Validation of Pyrosequencing for the Analysis of KRAS Mutations in Colorectal Cancer

The use of antibodies against epidermal growth factor receptor( EGFR) in conjunction with conventionalchemotherapy for metastatic colorectal cancer (CRC) in patients with KRAS wild-type tumors has beenproven to be efficacious. Recently, KRAS testing prior to anti-EGFR therapy has become mandatory formetastatic CRC patients. Although newly developed pyrosequencing is expected to be one of the highthroughput procedures detecting such mutations, the accuracy of the procedure has not been well evaluated.In the present study, we aimed to validate the accuracy, especially the potential for a false-negative result,in detecting KRAS mutations by pyrosequencing using cultured tumor cells. DNA extracted from culturedìNOZî gallbladder cancer cells( known to contain KRAS mutation G12V) at concentrations of 1%, 5%, 10%, and 25%, as well as 2 DNA samples extracted from a resected CRC specimen( known to contain anotherKRAS mutation, G12C) at concentrations of 5% and 25%, were prepared. We analyzed KRAS mutationalstatus and nonexistent and/or nonfunctional mutations of these 6 samples using pyrosequencing. TheKRAS mutation detection rates in the 4 NOZ samples( 1%, 5%, 10%, and 25%) were 0.37%, 2.79%, 5.28%,and 13.85%, respectively. Some artifacts of KRAS mutations unlikely to be present were detected in 1%samples of NOZ at a rate similar to that of the G12V mutation( G12C, 0.29%;G13C, 0.42%). Although theKRAS mutation G12C was detected at rates of 1.26% and 6.49% in samples with 5% and 25% DNA extractedfrom resected CRC specimen, respectively, no other type of KRAS mutation was detected in suchsamples. Pyrosequencing could not detect KRAS mutations correctly in the sample containing 1% DNA.This might cause false negatives. A sample mutated DNA concentration of at least 5% was necessary forprecise analyses by this procedure

Pathological Approach for Surveillance of Ulcerative Colitis-associated cancer:Usefulness of Immunohistochemistry for p53

The patients with long-standing ulcerative colitis( UC) have a high-risk of neoplastic lesions in the colonicmucosa. The UC-associated neoplastic lesion is difficult to detect by endoscopic examination or diagnosehistologically. In the present study, we aimed to clarify whether immunohistochemistry for p53 is useful todiscriminate the UC-associated neoplasia from inflammed regenerating epithelium. Tissue samples were obtainedfrom colorectomy specimens from 20 patients with long-standing UC (range 6-29 years). The surfaceof microstructure of the tissues was observed by stereomicroscopy, and the sections were examined usingimmunohistochemistry for p53. All of T2-4 carcinomas were detectable by endoscopic examination beforesurgery, whereas considerable number of dysplasias (52.5%), Tis carcinomas (33.3%), and T1 carcinomas(60.0%) were undetectable. Fifty-three of 67 UC-associated neoplastic lesions (79.1%) were of flat-typemacroscopically. The detection rate of flat-type neoplasias( 45.3%) was significantly lower than that of protrudingones (100%). The positivity of p53 overexpression was 0 % in UC-II, 52.5 % in UC-III, and 70.4 %in UC-IV, respectively. UC-II lesions had lower positivity of p53 overexpression than UC-III( P=0.027) or-IV lesions( P=0.003). Immunohistochemical analysis of p53 protein is useful to discriminate the UC-associatedneoplasia from inflammed regenerating epithelium

イ ネンマク ジョウヒ サイボウ ノ トレフォイル ファクター (TFF) オヨビ ムチンコア タンパク (MUC) ハツゲン ニ タイスル SOX2 ノ エイキョウ

