Published papers

ウサギモノクローナル抗ヒト神経膠腫関連癌遺伝子ホモログ(GLI)1抗体(C68H3)を用いて、基底細胞癌(BCC)の196例を含む皮膚上皮腫瘍382例におけるGLI1の免疫染色について検討した。皮膚上皮腫瘍382例から得られた腫瘍検体378検体を対象とした。BCC症例はいずれも原発腫瘍で、99%がGLI1陽性を示した。5つの組織学的サブタイプのうち、結節型100%(101/101)、表在型98.2%(57/58)、微小結節型95.8%(23/24)、硬斑型100%(4/4)、浸潤型100%(4/4)は核染色パターンを示した。腫瘍巣の周辺に柵状のパターンを示した腫瘍細胞は、いずれも陽性に染色された。GLI1発現は正常毛包で分析し、下バルジ領域および毛芽は染色陽性であった。毛芽腫症例全例(4/4)、扁平上皮癌症例のうち12.5%(4/32)、ボーエン病症例のうち15.1%(5/33)、日光角化症症例のうち3.5%(1/28)は、GLI1の核染色パターンを示したが、すべてのporoid新生物と脂漏性角化症は陰性に染色された。

膵癌微小環境における神経周囲浸潤について間葉-上皮移行(MET)が果たしている役割について検討した。神経周囲浸潤部における膵管腺癌(PDAC)の組織学的所見である限局性分化の特徴を調べ、腫瘍METを誘発する際のシュワン細胞(SC)の役割を調べた。168のPDAC組織で、E-カドヘリン(CDH)、SMAD3およびビメンチンの神経周囲浸潤部における腫瘍分化と免疫組織化学的発現を調べた。168の腫瘍のうち124(74%)は、CDH膜の発現の増強とSMAD3の核内蓄積の減少により限局性分化を示した。分化度1/2腫瘍を有する115のPDACの中で、限局性分化を示す腫瘍は示さないものより生存率が低かった。SCと共培養したPDAC細胞は、シート様の外観、CDH発現の増加、SMAD3およびビメンチンの発現の減少ならびに細胞運動性の低下を示した。以上より、SCはMET様の変化を誘発し、腫瘍細胞内のMET機構の活性化により膵臓神経におけるPDACコロニー形成に関与すると考えられた。

肝細胞癌から抽出したDNAを用いて、TERTプロモーター変異(TPM)と肝細胞癌、免疫組織化学的側面から臨床学的特徴との関連性を調査した。2004年および2007～2016年にかけ、慶応大学病院で外科的切除を受けた患者93例(男性76例、女性17例、65歳以上は69例)から得た、肝細胞癌結節標本97標本と非癌部隣接肝組織標本92標本からゲノムDNAを抽出し、PCR法により、TERTプロモーター領域を増幅させた。その結果、97個の肝細胞癌のうち53個(55%)にTPM97変異が検出され、TPMと年齢およびHCV感染との有意な関連性が示された。また、胆管/幹細胞マーカー陽性のサブグループではTPM保有率が低かった一方(29%)、Wnt/β-カテニンシグナル伝達マーカー陽性サブグループではTPM保有率が高く(60%)、TPM陽性肝細胞癌では、TPM陰性肝細胞癌に比べ、均一性指数で低値が示されたことから、腫瘍内不均一性が高いことが明らかにされた。なお、2つ以上の不均一な組織パターンや、早期もしくは高分化型の組織パターンが観察された肝細胞癌では、高率にTPMが認められた。以上の結果から、腫瘍内不均一性とTPM陽性肝細胞癌との有意な関連性が示され、肝細胞癌の病勢進行や診断において有用な情報になり得ると判断された。

