胰腺導管腺癌局部治療聯合免疫治療研究進展

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(1復旦大學附屬中山醫院介入治療科 上海 200032; 2上海市影像醫學研究所 上海 200032)

胰腺導管腺癌(pancreatic ductal adeno-carcinoma,PDAC)是我國發病率排行前十位的惡性腫瘤,其死亡率與發病率之比大于85%,且發病率呈現上升趨勢[1]。目前外科手術切除是唯一可能治愈PDAC的治療方式,但80%以上的患者在確診時已經失去手術機會。放療及以吉西他濱為主的化療是不可手術切除的PDAC患者的主要治療方式。盡管PDAC診療技術不斷提高,患者5年生存率仍然不超過5%[2]。傳統的放療是局部進展期PDAC的主要治療方式之一[3]。目前包括立體定向放射治療(stereotactic body radiotherapy,SBRT)、質子放療、放射性粒子植入、射頻消融治療、電穿孔治療(irreversible electroporation,IRE)、高強聚焦超聲(high intensity focused ultrasound,HIFU)在內的局部治療有望成為PDAC患者的新選擇。雖然免疫治療已經在血液系統腫瘤、黑色素瘤、非小細胞肺癌、膀胱癌等實體瘤的治療中取得令人矚目的進展,但是PDAC的免疫治療還處于探索階段。本文主要聚焦不可手術切除的PDAC局部治療聯合免疫治療的基礎及臨床研究進展。

PDAC局部治療研究概況放療是局部進展期PDAC的主要治療方式之一。近年來,隨著影像導引技術的提高,高劑量且更加精準的SBRT為PDAC的放療提供了新的手段[4-7]。質子放療通過不同能量的帶電粒子Bragg峰展寬得到擴展Bragg峰(spread out bragg peak,SOBP),使病灶位于SOBP峰區,從而獲得靶區內高劑量。研究表明,質子放療聯合卡培他濱作為胰腺癌新輔助放化療安全、可行,并可以達到良好的腫瘤局部控制率[8-10]。質子放療可以覆蓋高危淋巴結而減少損傷周圍正常組織[11]。與調強適形放療(intensity-modulated radiation therapy,IMRT)及三維適型放療(three dimensional conformal radiation therapy,3DCRT)比較,質子放療較少損傷周圍正常組織[12]。目前由于質子放療資源緊張,其大規模的推廣應用受限。通過術中或者影像導引下經皮穿刺植入放射性粒子,這種組織內放療可提高腫瘤局部的控制率[13-16]。早期報道放射源的植入多通過開腹途徑,隨著CT導引的應用,經皮穿刺途徑具有更高的安全性及微創性。同樣,新器械的出現、CT等導引系統的應用突破了原來被認為是“穿刺禁區”的胰腺,使經皮射頻/微波消融治療PDAC的安全性進一步提高[17-20]。2016年,復旦大學附屬中山醫院率先以21G經皮穿刺針穿刺PDAC,引入Habib消融導絲進行PDAC射頻消融治療(圖1)。該穿刺系統較傳統穿刺系統直徑更小,提高了穿刺的安全性。首例患者30天內未見嚴重不良反應,但是否能夠獲得生存獲益需要進一步臨床試驗證實。相比較于射頻消融,IRE對于結締組織的損傷較小且無熱沉效應,理論上更加適用于周圍血管較多的胰腺。2012年,Bagla等[21]首次報道1例IRE治療侵犯腹腔動脈及腸系膜上動脈的局部進展期PDAC患者,初步顯示出其可行性及良好的安全性。一項多中心臨床試驗表明IRE治療局部進展期PDAC安全可行,與傳統放療、化療比較IRE可提高患者的腫瘤局部/遠處無進展時間(14個月/6個月,15個月/9個月)及生存時間(20個月/13個月)[22]。目前最大樣本量的研究是Martin等[23]于2015年報道的200例局部進展期PDAC患者在傳統治療的基礎上增加IRE,在29個月的中位隨訪時間中只有 6 例(3%)出現局部復發,患者的中位生存期為24.9個月。該研究中IRE在開腹后超聲導引下進行。初步臨床研究表明超聲或CT導引下經皮穿刺IRE安全可行[24-25]。磁共振導引下HIFU治療不可手術切除PDAC的初步臨床研究表明,這種無創的局部治療可能在控制腫瘤發展的同時減輕患者疼痛[26-27]。PDAC的局部治療從原來的開腹巨創到現在的微創,未來正向更加精準、超微創甚至無創的方向發展。

圖1 CT導引下經皮穿刺PDAC射頻消融術Fig 1 CT-guided percutaneous radiofrequency ablation for PDAC

