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直腸癌に対する化学放射線療法におけるCPT-11の効果とFDG-PETによる効果・予後の予測

奥野, 貴之 東京大学 DOI:10.15083/0002002471

2021.10.15

概要

【研究の目的】
 直腸癌に対しては術前化学放射線療法(Chemoradiotherapy: CRT)と外科的手術を中心とした集学的治療が行われるが、予後の改善と骨盤機能温存を目的とした研究が盛んである。近年ではCPT-11を併用した術前CRTの効果に期待が集まりつつあるが、基礎研究による裏付けはあまりない。そこで第1章では大腸癌細胞を用いて、CPT-11の活性代謝物であるSN-38のⅩ線増感作用について解析した。また現在、術前CRTの効果は手術後の切除検体により評価されているが、手術前にその効果や予後を予測することができれば、骨盤機能温存や適切な個別化治療の選択に有効である。第2章では実臨床のデータを用いて、fludeoxyglucose(FDG)–positron emission tomography(PET)/computed tomography(CT)検査による術前CRTの効果や予後の予測能について明らかにした。

第1章大腸癌細胞におけるSN-38のⅩ線増感効果
【背景】
 CPT-11は肝臓でSN-38へと代謝され抗腫瘍効果を発揮する、大腸癌治療のキードラックである。近年は直腸癌に対する術前CRTのレジメンとしてもその効果が期待され、当科で実施した第I、II相臨床試験でも病理組織学的完全奏効(Pathological complete response: pCR)率が22.7%と高い抗腫瘍効果を示した。しかしこれまでCPT-11やSN-38のX線増感作用につき解析した基礎研究はあまりない。一方で直腸癌などの固形腫瘍においては、内部の低酸素環境がX線治療や化学療法への抵抗性を示すことが知られている。低酸素環境下の細胞ではHypoxia-inducible factor(HIF)-1αというタンパク質が発現し、癌細胞の生存能の向上や治療抵抗性の獲得などの悪性度に寄与する。当科では以前に、SN-38が低酸素環境下の大腸癌細胞に発現したHIF-1αを抑制することを報告した。さらに近年では他癌種細胞においてX線照射によりHIF-1αが発現することが報告されているが、大腸癌細胞における報告はこれまでにない。そこで第1章では、①大腸癌細胞においてⅩ線照射によりHIF-1αが発現する、②SN-38はⅩ線照射により発現したHIF-1αを抑制する、③SN-38がX線増感剤として作用する、という仮説をたて、これを明らかにすることとした。
【実験方法】
 ヒト大腸癌細胞(HT29, SW480)に対してX線照射(0-8Gy)を行い、HIF-1αの発現量をフローサイトメトリーとウェスタンブロッティングにより解析した。X線照射後の時間経過に伴うHIF-1αの発現量の推移についてもフローサイトメトリーにより解析した。次いでX線照射の直後にSN-38(0-4μM)、5-fluorouracil(0-20μM)、Oxaliplatin(0-20μM)を投与し、48時間後のHIF-1αとその下流のVascular endothelial growth factor(VEGF)の発現をフローサイトメトリーとウェスタンブロッティングにより解析した。最後にX線照射なし、または4GyのX線照射を施行したHT29細胞に各濃度のSN-38(0-4μM)を投与し48時間後の細胞数をカルセインアッセイにより測定して、X線照射による細胞増殖抑制効果のSN-38による増強について解析した。さらにAnnexin V/Propidium Iodideの2重染色とトリパンブルー染色によりアポトーシスを、FITC標識BrdU細胞周期キットにより細胞周期を解析し、SN-38によるX線増感作用の機序を解析した。
【結果】
 2-8GyのX線照射により、HT29、SW480の両細胞で線量依存性にHIF-1αの発現量の増加を認めた。4Gyと8GyのX照射後のHIF-1α発現量は同等であった。HIF-1αはX線照射12時間後から発現し、24-48時間で発現量はピークとなり、72時間以降は照射前の発現量に戻った。また4GyのX線照射によりHIF-1αに加えてVEGFも発現をみとめ、照射直後に0.25-4μMのSN-38を投与するとそれらの発現量は濃度依存性に抑制された。5-fluorouracilやOxaliplatinを投与してもHIF-1αの抑制は認められなかった。さらに4GyのX線照射をおこなったHT29細胞に0.25-2μMのSN-38投与を併用すると、X線照射による細胞増殖抑制効果のSN-38による増強と考えられる細胞数の低下を認め、特に1μMのSN-38の投与により最も強くX線増感効果を認めた(X線照射 vs. Ⅹ線照射とSN-38投与: 232vs.86)。コントロール、X線照射(4Gy)、SN-38投与(1μM)、X線照射とSN-38投与の4治療群間でアポトーシス細胞の割合に差はなかった。一方で細胞周期については、X線照射によりG2/M期、SN-38投与によりS期とG2/M期の細胞の割合の増加を認め、X線照射とSN-38の併用によりS期とG2/M期のさらなる細胞の割合の増加を認め(コントロール vs. X線照射とSN-38の併用: 44.2% vs. 71.4%)、成長期であるG1期の細胞の割合が大きく減少した(コントロール vs. X線照射とSN-38の併用: 55.4% vs. 24.3%)。
【第1章の結論】
 SN-38はX線照射により発現したHIF-1αを抑制し、大腸癌細胞においてX線増感剤として作用した。その機序はS期とG2/M期の細胞周期停止であることが示唆された。

