摘要
背景：原发性肝细胞癌(hepatocellular carcinoma,以下简称肝癌)是目前最常见的恶性肿瘤之一,发病率和死亡率逐年上升。传统化疗仍是晚期肝癌主要的治疗手段,但多数患者合并有失代偿性肝硬化、肝功能异常,难以耐受常规剂量化疗,是目前肝癌治疗失败、患者无法临床获益的主要原因。因此,寻找更加高效、低毒的治疗模式以提高总体疗效成为目前肝癌治疗研究的重点。分子靶向药物—索拉非尼(Sorafenib)具有直接抑制肿瘤增殖和阻断肿瘤新生血管形成的双重抗肿瘤的作用,使得肝癌治疗效果有所改善；节拍式化疗(metronomic chemotherapy)以血管内皮细胞为作用靶点,与传统化疗相比,具有患者依从性高、耐药现象减少、免疫功能增强、毒副反应小等明显优势。两者联合,作用机制不同,理论上具有协同增效作用。前期实验已证实新一代抗肿瘤药物伊立替康(CPT-11)对肝癌治疗有效,细胞毒作用强于临床常规用药5-FU、DDP。因此本实验选择将Sorafenib与伊立替康(CPT-11)节拍式化疗联合作用于小鼠H22肝癌移植瘤模型,进行疗效评价及基础性研究,为其可能的临床应用提供实验依据。
目的：观察伊立替康(CPT-11)节拍式化疗联合索拉非尼对小鼠移植瘤生长的抑制作用,通过检测血管内皮生长因子(VEGF)及微血管密度(MVD)的表达探讨其可能的相关机制。
方法：
1.建立小鼠H22肝癌移植瘤模型。
2.随机分组：空白对照组(A)：0.9%生理盐水腹腔注射0.2ml,qd,(d1-28)+0.9%生理盐水胃管灌入0.2ml,qd,(d1-20)；Sorafenib组(B)：0.9%生理盐水腹腔注射0.2ml,qd,(d1-28)+索拉非尼50mg／Kg,胃管灌入0.2ml,qd,(d1.-20)：CPT-11节拍式给药(LDM)组(C)：CPT-114mg／Kg腹腔注射0.2ml,qd(d1·28)+0.9%生理盐水胃管灌入0.2ml,qd,(d1-20)；CPT-11常规剂量(TDM)组(D)：CPT-1175mg／Kg腹腔注射0.2ml,2次／W,(d1-28)；Sorafenib+CPT-11(LDM)组(E)：CPT-114mg／Kg腹腔注射0.2ml,qd,(d1-28)+索拉非尼50mg／Kg,胃管灌入0.2ml,qd,(d1-20)。
3.停药24小时后处死小鼠,测量瘤体积及瘤重,计算抑瘤率,并用免疫组化SP法测定肿瘤组织中的VEGF和CD31的表达。
结果：
1.索拉非尼、CPT-11对小鼠H22肝癌的生长均有抑制作用,用药后各实验组肿瘤体积均小于对照组；
2.A、B、C、D、E各组用药后平均瘤重分别为：5.20g、3.53g、3.54g、3.63g、2.34g,统计学分析后,除B、C、D组间无差异外,其余组间比较均有差异,P均<0.05。与对照组相比,以联合用药组肿瘤负荷最低；B、C、D、E组相应抑瘤率分别为：32.01%、31.86%、30.14%、54.9%。各组抑瘤率比较,AB、AC、AD组间无差异,而E组抑瘤率明显高于其他四组,AE组比较P<0.01；
3.小鼠一般状态观察：A、E组小鼠出现进食饮水减少、精神萎靡、体重下降等症状较其他实验组更为明显,并且E组小鼠出现明显腹泻；
4.HE染色：A组大部分肿瘤细胞生长旺盛,核分裂相多见,只有少量细胞出现坏死。B、C、D、E组瘤体内坏死组织多,多数肿瘤细胞有细胞核结构不清、核固缩、核碎裂及核溶解现象,残存细胞核分裂减少,以E组肿瘤细胞坏死最为明显；
5.免疫组化检测VEGF,与A组相比,B、C、E组VEGF表达显著减少,(P均<0.05),其中以E组最为明显。AD组间无明显差异；
6.免疫组化检测CD31,高倍镜下,A、D组可见大量染成棕黄色的血管内皮,而B、C、E组被着色的血管内皮较少。计数各组MVD值并做组间比较,AB、AC、AE组间差异显著,P均<0.05,AD间无差异。BE、CE组间差异显著,P均<0.01。
结论：
1.索拉非尼和伊立替康节拍式化疗均可抑制小鼠H22肝癌移植瘤生长,联合用药抑制作用更强,表现为协同效应。
2.两药联合能够协同抑制VEGF、MVD表达,抑制肿瘤血管形成
3.两药联合不增加药物的毒副作用。
Background:HCC (hepatocellular carcinoma) is one of the most common malignant tumor in worldwide,and medical chemotherapy still is a major treatment in advanced hepatocellular carcinoma. Most patients associated with decompensated cirrhosis and liver function deterioration,all of these had increased the difficulty of HCC treatment and leads to the failure of chemotherapy.Therefore,searching for a new way with more efficient and low toxicity to improve the overall efficacy of the HCC treatment had became the current focus.The emergence of molecular targeted drugs-Sorafenib give hope to the treatment of HCC,but the overall effect was unsatisfactory.The rapid proliferation of vascular endothelial cells were viewed as the target of metronomic chemotherapy.Compared with traditional chemotherapy,it has many advantages,such as:high compliance of patients, reduction of drug resistance,enhancing immune function, little toxicity,et al.These two methods have different mechanisms,so the combination of them may has synergism in theory.In the other hand, Irinotecan(CPT-11) has been confirmed that it has the effect of HCC treatment,and the cytotoxicity is stronger than 5-Fu and DDP in the early experiments.So in this study,Sorafenib combined with metronomic Irinotecan(CPT-11) chemotherapy were used on H22 liver cancer tumor-bearing mice,confirmed that the two combined had a synergistic anti-tumor effect and provided experimental evidengce for possible clinical application.It was.significant for improving the overall efficacy of the HCC treatment.