PDAC免疫治療概況自19世紀90年代用Coley毒素治療腫瘤開始,人們不斷探索各種免疫療法用于治療腫瘤。目前腫瘤的免疫治療主要包括:免疫檢查點阻斷治療(immune checkpoint blockade,ICB)、基因改造T細胞治療、腫瘤疫苗、免疫刺激因子、溶瘤病毒及基因治療。近年來ICB已在黑色素瘤、非小細胞肺癌、腎癌、非霍奇金淋巴瘤的治療中取得重大突破。

ICB的療效與腫瘤自身的免疫微環境有關。基因改造的PDAC小鼠模型中,腫瘤惡性程度越高,CD4+Treg細胞比例越高。這表明細胞毒T淋巴細胞相關抗原4 (cytotoxic T lymphocyte-associated antigen 4,CTLA-4)抗體可能對PDAC有效[28]。然而,Ipilimumab (CTLA-4抗體) 3 mg/kg單藥治療局部進展期或轉移性PDACⅡ期臨床試驗未見提高腫瘤緩解率。但是,值得注意的是研究中1例患者具有較長的生存時間,說明在特殊情況下CTLA-4單抗可能有效[29]。而另一方面,程序性死亡配體1(programmed death-ligand 1,PD-L1)陽性的PDAC患者的預后顯著差于PD-L1陰性患者,這說明阻斷PD-1/PD-L1通路可能使PDAC患者獲益[30]。令人沮喪的是PD-L1抗體的Ⅰ期臨床試驗中,14例PDAC患者并沒有出現腫瘤緩解[31]。這給ICB治療PDAC的前景蒙上了一層陰影。究其原因,PDAC免疫阻斷治療的抵抗性可能與PDAC基質豐富及腫瘤內自身CD8+T細胞數量少有關。動物實驗表明去除纖維細胞活化蛋白(fibroblast activation protein,FAP)陽性的癌相關成纖維細胞能增加PD-L1抗體的療效[32]。抑制黏附激酶(focal adhesion kinase,FAK)的同時應用吉西他濱、PD-1及CTLA-4抗體能顯著減小腫瘤負荷、延長小鼠生存時間[33]。目前聯合FAK抑制劑及PD-1抗體治療包括PDAC在內的實體性腫瘤的臨床試驗正在進行中(NCT0275858)。放療、化療、射頻消融可以引起具有免疫反應的腫瘤細胞死亡(immunogenic cell death,ICD),釋放損傷相關模式分子(damage-associated molecular pattern,DAMP),增加腫瘤特異性抗原(tumor-associated antigen,TAA)的暴露,加強DC細胞及T細胞的浸潤,從而使原來對ICB無效的腫瘤重新變得敏感[34-36]。這種使得原來免疫“冷”瘤重燃為免疫“熱”瘤是局部治療聯合免疫腫瘤的重要理論,具體于下文詳述。Jaffee等[38]于2001年報道了粒細胞-巨噬細胞集落刺激因子疫苗(又稱為GVAX疫苗)治療PDAC的Ⅰ期臨床試驗,證實了該疫苗的安全性[37]。2015年的Ⅱ期臨床試驗結果顯示,Cy/GVAX+CRS270顯著提高轉移性PDAC患者的中位生存時間(試驗組vs.對照組:9.7個月vs.4.6個月)。以腺病毒為載體表達HSV-tk基因治療PDAC安全可行,并可提高CD8+T細胞浸潤及PD-L1的表達[39]。溶瘤病毒治療有望成為晚期PDAC的治療方式,但是目前尚處于基礎研究階段[40]。基因改造T細胞能夠靶向殺傷腫瘤細胞,而較少殺傷正常組織。以癌胚抗原(carcino-embryonic antigen,CEA)、黏蛋白(mucoprotein 1,MUC1)、間皮素(Mesothelin)為靶點基因改造T細胞治療PDAC在動物試驗中顯示出良好的安全性及有效性[41-43]。目前針對CEA、人表皮生長因子受體2(human epidermal growth factor receptor 2,Her-2)、前列腺干細胞抗原(prostate stem cell antigen,PSCA)、間皮素、MUC1、CD-133靶點的基因改造T細胞治療的臨床試驗正在進行中(如NCT02349724、NCT02713984、NCT02744287、NCT01897415、NCT02706782、NCT02587689、NCT02541370),包括PDAC在內的多種實體腫瘤。瘤外靶向損傷及細胞因子風暴(cytokine release syndrome,CRS)是基因改造T細胞治療的主要不良反應[44]。其中CRS指的是T細胞輸入后釋放IFN-γ、IL-6等,產生高熱、低血壓、低氧、心衰、腎衰、電解質紊亂等臨床癥狀。雙靶點、抑制性因子的敲入、自殺基因的敲入可以增加細胞治療的精準性及可控性。另外采用局部注射的方式有望在產生局部及全身療效的同時減少細胞的用量,從而減少不良反應。如何增加輸注的基因改造T細胞定植、減少腫瘤微環境對其的抑制是細胞治療的另一個重要方面。局部治療可釋放CCL4等細胞趨化因子,在理論上可以增加T細胞定植[45]。聯合ICB治療可能進一步提高基因改造T細胞治療的療效。在單種細胞因子治療PDAC方面,Ⅲ期臨床實驗表明TNFerade不能提高局部進展期PDAC患者的生存時間,而且與經皮穿刺途徑比較,通過內鏡經食道途徑是無疾病進展時間較短的風險因素[46]。由此提示,細胞因子對腫瘤細胞、機體整體及其不同濃度細胞因子對腫瘤的作用有待進一步研究。