＜文献概要＞73歳,男性.約47年間アスベスト曝露歴があった.当院受診1年前に左気胸を発症し,左胸腔にトロッカーを挿入されて手術を受けた.半年前から左胸部のトロッカー挿入部の瘢痕に一致して皮下腫瘤が出現した.3ヵ月前から咳嗽が出現し,他院で胸部X線上透過性低下を指摘され,気管支炎として加療されたが改善せず,当院内科を受診し,皮下腫瘤につき当科を紹介受診した.腫瘤の生検病理組織では異型細胞が真皮膠原線維間に増生する像を認めた.異型細胞はカルレチニンおよびD2-40陽性で,p40,p63,TTF-1,ナプシンAはいずれも陰性であった.気管支擦過細胞診,肺生検でカルレチニン陽性の異型細胞を認め,皮下腫瘤はトロッカー挿入による悪性胸膜中皮腫の皮膚転移と診断した.悪性胸膜中皮腫は肺癌と比較して経皮的医療行為による播種が多く,穿刺や手術部位に一致して皮下腫瘤が出現した場合は皮膚転移を疑い,積極的に精査を行うべきである.

肝細胞癌(HCC)患者154例に認められた165病変を大滴性脂肪化を伴うHCC(MaS-HCC)、小滴性脂肪化を伴うHCC(Mis-HCC)および標準的なHCC(cHCC)に分類し、これら病変の臨床病理学的相違を解析した。その結果、MaS-HCCでは、MiS-HCCおよびcHCCと比べ門脈浸潤や累積再発率のリスクが低く、腫瘍内線維化巣の比率が高いことが示された。また、MaS-HCCとMiS-HCCではいずれも、cHCCに比べウイルス性肝炎の発症率が低く、術前HbA1c値が高いことも示され、さらに、背景肝組織に観察される脂肪化のグレードや線維化を伴う病変においても、cHCCに比べMaS-HCCでは有意に高値が認められた。次に、脂肪滴膜上に発現するペリリピンおよびアディポフィリンの免疫組織学的検討を行ったところ、ペリリピンはHCCに観察される大半の脂肪滴では陰性が示されたが、背景肝の脂肪滴では陽性が示され、一方、アディポフィリンはHCCと背景肝の脂肪滴でともに陽性が認められた。以上の所見から、MaS-HCCとMiS-HCCが代謝異常と深く関連することが示されたが、生物学的には異なる挙動を示し、HCCの脂肪滴は背景肝の脂肪滴とは病態生理学的に異なることが明らかにされた。

症例は、70歳代女性。肝S4の約2cm大、術前診断で悪性が考えられた結節性病変に対して、肝左葉切除術が実施された。摘出検体の割面では、黄白色調で隆起を伴わず、八つ頭状・境界明瞭な病変を認め、組織学的には線維化、脂肪化、ballooning、Mallory-Denk体形成の目立つ硬化型肝細胞癌であった。背景肝にも軽度の脂肪化、ballooning様の細胞変性像、中心静脈域のpericellular fibrosisを認め、軽度の非アルコール性脂肪性肝炎(NASH)と判断された。近年、肝細胞癌の新たな組織型として生活習慣病との関連が強いとされるsteatohepatitic typeが提唱されているが、いまだ臨床病理学的な検討は十分とは言い難く、定義も含め一定の見解には至っていない。今回、NASHを背景として生じたsteatohepatitic typeの肝細胞癌と考えられる比較的典型的な1症例を経験したので報告する。(著者抄録)

Epithelial-mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal-epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding "focal differentiation" in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E-cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co-cultured with Schwann cells to investigate cell morphology, motility, or EMT-related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E-cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co-cultured with Schwann cells demonstrated a sheet-like appearance, increased E-cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET-like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.

