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上海交通大學附屬新華醫院普外科專家解讀膽囊癌文獻~

1OPT-IN研究：術后確診膽囊癌的治療策略（IF：5.343）

龔偉教授點評：

目前臨床上確診的膽囊癌約50%以上是術前診斷為膽囊良性疾病行腹腔鏡膽囊切除術時在術中或術后經病理檢查發現而確診的（所謂的“意外膽囊癌”）。

對于分期在T1b以上的膽囊癌，往往需要在膽囊切除的基礎上進行再次手術清掃淋巴結和膽囊床局部肝組織。但在臨床實踐中往往有高達75%患者在第二次手術時已出現淋巴結轉移。

對于T2期以上的腫瘤，即使進行了二次根治性手術，50%的患者在1年左右出現腫瘤復發。因此，如何改進治療策略，提高這類患者的治療效果非常重要。

OPT-IN研究提出了對T2和T3期的“意外膽囊癌”進行術前新輔助化療再進行第二次手術的理念。

對照組采用傳統的策略直接行根治性手術，研究組則進行4周期的吉西他濱聯合順鉑新輔助化療后行根治性手術。兩組術后都進行輔助化療。主要觀察目標為5年的生存期率，次要觀察目標為兩組再次手術時的殘余腫瘤率、臨床指標、無瘤生存率的差別。

“意外膽囊癌”的新輔助治療已有文獻報道，但數據較為零散并且方案并不一致。雖然OPT-IN中的化療方案還是延續ABC-02研究的基于吉西他濱聯合順鉑的方案，而且對于T2或T3期膽囊癌肝的切除范圍沒有做出明顯的界定。

但它是目前所知第一項針對“意外膽囊癌”行新輔助治療的大規模的多中心隨機對照研究，期待此項研究數據有助于改變目前“意外膽囊癌”的治療策略并提升治療效果。

2單細胞測序揭示膽囊癌免疫微環境特征（IF：25.080）

摘要（上下滑動查看）：

We employed single-cell RNA sequencingand functional assays to reveal transcriptomic heterogeneity and intercellularcrosstalk in GBC patients.GBC cancers were characterized with heterogeniccellular ecosystem in which the ErbB pathway mutations were associated withanti-cancer immunity and cancer development.Increased MDK from cancer cells ofErbB pathway mutation tumors resulted in interaction with its receptor LRP1expressed by tumor infiltrating macrophages and promoted immuno-suppressivemacrophage differentiation.In addition,the crosstalk betweenmacrophage-secreted CXCL10 and its receptor CXCR3 on Tregs was induced in GBCwith ErbB pathway mutations,which contributes to GBC progression.

張一鑒研究員點評：

這是劉穎斌教授團隊繼2014年NG（Nature Genetics，Whole-exome and targetedgene sequencing of gallbladder carcinoma identifies recurrent mutations in theErbB pathway）、2017年GUT（Gut，Genomic ERBB2/ERBB3 mutations promotePD-L1-mediated immune escape in gallbladder cancer:a whole-exome sequencinganalysis）后又一姊妹篇。

前兩項研究是運用全基因組外顯子和靶向測序技術國際上首次系統闡述膽囊癌基因突變譜，發現驅動膽囊癌發生發展的關鍵ErbB信號通路，并進一步發現ERBB2/3熱點突變通過上調PD-L1促進膽囊癌細胞的免疫逃逸機制。

而該項研究是在此基礎上進一步探索帶有ErbB信號通路突變的膽囊癌微環境中，各個細胞類型間是通過怎樣的交互、如何促進免疫逃逸的，可以說是層層遞進，系統深入性的探索膽囊癌的發生發展機制。

受限于動物模型和臨床樣本的較難獲取，GBC的發生發展機制尤其是免疫微環境一直是該領域的研究難點。與免疫治療在其它腫瘤中快速發展相比，對GBC免疫微環境認識相對較少。

該項目的設計初心是想在前期成果的基礎上進一步探索GBC免疫微環境的特征及免疫治療的應用基礎，因此選擇了明確ERBB通路突變信息的13例GBC患者的癌和癌旁組織進行單細胞轉錄組測序分析，探索組織異質性和細胞間的相互作用。