總之,新興的局部治療的臨床有效性需要進一步的臨床試驗來證明。雖然GVAX疫苗治療晚期PDAC已取得重要成就,但是免疫治療單獨應用于PDAC的治療效果較差。如何打破PDAC免疫治療抵抗需要進一步的研究。

局部治療后PDAC免疫微環境的改變及聯合治療探索PDAC組織內CD8+T細胞稀少,而調節性T細胞(Treg)及骨髓源性抑制細胞(myeloid-derived suppressor cells,MDSC)豐富[28,30]。豐富的基質減少了癌細胞與T細胞的接觸[32]。腫瘤免疫治療的作用主要取決于:(1)腫瘤特異性抗原的表達;(2)有效的抗原呈遞;(3)與腫瘤細胞接觸的功能完整的T細胞。從這些角度看,PDAC屬于免疫“冷”瘤,單獨針對某一環節的免疫治療難以起效。如何打破腫瘤“冷”的格局是免疫治療成功的關鍵。PDAC的局部治療主要通過物理作用(如輻射、熱、電穿孔)直接殺傷腫瘤細胞。與機體默認無免疫反應的程序性凋亡不同,這些局部治療可以導致ICD。

Filatenkov等[47]研究發現高劑量的放療后DC細胞活化,釋放INFγ,增加CD8+T細胞的浸潤,同時還可以減少MDSC,重新塑造腸癌的腫瘤微環境。DC細胞的活化主要通過2條通路:(1)放療后HMGB1釋放,通過Toll樣受體4 (toll-like receptor 4,TLR-4)、髓樣分化因子(myeloid differentiation factor 88,MYD-88)激活DC細胞從而影響療效[48];(2) DC細胞感受外來雙鏈DNA的干擾素刺激基因(stimulator of interferon genes,STING)途徑,釋放Ⅰ型干擾素,從而增強T細胞的活化[49-50]。理論上,放療可以增加細胞質中的雙鏈DNA,再者氧自由基的作用可減少雙鏈DNA的降解。但這種雙鏈DNA如何進入DC細胞的細胞質需要進一步的研究。PDAC小鼠皮下瘤模型中,放療聯合局部注射環二核苷酸(cyclic dinucleotide,CDN)類似物進一步激活STING通路,導致T細胞依賴的腫瘤壞死[51]。單次高劑量放療可通過激活T細胞殺傷腫瘤,傳統的分次放療則有可能抑制這種放療后的抗腫瘤免疫。且聯合ad-LIGHT免疫治療可進一步提高療效[52]。碘-125粒子能夠長期釋放低劑量的射線,其是否能夠通過類似的途徑增強細胞免疫尚需進一步研究。SBRT聯合CTLA-4抗體的Ⅰ期臨床試驗研究中納入22例患者,根據實體瘤療效評估標準(response evaluation criteria in solid tumor,RECIST),有18%的患者腫瘤部分緩解,此后的動物試驗也只能達到類似的結果。RNA測序分析結果顯示,治療后PD-L1明顯升高。聯合PD-1抗體后的動物試驗顯示出令人震驚的療效(總體生存率100%),其中機制為SBRT增加T細胞抗原受體(T cell receptor,TCR)的多樣性、抗CTLA-4減少Treg細胞、抗PD-1/PD-L1逆轉T細胞失能三者相輔相成[53]。另外,聯合PD-1抗體、TIM-3抗體及放療能夠使小鼠神經膠質瘤體積縮小,這表明聯合多通路的ICB治療有望進一步提高療效[54]。Zheng等[55]建立表達SIY抗原的Pan02PDAC小鼠模型,放療聯合疫苗(SIY多肽+TLR-3激動劑Poly I:C)能夠增加CD8+T細胞浸潤;聯合PD-L1抗體后顯著增加腫瘤的緩解率,延長小鼠生存期。