Objective:
To observe the inhibition effects of low dose CPT-11 Combined with Sorafenib on the growth of tumor transplanted from a mouse and its effect on the expression of vascular endothecial growth factor(VEGF) and microvessel density(MVD).
Methods:
1. mouse tumor model was set up by implanting H22 mouse liver cancer cells under the skins of mouse.
2. Seventy-five experimental mice were randomly divided into five groups:control group(A),Sorafenib-treated group(B),CPT-11(LDM)-treated group(C),CPT-11(TDM)-treated group(D) and combination-treated group(E). Evaluate volume of tumor in five groups before and after treatment.
3. Execute the mice at the twenty-eight day after treatments,to strip and weigh the neoplasm for evaluating the inhibition of the anti-tumor. Determine tumor tissue VEGF protein expressions and micro-vessel density(MVD),(assessed by CD31 immunohistoehemistry).
Results:
1. Both Sorafenib and CPT-11 affected the growth of tumor,The mean tumor volume of control group is the largest one in all groups after treatments.
2. The mean tumor quality was 5.20g,3.53g,3.54g,3.63g,2.34g and the inhibition rate was 32.01%,31.86%,30.14%,54.9%in B,C,D,E respectively. The combination group had lower tumor burden and higher inhibition rate than the other groups.
3. The mice of A,E appear to reduce the consumption of drinking and food, low spirit,weight loss more obvious than other groups.Even there were significant diarrhea in mice of E.
4. HE staining:Most tumor cells of A group had vigorous growth and mitotic more common,only a few cells were necrotic.In the other groups, most tumor tissue were necrotic,and the phenomenon of nuclear condens-ation,nuclear fragmentation and nuclear dissolution were more common, especially in E group.
5.Immunohistochemical detection of VEGF:compared with A,B,C,D groups significantly red(?)ced VEGF expression,(P all<0.05),of which the most significant E group.No significant difference between the AD group;
6.Immunohistochemical detection of CD31:In high magnification,Aand Dgroup showed a lot of dyed brown vascular endothelium,while B,C,E groups were less colored vascular endothelial.Counted MVD in each group, AB,AC,AE,BE,CE had significant differences between groups,while AD had no difference.
Conclusion:
Metronomic CPT-11 chemotherpy combined with Sorafenib have significant inhibitory effect on the growth of the tumors and the expression of the VEGF and MVD in H22 liver cancer tumor-bearing mice.Metronomic chemotherpy combined with molecularly targeted therapy may be a new way of anti-tumors.
引文
1. Caballero M,Grau J J, Blanch,et al.Serum vascular endothe-lial growth fact or as a predictive factor in metronomic(weekly) Paclitaxel treatment for advanced head and neck cancer[J].Arch Otolaryngol Head Neck Surg,2007,133(11):1143-1148.