本研究首次構建膽囊癌微環境單細胞轉錄圖譜，展示膽囊癌組織異質性和細胞間相互作用，為了解膽囊癌的微環境提供了重要數據信息。

膽囊癌的研究重點多關注腺癌，對比較難獲取組織樣本的腺鱗癌、鱗癌等了解甚少，該項研究發現了鱗癌主要上皮細胞類型，并篩選出膽囊鱗癌細胞特有的分子標志物，為今后針對性檢測提供了可靠的實驗依據。

進一步深入分析后，揭示帶有ErbB信號通路突變的膽囊癌含有更多的1型/2型上皮細胞、Tregs和M2型巨噬細胞；并通過分泌MDK與巨噬細胞上表達的LRP1相互作用，促進巨噬細胞由M1型向M2型分化；

而M2型巨噬細胞通過分泌更多的細胞因子CXCL10，與Treg細胞上的CXCR3相互作用，促進膽囊癌細胞的免疫逃逸。同時，分析臨床組織樣本后顯示MDK的高表達與膽囊癌患者不良預后顯著相關。

該研究結果全景式分析了膽囊癌微環境中的腫瘤細胞和免疫細胞特征，揭示了這些細胞共同驅動腫瘤的惡性轉化機制，為膽囊癌的診斷和新的免疫療法開發與應用奠定了重要基礎；也對未來進一步改善膽囊癌治療現狀、開展膽囊癌綜合治療具有很大的引領作用。

3單細胞測序揭示膽囊癌多樣性及瘤內異質性（IF：11.491）

摘要（上下滑動查看）：

Background:Gallbladder cancer(GC)is amalignant disease characterized with highly cellular heterogeneity and poorprognosis.Determining the intratumoral heterogeneity and microenvironment(TME)can provide novel therapeutic strategies for GC.

Methods:We performed the single-cell RNAsequencing on the primary and lymph node metastatic gallbladder tumors and theadjacent normal tissues of five patients.The transcriptomic atlas andligand-receptor-based intercellular communication networks of the single cellswere characterized.

Results:The transcriptomic landscape of24,887 single cells was obtained and characterized as 10 cellular clusters,including epithelial,neuroendocrine tumor cells,T&NK cells,B cells,RGS5+fibroblasts,POSTN+fibroblasts,PDGFRA+fibroblasts,endothelial,myeloid cells,and mast cells.Different types of GC harbored distinctepithelial tumor subpopulations,and squamous cell carcinoma could bedifferentiated from adenocarcinoma cells.Abundant immune cells infiltratedinto adenocarcinoma and squamous cell carcinoma,rather than neuroendocrineneoplasms,which showed significant enrichment of stromal cells.CD4+/FOXP3+T-reg and CD4+/CXCL13+T helper cells with higher exhausting biomarkers,aswell as a dynamic lineage transition of tumor-associated macrophages fromCCL20hi/CD163lo,CCL20lo/CD163hi to APOE+,were identified in GC tissues,suggesting the immunosuppressive and tumor-promoting status of immune cells inTME.Two distinct endothelial cells(KDR+and ACKR1+),which were involved inangiogenesis and lymphangiogenesis,showed remarkable ligand-receptorinteractions with primary GC cells and macrophages in gallbladder tumors.

Conclusions:This study reveals awidespread reprogramming across multiple cell populations in GC progression,dissects the cellular heterogeneity and interactions in gallbladder TME,andprovides potential therapeutic targets for GC.