熱、冷、電穿孔等局部治療也可導致ICD。其潛在機制為術后DNA、RNA、高遷移率族蛋白1(high mobility group box-1 protein,HMGB1)、熱休克蛋白70等升高,促進DC細胞、T細胞活化。再者,大量的腫瘤細胞壞死、TAA暴露,相當于在腫瘤原位建立類似于腫瘤疫苗的腫瘤抗原庫。由于熱消融、冷消融、電穿孔導致細胞損傷的方式不同,術后細胞因子的釋放、細胞抗原的保存不盡相同[56]。動物實驗表明射頻消融術后凝固性壞死區周圍可見大量的中性粒細胞、DC細胞浸潤,能促進針對腫瘤的抗原特異性免疫[35,57]。HIFU治療后腫瘤特異性T淋巴細胞、DC細胞增加[58-59]。Zerbini等[60]研究發現,22例接受射頻消融的肝癌患者外周血單核細胞能激活患者自身消融術后獲取的腫瘤組織并釋放INF-γ,但是這種反應并未有效抑制腫瘤的復發。消融術后可以增加T細胞、DC細胞浸潤來增強免疫,但另一方面消融術后腫瘤免疫微環境的改變可能導致的免疫抑制會影響這種腫瘤特異性免疫的持續性。Zeng等[61]研究發現肝癌患者消融術后PD-1、PD-L1升高,且與患者預后相關。消融術后抗原持續暴露、缺氧、前列腺素E2 (prostaglandin E2,PGE2)、腺苷(adenosine)及PD-1/PD-L1升高,腫瘤發生EMT,從而導致T細胞失能、腫瘤免疫逃逸。聯合ICB治療逆轉消融術后的T細胞失能,有可能提高療效。Waitz等[62]報道冷凍消融聯合CTLA-4抗體能夠產生局部及遠處病灶CD8+T細胞依賴的腫瘤特異性免疫。Shi等[63]報道腸癌肝轉移患者轉移灶射頻消融術后,原發病灶切除標本CD8/4+T細胞增加,同時PD-L1升高。進一步動物試驗表明,射頻消融處理后遠處的病灶也可觀察到這種現象。射頻消融聯合PD-L1抗體能通過CD8+T細胞依賴的途徑顯著延長小鼠的生存時間[63]。這表明局部治療在控制局部腫瘤的同時改變了局部及遠處病灶的免疫微環境,聯合ICB治療不僅增強局部的療效,而且對遠處病灶產生T細胞依賴的腫瘤殺傷作用。這種消融術后對于遠處病灶免疫微環境的“遠隔效應”格外引人注目,有病例報道消融術后遠處病灶自行緩解甚至消失[64-65]。這使得人們對于局部治療聯合免疫治療腫瘤的未來充滿期待。目前一項冷凍、射頻消融或者經動脈化療栓塞聯合PD-1抗體治療肝細胞肝癌的臨床試驗正在進行中(NCT02821754)。近年來局部治療聯合免疫治療PDAC的臨床研究逐漸增加。大部分的臨床試驗為放療聯合免疫治療,最常用的聯合模式是放療聯合ICB(表1)。

表1 進行中的局部治療聯合免疫治療PDAC的臨床研究Tab 1 Ongoing clinical trials of immunotherapy plus loco-regional therapy for PDAC

GMCI:Gene-mediated cytotoxic immunotherapy;adenovirus expressHSV-tkgene; Tadalafil:PDE5 inhibitor.

結語PDAC免疫治療的研究在不斷探索中前行,局部治療顯示出良好的安全性及有效性。局部治療能夠改變腫瘤局部及遠處轉移灶的免疫微環境,增加免疫治療的敏感性,有望減少免疫治療的不良反應。聯合治療的理論及基礎研究為其未來的臨床轉化提供了充分的證據,近年來注冊的臨床研究結果值得期待,尤其是放療聯合ICB治療的臨床研究。目前胰腺局部消融治療聯合局部免疫治療尚無相關研究報道,其機制仍不明確且缺乏相應的循證醫學證據。消融、IRE等聯合ICB治療PDAC值得進一步研究。目前尚不清楚局部治療能否促進基因修飾T細胞的定植、擴增并加強其細胞毒作用。本文未就溶瘤病毒、多肽疫苗、細菌治療等其他免疫療法展開闡述。總之,局部治療與免疫治療相互結合,可能達到1+1>2的效果。

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