SOX2 は SRY-related HMG box ファミリーに属する転写因子であり,SOX2の胃粘膜上皮細胞における発現は以前より報告されているが,胃特異的遺伝子発現へのSOX2の影響は十分には解明されていない.本研究では胃癌由来細胞株(MKN45細胞,AGS細胞)を用い,胃粘膜上皮細胞におけるTFF およびMUC の発現に対するSOX2の影響について検討を行った.両細胞株においてSOX2蛋白の発現をウエスタンブロット法で確認し,TFF(TFF1,TFF2,TFF3)およびMUC(MUC2,MUC5AC,MUC6)それぞれのレポーター遺伝子を用いた解析を行った.SOX2の過剰発現で影響を受けたのはTFF1とMUC6であり,TFF1発現は有意に低下,逆にMUC6発現は有意に増加することが認められた.リアルタイム定量的RT-PCR により,内因性のTFF1およびMUC6 mRNAもSOX2過剰発現により同様の効果を受けることを確認した.本研究の結果は,SOX2が消化管において胃粘膜に特異的に発現しているTFF(TFF1,TFF2)およびMUC(MUC5AC,MUC6)の発現を一様に誘導しているのではなく,異なった影響を与えていること,また,本来,消化管において腸粘膜に特異的に発現しているTFF3,MUC2の胃粘膜における発現の抑制に機能しているのではないことを示唆している.今後,他の様々な因子との相互作用を念頭に置いた上で,胃粘膜上皮におけるSOX2の機能をさらに解明していく必要があると考えられる.Although previous studies showed the expression ofSOX2, a SRY-related HMG box family transcription factor,in gastric epithelial cells, little is known about the regulationof stomach-specific gene expression by SOX2. In this study,by using gastric cancer cell lines (MKN45 and AGS) as amodel system, we examined the effects of SOX2 on the expressionof trefoil factor family peptides( TFF1, TFF2, andTFF3) and mucin core proteins (MUC2, MUC5AC, andMUC6). Reporter gene analyses showed that forced overexpressionof SOX2 proteins significantly down-regulatesthe transcription of TFF1 and up-regulates the transcriptionof MUC6. TFF2 and MUC5AC transcription wasslightly repressed by SOX2. TFF3 and MUC2 transcriptionwas slightly up-regulated by SOX2 in MKN45 cells butslightly down-regulated in AGS cells. Real-time quantitativeRT-PCR also confirmed the down-regulation of endogenousTFF1 mRNA expression and up-regulation of MUC6mRNA expression by SOX2 overexpression. These resultssuggest that SOX2 differentially affects the expression ofstomach-specific TFFs (TFF1 and TFF2) and MUCs(MUC5AC and MUC6), and that SOX2 may not be a keyfactor for the repression of the expression of intestine-specificTFF (TFF3) and MUC(MUC2) in gastric epithelialcells

カン サイボウガン ニ タイスル ラジオハ ショウシャク リョウホウ ゴ ノ キョクショ サイハツ ヨソク インシ ニ ツイテノ ケントウ

肝細胞癌(HCC)に対する経皮的ラジオ波焼灼療法(RFA)後の局所再発因子について検討した.対象はHCC43例,45結節,平均年齢66.5±10.3歳,男性29例,女性14例であった.病因はHBV4例,HCV38例,原因不明1例.平均腫瘍径は2.2±0.7(1.0～4.5)cm,単発例が14例,多発例が29例であった.RFA単独治療群が20結節,他の内科的治療併用群が25結節.治療後にダイナミックCTを施行し,遺残なしと判定された結節について多変量解析にて局所再発因子を検討した.局所再発率の算出にはKaplan-Meier法を用いた.効果判定のCTは43例45結節中,腎不全合併例2例2結節を除く43結節に施行し,39結節(90.7%)が遺残なしと判定された.遺残が疑われた4例は,他疾患合併などの理由から追加治療は施行されなかった.遺残なし群(39例)の1年,2年,3年の局所再発率は,20.5%,27.5%,27.5%,これらのうち単発例14結節の局所再発率は1,2,3年ともに16.3%であった.39例における多変量解析の結果,年齢,性差,腫瘍径,臨床病期,併用療法の有無,治療前のAFP値はいずれも統計学的には局所再発に寄与せず,治療前のPIVKA-II値のみに統計学的な有意差を認め,HCCの局所再発への関与が示唆された.We have investigated the factors underlying the local recurrence of hepatocellular carcinoma (HCC) after percutaneous radiofrequency ablation (RFA). Forty-five nodules in 43 HCC patients, consisting of 29 men and 14 women with a mean age of 66.5±10.3 years, were studied. The cause of HCC was HBV in 4 patients, HCV in 40, and cryptogenic in 1. The mean tumor diameter was 2.2±0.7cm (1.0-4.5). Fourteen patients had single HCC nodule and 29 patients had multiple HCC nodules. Two treatment groups were set up: the RFA alone group of 20 nodules and the combined group of 25 nodules that were treated with another medical therapy together with RFA. After treatment, all nodules were evaluated by dynamic CT, and those judged as having "no residual tumor" were examined for local recurrence factors using multivariate analysis. The recurrence rate was calculated by the Kaplan-Meier method. CT for outcome assessment, carried out in 43 nodules in 41 patients excluding 2 patients (2 nodules) with renal failure revealed that 39 nodules (90.7%) had no residual tumor. The 4 nodules, suspected of having a residual tumor, were not additionally treated because of the presence of complications. The local recurrence rates at 1, 2 and 3 years after treatment in the "no residual tumor" group (n=39) were 20.5, 27.5 and 27.5%, respectively. The multivariate analysis revealed that neither of age, sex, tumor diameter, clinical stage, combined therapy, nor AFP value statistically contributed to local recurrence. Only PIVKA-II value was a statistically independent factor for local recurrence of HCC. In conclusion, detailed examination with dynamic CT appears necessary for the assessment of RFA treatment for HCC. PIVKA-II value is likely the most important factor to predict the local recurrence of HCC after RFA