劉法濤研究員點評：

本研究針對膽囊癌這一細胞異質性高、預后差的惡性腫瘤，利用單細胞測序技術研究了腫瘤內異質性和微環境（TME）。研究的主要結論包括整體細胞亞群大類的描述、不同病理類型膽囊癌TME的異同以及上皮細胞和免疫細胞之間的受體-配體相互作用等。

本研究納入了5例患者的樣本，但覆蓋的膽囊癌病理類型比較全面，包括了腺癌、鱗狀細胞癌和神經內分泌癌，也覆蓋了對應的淋巴結轉移樣本。

本研究測序技術采用的是BD的平臺。目前市場上10X Genomics以及BD Rhapsody是兩個單細胞測序的主流技術，從技術原理上都是基于UMI（Unique Molecular Identifier）+CL（Cell Label）的技術。發布時間上，10X Genomics技術稍占優勢，但兩種技術目前都有高質量的論文支撐。

本研究的后續研究可以針對文中的新發現開展下游的功能學機制探究。后續研究中也可以結合多組學研究，比如與膽囊癌基因組突變譜、bulk-RNAseq、蛋白組學等的聯合等，可能提供更豐富的結果。

此外，本文討論部分也提到，由于受限于樣本量，膽囊神經內分泌癌的TME特征等結論，需要在更大的樣本隊列中進行驗證。

總的來講，本研究對膽囊癌的單細胞水平特征和免疫微環境進行了闡述，為后續的深入研究以及臨床轉化提供了參考。

4

Ⅱ期研究：mFOLFIRINOX在晚期膽囊癌中的療效（JCO新子刊，暫無IF）

摘要（上下滑動查看）：

Materials and methods:We conducted aprospective,phase II single-arm pilot study.Inclusion criteria werehistologically proven GBC and Eastern Cooperative Oncology Group 0-1.Primaryend points were overall response rates and overall survival.The followingtreatment was given:oxaliplatin 85 mg/m2,leucovorin 400 mg/m2,and irinotecan150 mg/m2,all once on day 1,fluorouracil 2,400 mg/m2 continuous intra-venousinfusion over 46 hours repeated every 2 weeks,and maximum 12 doses,withprimary granulocyte colony-stimulating factor prophylaxis.

Results:Between February 2019 and July2020,29 patients with unresectable GBC were enrolled.The median age was 52years,and 18 were females.The Eastern Cooperative Oncology Group was 0 in 4.Five had bilirubin>normal,and 15 each had high serum alkaline phosphataseand carbohydrate antigen 19-9.Twenty-five patients had stage IV disease,andremaining unresectable locally advanced disease.A median of 8.5 cycles wasgiven,and 11 completed treatment.Nine stopped chemotherapy because ofprogression,and one because of toxicity,and treatment is ongoing in three.Twenty-two required dose reduction.A treatment delay of 1-2 weeks was seen in25 patients.Best response was complete response 1,partial response 13(overall response rate 48.2%),and stable disease 9.Four patients withmetastatic disease underwent R0 resection.As on cutoff date,nine aresurviving(three without disease).Eighteen died of PD,and in two,cause wasunknown.There was no toxic death.The median overall survival andprogression-free survival were 309 and 252 days,respectively.Twenty-threepatients experienced grade III or IV toxicity,and common were diarrhea(13),vomiting(12),and anemia(7).

Conclusion:First-line modifiedflourouracil,oxaliplatin,and irinotecan is feasible in unresectable GBC withencouraging responses.Toxicities are higher but manageable.Higher responserates make this an option to explore in borderline resectable cases.

李茂嵐教授點評：

膽囊癌起病隱匿，早期無明顯癥狀，大部分患者確診時已處于疾病晚期，失去手術機會。化療是晚期膽囊癌患者治療的主要方式。

NCCN指南推薦吉西他濱聯合奧沙利鉑（GEMOX）為晚期膽囊癌的一線治療策略，然而臨床客觀緩解率僅30%左右，無法滿足患者的治療需求，臨床上亟需尋找新的化療方案改善膽囊癌的不良預后。

mFOLFIRINOX方案（氟尿嘧啶+伊利替康+奧沙利鉑）是轉移性胰腺癌患者的一線化療方案，與GEMOX相比，FOLFIRINOX在轉移性胰腺癌中顯示出更高的生存率。