Gamma-synuclein (SNCG) promotes invasive behavior and is reportedly a prognostic factor in a range of cancers. However, its role in biliary tract carcinoma (BTC) remains unknown. Consequently, we investigated the clinicopathological significance and function of SNCG in BTC. Using resected BTC specimens from 147 patients with adenocarcinoma (extrahepatic cholangiocarcinoma [ECC, n = 96]; intrahepatic cholangiocarcinoma [ICC, n = 51]), we immunohistochemically evaluated SNCG expression and investigated its correlation with clinicopathological factors and outcomes. Furthermore, cell lines with high SNCG expression were selected from 16 BTC cell lines and these underwent cell proliferation and migration assays by siRNAs. In the results, SNCG expression was present in 22 of 96 (22.9%) ECC patients and in 10 of 51 (19.6%) ICC patients. SNCG expression was significantly correlated with poorly differentiated tumor in both ECC and ICC (p = 0.01 and 0.03, respectively) and with perineural invasion and lymph node metastases in ECC (p = 0.04 and 0.003, respectively). Multivariate analyses revealed that SNCG expression was an independent poor prognostic factor in both OS and RFS in both ECC and ICC. In vitro analyses showed high SNCG expression in three BTC cell lines (NCC-BD1, NCC-BD3, and NCC-CC6-1). Functional analysis revealed that SNCG silencing could suppress cell migration in NCC-BD1 and NCC-CC6-1 and downregulate cell proliferation in NCC-CC6-1 significantly. In conclusion, SNCG may promote tumor cell activity and is potentially a novel prognostic marker in BTC.

Background & Aims: There is a major unmet need to assess the prognostic impact of antifibrotics in clinical trials because of the slow rate of liver fibrosis progression. We aimed to develop a surrogate biomarker to predict future fibrosis progression. Methods: A fibrosis progression signature (FPS) was defined to predict fibrosis progression within 5 years in patients with hepatitis C virus and nonalcoholic fatty liver disease (NAFLD) with no to minimal fibrosis at baseline (n = 421) and was validated in an independent NAFLD cohort (n = 78). The FPS was used to assess response to 13 candidate antifibrotics in organotypic ex vivo cultures of clinical fibrotic liver tissues (n = 78) and cenicriviroc in patients with nonalcoholic steatohepatitis enrolled in a clinical trial (n = 19, NCT02217475). A serum protein–based surrogate FPS was developed and tested in a cohort of compensated cirrhosis patients (n = 122). Results: A 20-gene FPS was defined and validated in an independent NAFLD cohort (adjusted odds ratio, 10.93; area under the receiver operating characteristic curve, 0.86). Among computationally inferred fibrosis-driving FPS genes, BCL2 was confirmed as a potential pharmacologic target using clinical liver tissues. Systematic ex vivo evaluation of 13 candidate antifibrotics identified rational combination therapies based on epigallocatechin gallate, which were validated for enhanced antifibrotic effect in ex vivo culture of clinical liver tissues. In patients with nonalcoholic steatohepatitis treated with cenicriviroc, FPS modulation was associated with 1-year fibrosis improvement accompanied by suppression of the E2F pathway. Induction of the PPARα pathway was absent in patients without fibrosis improvement, suggesting a benefit of combining PPARα agonism to improve the antifibrotic efficacy of cenicriviroc. A 7-protein serum protein–based surrogate FPS was associated with the development of decompensation in cirrhosis patients. Conclusion: The FPS predicts long-term fibrosis progression in an etiology-agnostic manner, which can inform antifibrotic drug development.