第2章直腸癌に対する術前化学放射線療法におけるFDG-PET/CT volumetryによる効果・予後予測能
【背景】
 直腸癌術前CRTの効果は症例により大きく異なるため、症例ごとに適切な治療を行うことを目的として、手術前にCRTの効果や予後を予測するマーカーが研究されている。そこで細胞の代謝をstandardized uptake value(SUV)として反映し、癌細胞の遺残や悪性度などを評価するFDG-PET/CT検査に着目した。これまで直腸癌術前CRT後のmaximum standardized uptake value(SUVmax)がCRTの効果と相関することが報告されているが、予後の予測能については明らかではない。さらに近年では腫瘍内の1ピクセルのSUVの集積であるSUVmaxよりも、腫瘍体積の全ボクセルのSUVの総和であるFDG-PET/CT volumetryのほうが、より前治療の効果や予後を反映しうる可能性が報告されている。そこで直腸癌に対する術前CRT前後のFDG-PET/CT検査において、①SUVmaxにFDG-PET/CT volumetryを加えることでCRTの効果の予測能がより向上する、②FDG-PET/CT volumetryは予後を予測し得る、いう仮説をたてて、これを明らかにすることとした。
【対象と方法】
 2005年1月から2015年12月まで、東京大学医学部附属病院大腸肛門外科にてcT3-4の下部直腸腺癌と診断され、術前CRTと根治的切除を行った91例を対象とした。遠隔転移を有する症例は除外した。CRTは50.4Gy/28Frの放射線照射と5-FUベースの化学療法を行い、FDG-PET/CT検査は同一のPET/CTスキャナーを用いて撮影した。PET/CT画像の解析は核医学放射線科医と外科医でそれぞれ腫瘍の境界線を作成し、設定した閾値(SUV=2、2.5、3、SUVmaxの50%)を用いてvolume of interest(VOI)を測定し、VOI中のSUVmaxとFDG-PET/CT volumetry(Metabolic tumor volume: MTV, Totallesionglycolysis: TLG)、Δ(%)(CRT前後におけるPET/CTの値の減少率)を算出した。PET/CTの値と臨床病理学的因子の相関はStudent t検定、予後(Relapse free survival: RFS, Overall survival: OS)との相関はLog-rank検定とcox比例ハザードモデルを用いた多変量解析により解析した。
【結果】
 pCRは14例(15%)に認められた。pCRとはCRT後のSUVmax(pCR vs. non-pCR: 3.1 vs. 4.7)とΔSUVmaxが相関していたが、FDG-PET/CT volumetryは相関を認めなかった。またypTとはCRT後のSUVmax(T0-2 vs. T3-4: 3.4 vs. 4.9)、ΔSUVmax、CRT後のMTV、CRT後のTLG(T0-2 vs. T3-4: 31.1 vs. 74.4)が相関していた。一方でypNとはCRT後のMTV、CRT後のTLG(N0 vs. N1-2: 37.9 vs. 81.6)のみが相関していた。最後にSUVmaxとTLGを平均値より高い群と低い群に分け、予後との相関を解析した。CRT後のSUVmaxが高い群は局所無再発生存期間(p=0.008)、遠隔転移無再発生存率(p<0.001)、RFS(p<0.001)が不良であったが、OSとの相関を認めなかった。一方で、CRT後のTLGの高い群ではOSを含むいずれの予後とも不良な相関を認めた(局所無再発生存期間: p=0.002、遠隔転移無再発生存期間: p<0.001、RFS: p<0.001、OS: p=0.02)。さらにRFSについて単変量解析で有意であったypT、ypN、リンパ管侵襲、静脈侵襲、病理組織学的効果判定、組織型、CRT後のSUVmax、CRT後のTLGについて多変量解析を行うと、CRT後のTLGの高値が不良なRFSを予測する独立因子であった(ハザード比: 4.718、p=0.04)。
【第2章の結論】
 直腸癌に対する術前CRT後のFDG-PET/CTでFDG-PET/CT volumetryが高値の症例のRFSやOSは不良であり、CRT後のTLGは独立した予後予測因子である。

【全体の結論】
 CPT-11は直腸癌に対する術前CRTに併用する薬剤として候補たり得る、またFDG-PET/CT volumetryは直腸癌患者の予後の予測に有用な可能性があると考えられた。

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