イネンマク ジョウヒ サイボウ ノ trefoil factor family ハツゲン ニ オヨボス indomethacin ノ エイキョウ

Trefoil factor family(TFF)に属するペプチド(TFF1,TFF2,TFF3)は,消化管粘膜の健常性維持,傷害修復過程に重要な役割を果たしているが,その発現調節については不明の部分が多い。本研究では,胃癌由来細胞株であるMKN45細胞を用いて,胃粘膜傷害の起因因子であるNSAIDsのTFF発現に対する影響をリアルタイム定量的RT-PCR法で解析した。MKN45細胞では,TFF1の発現レベルが最も高く,次いでTFF2であり,TFF3の発現レベルは非常に低かった。IndomethacinはTFF1,TFF3の発現に対しては有意な影響を与えず,TFF2の発現は濃度依存性に上昇させた。IndomethacinによるTFF2発現誘導は他の胃癌由来細胞株(AGS,JR)でも認められ,また,他のNSAIDs (aspirin,NS-398)もMKN45細胞においてTFF2発現を上昇させた。このindomethacinの効果は外来性のPGE2の投与では拮抗されなかった。以上の結果から,NSAIDsはそのCOX抑制作用とは別の機構を介して胃粘膜上皮細胞のTFF2発現を誘導していることが示唆された。NSAIDsの投与によるTFF2発現レベルの上昇は,NSAIDs起因性胃粘膜傷害の発生をある程度防御している可能性が考えられるAlthough trefoil factor family (TFF) peptides (TFF1, TFF2, and TFF3) are assumed to play important roles in the protection of gastrointestinal mucosa, regulatory mechanisms of TFF expression are poorly understood at present. In the present study, we examined the effect of indomethacin, a non-selective inhibitor of cyclooxygenase (COX), on the expression of TFF mRNA expression in MKN 45 gastric cells by real-time quantitative RT-PCR method. In MKN 45 cells, relative expression level of TFF1, TFF2, and TFF3 mRNA was 617 : 12 : 1 in the control condition. Indomethacin up-regulated the expression level of TFF 2 in a dose-dependent manner, whereas TFF 1 and TFF 3 expression levels were not significantly affected. Luciferase reporter gene assay confirmed this stimulatory effect of indomethacin on TFF 2 expression. Other COX inhibitors, such as aspirin and NS -398, also up-regulated TFF 2 expression and indomethacin-induced up-regulation of TFF2 expression was also observed in other gatric cell lines, such as AGS and JR. Externally applied PGE2 did not antagonize the effect of indomethacin on TFF2 expression. Since TFF peptides play a critical role in gastric mucosal protection, indomethacin-induced TFF2 may reduce the degree of gastric mucosal damage induced by indomethacin. The present results also suggest that the effect of indomethacin on TFF2 expression is COX-independent

イ ネンマク ジョウヒ サイボウ ニオケル trefoil factor family 1 (TFF1) ハツゲン ニ エイキョウ オ アタエル インシ ニ カンスル ケントウ