Prediction of hepatocellular carcinoma (HCC) risk is an urgent unmet need in patients with nonalcoholic fatty liver disease (NAFLD). In cohorts of 409 patients with NAFLD from multiple global regions, we defined and validated hepatic transcriptome and serum secretome signatures predictive of long-term HCC risk in patients with NAFLD. A 133-gene signature, prognostic liver signature (PLS)-NAFLD, predicted incident HCC over up to 15 years of longitudinal observation. High-risk PLS-NAFLD was associated with IDO1+ dendritic cells and dysfunctional CD8+ T cells in fibrotic portal tracts along with impaired metabolic regulators. PLS-NAFLD was validated in independent cohorts of patients with NAFLD who were HCC naïve (HCC incidence rates at 15 years were 22.7 and 0% in high- and low-risk patients, respectively) or HCC experienced (de novo HCC recurrence rates at 5 years were 71.8 and 42.9% in high- and low-risk patients, respectively). PLS-NAFLD was bioinformatically translated into a four-protein secretome signature, PLSec-NAFLD, which was validated in an independent cohort of HCC-naïve patients with NAFLD and cirrhosis (HCC incidence rates at 15 years were 37.6 and 0% in high- and low-risk patients, respectively). Combination of PLSec-NAFLD with our previously defined etiology-agnostic PLSec-AFP yielded improved HCC risk stratification. PLS-NAFLD was modified by bariatric surgery, lipophilic statin, and IDO1 inhibitor, suggesting that the signature can be used for drug discovery and as a surrogate end point in HCC chemoprevention clinical trials. Collectively, PLS/PLSec-NAFLD may enable NAFLD-specific HCC risk prediction and facilitate clinical translation of NAFLD-directed HCC chemoprevention.

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

Obesity-induced chronic liver inflammation is a hallmark of nonalcoholic steatohepatitis (NASH)—an aggressive form of nonalcoholic fatty liver disease. However, it remains unclear how such a low-grade, yet persistent, inflammation is sustained in the liver. Here, we show that the macrophage phagocytic receptor TREM2, induced by hepatocyte-derived sphingosine-1-phosphate, was required for efferocytosis of lipid-laden apoptotic hepatocytes and thereby maintained liver immune homeostasis. However, prolonged hypernutrition led to the production of proinflammatory cytokines TNF and IL-1β in the liver to induce TREM2 shedding through ADAM17-dependent proteolytic cleavage. Loss of TREM2 resulted in aberrant accumulation of dying hepatocytes, thereby further augmenting proinflammatory cytokine production. This ultimately precipitated a vicious cycle that licensed chronic inflammation to drive simple steatosis transition to NASH. Therefore, impaired macrophage efferocytosis is a previously unrecognized key pathogenic event that enables chronic liver inflammation in obesity. Blocking TREM2 cleavage to restore efferocytosis may represent an effective strategy to treat NASH.

30代女性。Reed症候群疑いにて経過観察中、子宮平滑筋腫に対する術前PET検査にて肝S6に集積を有する腫瘤が指摘された。婦人科手術と同時に腹腔鏡下肝部分切除術が施行された。切除検体では12mm大の境界不明瞭な腫瘤性病変を認め、肉眼所見では限局性結節性過形成(FNH)を第一に考えた。組織学的にはFNH様病変とともに、拡張した類洞内に腫大した類上皮細胞様の内皮細胞が微細な管腔構造や原始血管腔を形成する類上皮血管腫(EHE)様の所見が認められた。しかしながらFNH、EHEのいずれとしても典型的ではなく、その病理学的診断や解釈に苦慮した。病理組織学的に貴重な症例と考えられるため報告する。(著者抄録)

症例は87歳男性。検診で増大する肝腫瘤を指摘され当科紹介受診となった。画像所見から肝内胆管癌を含む肝原発悪性腫瘍が疑われたが、胆管浸潤を示唆する所見はなく、高齢で基礎疾患を多数有するため、ラジオ波焼灼療法(RFA:radiofrequency ablation)を行なった。治療時に施行した腫瘍生検の結果、肝内胆管癌と診断されたが、同時に採取された背景肝に1930-50年にかけて使用された造影剤であるトロトラストの沈着が疑われた。再発病変に対して2度RFAを施行し、初回治療後約3年間病勢が制御されている。今回我々は、RFAにより良好な病勢制御が得られたトロトラスト沈着症による肝内胆管癌の一例を経験した。トロトラスト晩発障害による発癌症例は肝内多中心性の発癌様式を示すため、本症例のように高齢で耐術能の低い胆管細胞癌症例においては、RFAなどの局所療法が有用な治療選択肢となりうると考えられた。(著者抄録)