由于胰腺腫瘤和膽道腫瘤在組織學、生物學以及治療方法的相似性，mFOLFIRINOX應用于晚期膽囊癌治療受到越來越多中心的關注。

本研究中系單臂II期臨床實驗，該研究招募了29例不可切除或轉移性膽囊癌患者，行mFOLFIRINOX方案化療，直到疾病進展、無法耐受的毒性或治療時間達6個月。

其客觀緩解率和疾病控制率分別為48.3%和79%，中位總生存期和中位無進展生存期分別為10.3個月和8.4個月。不良反應主要表現為腹瀉、嘔吐和貧血等。

我們中心也有類似的結果，相比于GEMOX方案，mFOLFIRINOX方案顯著延長了晚期膽囊癌患者的PFS（5.8 vs 3.3m）和OS（12.8vs 8.4m）。這些結果表明mFOLFIRINOX方案在晚期膽囊癌中是安全可行的，并且表現出較好的反應率和生存率。

期待進一步的多中心RCT研究，為mFOLFIRINOX方案在膽囊癌中的應用提供更有說服力的數據，改變目前晚期膽囊癌的不良預后。

5膽囊管癌新分型（IF：6.242）

摘要（上下滑動查看）：

Background:Cystic duct carcinoma(CDC)isa rare biliary malignancy with a low incidence and poor prognosis.However,theclinical landscape of the disease has not been clarified and no widelyapplicable classification system has been developed.

Methods:Sixty-two patients with CDC wereincluded in this retrospective study,and a new classification system wasestablished using imaging data.Blood indices,radiological characteristics,pathological features,surgical procedures,and overall survival data were collected.The efficacy of the new classification in predicting resectability wasevaluated using receiver operating characteristic(ROC)curves,and K-meansclustering and t-distributed stochastic neighbor embedding were applied toverify the conclusion.

Results:The pT stage of patients with typeII CDC was significantly worse than that of type I.Patients with type II CDCwere more likely to experience distant metastasis and invasion of the nervoussystem,vascular system,and liver.The resectability of patients with type IICDC was significantly worse than that of patients with type I CDC.Patientswith type II CDC had worse prognoses.ROC curve analysis and K-means clusteringrevealed that the new classification could better categorize patients with CDCthan currently available systems.

Conclusion:Patients with type II CDC havesignificantly worse clinicopathological outcomes.The new classification systemhas better accuracy in grouping patients with CDC.

吳向嵩教授點評：

榮幸參加本期點評，此項研究以膽囊管癌為研究對象，基于腫瘤解剖位置特點提出了相關新分型，并分析了新分型下膽囊管癌的臨床病理特點、手術方式差異以及預后，同時還同已存在的幾個分型系統進行了可切除性的比較。

不可否認，膽囊管癌或膽囊頸管癌屬于膽囊癌的一個亞型，病例數相對較少，針對其進行大樣本回顧性研究困難重重。

但由于該位置膽囊癌容易侵犯膽總管，引起梗阻性黃疸，可較早發現，其預后要優于其他部位的膽囊癌。因此，如何有效的進行可切除性的判斷以及手術方案的規劃就顯得格外重要。該分型的提出對于基層醫院進行膽囊頸管的手術具有一定的指導作用。

臨床工作中，膽囊癌侵犯左右肝管是手術切除的一個難點，而本項研究也將這一類型患者單獨分類，并進行了相關臨床病理數據分析，以期更好指導臨床工作，填補了已存在的相關分型的空白，但可能由于樣本量較小，部分生存分析結果未發現顯著性差異，后續仍需更進一步的多中心研究提供更好級別的循證醫學證據。

針對這類型的患者，各中心應充分評估膽管切緣、剩余肝體積、血管侵犯、淋巴結轉移等因素，在多學科討論的基礎上評估手術的可切除性以及相關的治療方案，根治性R0切除應該仍是這類患者治療的首要目標。

