control mean + 2 SD). Clinicopathological characteristics and positive rates of conventional tumor markers were evaluated for seropositive patients. Overall positive rate for serum anti-RalA antibodies was 18 %, which gradually increased with the tumor stages. Although the positive rate for serum anti-RalA antibodies was comparable with that of carcinoembryonic antigen (24 %) and CYFRA21-1 (21 %), it was lower than the rate for serum p53 antibodies (31 %) and squamous cell carcinoma antigen (37 %). Although serum anti-RalA antibodies were not associated with other serum markers, it was inversely associated with serum p53 antibodies. No clear association was observed between serum anti-RalA antibodies and RalA immunoreactivity. Presence of serum anti-RalA antibodies is associated with tumor stages, but not with conventional tumor markers. Serum anti-RalA antibodies may be candidate serum markers in combination with other serum markers for esophageal squamous cell carcinoma.

甲状腺刺激ホルモン(TSH)のハーモナイゼーションについて検討した。アーキテクトTSH、エクルーシスTSH、ルミパルスTSH-III、Eテスト「TOSOH」II(TSH)について検討した。NIBSC81/565調製液の濃度に対して、ルミパルスの測定値が最も低く平均94.3%、エクルーシスの測定値も平均97.1%と低値であった。アーキテクトは平均106.1%と高値、Eテスト「TOSOH」は平均118.7%と最も高値であった。臨床検査精度管理調査用試料では、ルミパルス、アーキテクト、Eテスト「TOSOH」の3試薬で比較し、ルミパルスが低値、Eテスト「TOSOH」が高値となった。エクルーシスは4試薬の中で最も高値を示した。各試薬での測定値とAPTMとの差は換算することにより減少した。また、試薬による測定値の変動も換算することにより減少した。

Ishige T, Nishimura M, Satoh M, Fujimoto M, Fukuyo M, Semba T, Kado S, Tsuchida S, Sawai S, Matsushita K, Togawa A, Matsubara H, Kaneda A, Nomura F, Scientific reports, 2016, vol. 6, pp. 21681, 2016

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or. rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by gamma H2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIR Delta exon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P = 0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

A 59-year-old man was admitted to our hospital for treatment of a 45 mm pancreatic mass found during a medical examination. Endoscopic ultrasound-guided fine-needle aspiration cytology showed polygonal cells with pseudopapillary structures. The tumor cells were positive for nuclear/cytoplasmic beta-catenin and CD10, and negative for chromogranin A. After a tentative diagnosis of a solid pseudopapillary neoplasm, middle pancreatectomy was performed. Histologically, polygonal cells with abundant eosinophilic cytoplasm formed in the trabeculae and were immunohistochemically positive for HepPar1 and protein induced by vitamin K absence or antagonist-II. The tumor was finally diagnosed to be pancreatic hepatoid carcinoma. No recurrence occurred for 12 months, even without adjuvant chemotherapy.