The limited performance of guideline-recommended abdominal ultrasound and serum alpha-fetoprotein (AFP) highlights the urgent, unmet need for new biomarkers for more accurate detection of early hepatocellular carcinoma (HCC). To this end, we have conducted a prospective clinical validation study to evaluate the performance of the HelioLiver Test, a multi-analyte blood test combining cell-free DNA methylation patterns, clinical variables, and protein tumor markers. A blinded, multicenter validation study was performed with 247 subjects, including 122 subjects with HCC and 125 control subjects with chronic liver disease. The performance of the HelioLiver Test was compared with AFP and the GALAD score as established HCC surveillance blood tests. The performance of the HelioLiver Test (area under the receiver operating characteristic curve [AUROC] = 0.944) was superior to both AFP (AUROC = 0.851; p < 0.0001) and GALAD (AUROC = 0.899; p < 0.0001). Using a prespecified diagnostic algorithm, the HelioLiver Test showed sensitivities of 85% (95% confidence interval [CI], 78%–90%) for HCC of any stage and 76% (95% CI, 60%–87%) for early stage (American Joint Committee on Cancer [AJCC] I and II) HCC. In contrast, AFP (≥20 ng/mL) alone and the GALAD score (≥−0.63) showed lower sensitivities of 62% (95% CI, 54%–70%) and 75% (95% CI, 67%-82%) for HCC overall, and 57% (95% CI, 40%–71%) and 65% (95% CI, 49%–79%) for early stage (AJCC I and II) HCC, respectively. The specificities of the HelioLiver Test (91%; 95% CI, 85%–95%), AFP (97%; 95% CI, 92%–99%), and the GALAD score (94%; 95% CI, 88%–97%) were similar for control subjects. The HelioLiver Test showed superior performance for HCC detection compared to with both AFP and the GALAD score and warrants further evaluation in HCC surveillance settings.

Background & Aims: Despite recent progress, long-term survival remains low for hepatocellular carcinoma (HCC). The most effective HCC therapies target the tumor immune microenvironment (TIME), and there are almost no therapies that directly target tumor cells. Here, we investigated the regulation and function of tumor cell-expressed Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HCC. Methods: HCC was induced in mice by Sleeping Beauty-mediated expression of MET, CTNNB1-S45Y, or TAZ-S89A, or by diethylnitrosamine plus CCl4. Hepatocellular TAZ and YAP were deleted in floxed mice via adeno-associated virus serotype 8-mediated expression of Cre. TAZ target genes were identified from RNA sequencing, confirmed by chromatin immunoprecipitation, and evaluated in a clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen. TEA domain transcription factors (TEADs), anillin (ANLN), Kif23, and programmed cell death protein ligand 1 were knocked down by guide RNAs in dead clustered regularly interspaced short palindromic repeats–associated protein 9 (dCas9) knock-in mice. Results: YAP and TAZ were up-regulated in murine and human HCC, but only deletion of TAZ consistently decreased HCC growth and mortality. Conversely, overexpression of activated TAZ was sufficient to trigger HCC. TAZ expression in HCC was regulated by cholesterol synthesis, as demonstrated by pharmacologic or genetic inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), or sterol regulatory element-binding protein 2 (SREBP2). TAZ- and MET/CTNNB1-S45Y–driven HCC required the expression of TEAD2 and, to a lesser extent, TEAD4. Accordingly, TEAD2 displayed the most profound effect on survival in patients with HCC. TAZ and TEAD2 promoted HCC via increased tumor cell proliferation, mediated by TAZ target genes ANLN and kinesin family member 23 (KIF23). Therapeutic targeting of HCC, using pan-TEAD inhibitors or the combination of a statin with sorafenib or anti–programmed cell death protein 1, decreased tumor growth. Conclusions: Our results suggest the cholesterol-TAZ-TEAD2-ANLN/KIF23 pathway as a mediator of HCC proliferation and tumor cell-intrinsic therapeutic target that could be synergistically combined with TIME-targeted therapies.