6

MCL1參與瘦素介導的線粒體融合并影響膽囊癌藥物敏感性（IF：8.314）

摘要：

Obesity is a risk factor for gallbladder cancer(GBC)development and correlates with shorter overall survival.Leptin,derivedfrom adipocytes,has been suggested to contribute to the growth of cancercells.However,the detailed mechanism of leptin in GBC drug resistance remainsuninvestigated.In this study,it is clinically relevant that GBC patients witha higher BMI(BMI≥24 kg/m2)(n=30)were associated with increased GBC risks,including survival.Moreover,obese NOD/SCID mice exhibited a higher circulatingconcentration of leptin,which is associated with GBC growth and attenuatedgemcitabine efficacy.We further revealed that leptin can inhibitgemcitabine-induced GBC cell death through myeloid cell leukemia 1(MCL1)activation.The transcription factor CCAAT/enhancer-binding protein delta(CEBPD)is responsive to activated signal transducers and activators oftranscription 3(pSTAT3)and contributes to MCL1 transcriptional activationupon leptin treatment.In addition,MCL1 mediates leptin-induced mitochondrialfusion and is associated with GBC cell survival.This study suggests theinvolvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrialfusion and survival.It provides a new therapeutic target to improve theefficacy of gemcitabine in GBC patients.

張飛博士點評：

我們都知道肥胖是膽囊癌發生發展的一個重要危險因素，然而肥胖影響膽囊癌患者預后的具體機制尚不清楚，研究人員首先通過研究肥胖與膽囊癌患者分期、腫瘤轉移、微血管浸潤以及生存期等方面相關性，發現肥胖的膽囊癌患者具有更差的預后和更短的生存時間。

通過高脂飲食建立肥胖小鼠模型發現膽囊癌細胞在肥胖小鼠體內生長速度更快。吉西他濱是膽囊癌一線化療藥物，作者通過將脂肪細胞與膽囊癌共培養發現脂肪細胞能增加膽囊癌細胞對吉西他濱的化療耐藥。

瘦素是脂肪細胞分泌的重要脂肪因子，研究發現脂肪細胞通過瘦素抑制膽囊癌細胞凋亡從而促進其對化療藥物的耐藥作用。

進一步機制研究發現瘦素通過激活膽囊癌細胞STAT3信號通路促進CEBPD轉錄表達，CEBPD上調MCL-1表達促進線粒體融合最終促進細胞存活和增強化療耐藥，這也給了我們提示：通過阻斷STAT3/CEBPD/MCL-1軸能抑制肥胖患者膽囊癌的進展和增強膽囊癌的化療敏感性，希望給膽囊癌的治療帶來新的思路。

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參考文獻：

[1] Perioperative Versus Adjuvant Chemotherapyin the Management of Incidentally Found Gallbladder Cancer(OPT-IN).Ann SurgOncol.2021 Jun 16.doi:10.1245/s10434-021-10277-7.Epub ahead of print.PMID:34132951.

[2]Single-cell RNA-sequencing atlas reveals anMDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladdercancer.J Hepatol.2021 Jun 22:S0168-8278(21)00442-6.doi:10.1016/j.jhep.2021.06.023.Epub ahead of print.PMID:34171432.

[3]Diversity and intratumoral heterogeneity inhuman gallbladder cancer progression revealed by single-cell RNA sequencing.Clin Transl Med.2021 Jun;11(6):e462.doi:10.1002/ctm2.462.PMID:34185421.

[4]Safety and Efficacy of Modified FOLFIRINOXin Unresectable or Metastatic Gallbladder Cancer:A Phase II Pilot Study.JCOGlob Oncol.2021 Jun;7:820-826.doi:10.1200/GO.20.00657.PMID:34086477.

[5]Cystic Duct Carcinoma:A New ClassificationSystem and the Clinicopathological Features of 62 Patients.Front Oncol.2021Jun 11;11:696714.doi:10.3389/fonc.2021.696714.PMID:34178696;PMCID:PMC8225998.

[6]MCL1 participates in leptin-promotedmitochondrial fusion and contributes to drug resistance in gallbladder cancer.JCI Insight.2021 Jun 22:135438.doi:10.1172/jci.insight.135438.Epub ahead of print.PMID:34156978.

本文來源：上海交通大學附屬新華醫院普外科

-End-

加硒教授微信：623296388，送食療電子書，任選一本