乳癌由来細胞株MCF-7でエストロゲン誘導遺伝子として見出されたTFF1は,生理的には胃粘膜に発現し重要な粘膜防御因子として機能している.本研究では,胃癌由来細胞株MKN45を用い,胃におけるTFF1発現調節機構について検討した.MCF-7ではTFF1発現はエストロゲン依存的であったが,MKN45ではTFF1の発現はエストロゲン非感受性であり,TFF1遺伝子プロモーター上のエストロゲン応答配列よりもさらに近位側の配列が,MKN45におけるTFF1発現に大きな影響を与えていた.MKN45にもエストロゲン受容体(ERα,ERβ)の発現は認められたが,MCF-7と比べるとERαの発現レベルが低く,ERαを強制的に発現させるとMKN45でもTFF1発現がエストロゲン感受性となった.胃粘膜でのTFF1発現は粘膜損傷時に上昇することが知られているので,このような誘導性のTFF1発現機構を検討する目的でphorbol ester (TPA),TNF-αの影響をみたところ,TPAはAP-1を介して,TNF-αはNF-κBを介してTFF1の発現を上昇させることが示唆された.これらの結果より,胃粘膜上皮細胞ではTFF1発現はエストロゲン非依存性であり,また,AP-1やNF-κBが誘導性の発現調節に関与していると考えられた.Trefoil factor family 1 (TFF1) is a protease-resistant polypeptide expressed at a high level in gastric epithelial cells and plays a critical role in the defense and repair of gastric mucosa. In the present study, we examined the regulatory mechanisms of gastric TFF1 expression using a gastric cancer cell line, MKN45. Since TFF1 was originally discovered as an estrogen-inducible gene in a breast cancer cell line, MCF-7, MCF-7 was also examined for comparison. The promoter sequence of human TFF1 gene (-953 to +34) was cloned into pGL3 basic vector to make a TFF1 reporter gene and several deletion mutant reporters were also made. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RT- PCR. In MCF- 7 cells, basal TFF1 expression was dependent on the presence of an estrogen-responsive element (ERE) (-406), while deletion of ERE had no significant effect on the reporter gene expression in MKN45 cells. In MKN45 cells, deletion of more proximal region (-393 to -365) significantly affected the reporter gene expression. Compared to estrogen receptor β (ER β), the expression level of estrogen receptor α (ER α) was low in MKN45 cells and we found that TFF1 expression became estrogen-sensitive when ER a was overexpressed in MKN45 cells. Phorbol-12-miristate-13-acetate (TPA) and tumor necrosis factor-α (TNF-α) up-regulated the expression of endogenous TFF1 mRNA and TFF1 reporter genes. A series of reporter gene experiments suggested that AP-1 site (-338) and NF-κB sites (-251, -230) are involved in the action of TPA and TNF-α, respectively. These results suggest that, compared to MCF-7 cells, the basal TFF1 expression is differently regulated in gastric epithelial cells, and that up-regulation of TFF1 expression, which is often observed at the site of gastric mucosal lesions, may be mediated by AP-1 and/or NF-κB

ガクセイ アンケート チョウサ ニヨル コウギ ヒョウカ ト ソノ モンダイテン

近年,医学教育改革が目まぐるしく行われている.その中で,学生による講義評価が行われはじめているが,まだその報告は少ない.獨協医科大学では学年委員会が主体となって,講義・実習に対するアンケート調査を第1学年から第4学年まで行った.その結果,講義に対して「十分に満足している」という学生は,どの学年においても多くはなかった.さらに,「わかりやすい講義」をしている教員の共通点は,板書・プリント・シラパスを活用しているという点であった.実習に関しては,「基礎医学」に関わる実習に対しての評価が高かった.今後の医学教育において,学生の意見の反映がよりよい講義につながるものと考えられる.Extensive innovations in medical education have been carried out in recent years in our country. Although student evaluations of lectures have been conducted, there have been few reports on this issue. Thus, our committee consisting of representatives of every grade played a leading role in making and evaluating a questionnaire investigation of the lectures and the experiments. The investigation covered students from freshmen to seniors. We found that there were few students in each grade who were satisfied with the lectures. Students gave high marks to lecturers who wrote in detail on the blackboard, distributed well-organized documents, and made practical use of the syllabus. Experiments concerning the preclinical course were also well accepted. We consider that this sort of investigation will be influential in improving the quality of lectures

ホンガク ニオケル シンリョウ サンカガタ リンショウ ジッシュウ ニ カンスル コウサツ : モデル・コア・カリキュラム ノ ドウニュウ ト シンリョウ サンカガタ リンショウ ジッシュウ ノ アリカタ

獨協医科大学では,医学教育モデル・コア・カリキュラムに準じた診療参加型臨床実習を実施する予定である.平成16年度以降の臨床実習形式は46週と従来より長くなり,診療参加型となる.これを円滑に導入するためには,臨床実習開始以前から学生の準備教育を充実させ,コ・メディカルを含めた実習にかかわるすべてのスタッフに,臨床実習の意義を徹底させることが必要である.現実的な問題点として,現状では実習教育にたずさわる教員数の絶対的な不足が挙げられ,マンパワーの補充を目的とした組織的な対応を早急に検討することが望まれる.(本論文は,第8回医学教育ワークショップCグループの発表内容を中心に加筆検討を加えたものである.)In introducing clinical clerkship incorporating a core curriculum at Dokkyo University School of Medicine, its problems and concrete plans were evaluated. For smooth introduction of this plan, improving the preliminary education of students before the beginning of clinical training and assuring the understanding of all the staff members involved in clinical training including co - medicals about its significance were considered to be necessary. As a practical problem, a lack of instructors who can be involved in clinical training was raised, and prompt evaluation of systematic measures to increase the staff was strongly recommended