2. Folkman J.Tumor angiogenesis:therapy Engl J Med,1971,285:1182-1186.
3. SandlerA, GrayR,Brahmer J.et al.Randomized phasetrial of paclitaxel plus carboplatin with or without bevacizumab in patients with advanced non-squamous non-small cell lung cancer:An Eastern Cooperative Oncology Group Trial E4599[J].ClinOnco,2005,23:2.
4. Xiuheng Oi,Zhenming Wu,Oi Liu,Shiyu Yu,Study of hepatic arterial infusion of Endostar combined with TACE on advanced hepatocellular carcinoma, Clinical Oncology,2008,10,P568-570.
5.刘秀峰,秦叔逵,王琳等,恩度与化疗联合治疗多种晚期恶性肿瘤的临床观察,临床肿瘤学,2007,7,241-250.
6. Blumenschein GRJr,Gatzemeier U,Fossella F,et al,Phase Ⅱ,Multicenter, Uncon-trolled Trial of Single-Agent Sorafenib in Patients With Relapsed or Refractory Advanced Non-Small-Cell Lung Cancer[J].Journal Of Oncology,2009,8.
7. Smith KJ,Haley H,Hamza S,et al,Eruptive Keratoacanthoma-Type Squamous Cell Carcinomas in Patients Taking Sorafenib for the Treatment of Solid Tumors[J]. Dermatol Surg,2009,4,29.
8. Takezawa K,Okamoto I,Yonesaka K,et al,Sorafenib inhibits non-small cell lung cancer cell growth by targeting B-RAF in KRAS wild-type cells and C-RAF in KRAS mut ant cells[J].Cancer Research,2009,8,6515-652.
9. Drevs J,Fatder J,Eisde S,et al.Antiangiogenic potency of vario chemotherapeutic drugs for metronomic chemotherapy[J].Anticance Res,2004,24(3a):175-63.
10. Bocci G.Nicolaou KC.KerbellL.Protracted 10 dose effects on human endothelia l cell proliferation and survival in vitro reveal a selective antiangiogenic window for various chemotherapeutic drugs[J].Cance Res,2002,62:6938-6943.
11. Kamat A A,Kim T J,Landen CN,et al.Metronomic chemotherpy enhances the eff icacy of antivascular therapy in ovarian canc[J].Cancer Res,2007,67(1):281-288.
12. Garda-Barros M,Parris F,Cordon-Cardo C,et al.Tumor response to radiotherapy regulated by endothelial cell apoptosis[J].Science,2003,300:1155-1159.
13. Stoelting S,Trefzer T.Kisro J,et al.Low-dose oral metronomic chemotherapy prevents mobilization of endothelial progenitor cells into the blood of cancer patients,In Vivo.2008,22(6):831-6.
14. Bertolini F,Paul S,Mancus o P,et al.Maximum tolerable dose and low dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells[J].Cancer Res,2003,63:4342-4346.
15. Dawson DW,Pearce SF,Zhong Retal Cd.36 mediates the in vitro inhibitory eff ects of thrombospondin-lonendothelial cells[J].Cell Biol,1997,138(6):707-717.
16.Bocci G,Francia G,Man S,et al.Thrombos pondin-1,a mediator of the antiangi ogenic effects of low dose metronomic chemotherapy[J]. Proc Natl Acad Sci USA, 2003,100:29172-12922.
17. Loven D,Becry E.Yernshalmi R,et al.Daily low-dose/continuous capecitabine combined with neo-adjuvanti chotolaryngol[J].Head Neck Surg,2007,133(11): 1143-1148.
18. Zhou Q,Guo P,Wang X,et al.Preclinical pharma cokinetic and pharma codynamic evaluation of metronomic and conventionaltemozolomide dosing regimens[J]. J Pharmacol Exp Ther,2007,321(1):265-275.
19. Caballero M,Grau J J,Blanch,et al.Serum vascular endothe-lial growth fact or as a predictive factor in metronomic(weekly)Paclitaxel treatment for advanced head and neck cancer[J].Arch Otolaryngol Head Neck Surg,2007,133(11):1143-1148.
20. Park ST.Jang JW.Kim GD et al,Beneficial effect of metronomic chemotherapy on tumor suppression and survival in a ratmodel of hepatocellular carcinoma with liver cirrhosis,2009,8.
21.Mitsuoka A,BabaM,Morikawa S.Enhancement of delayed hyper-sensitivity by depletion of suppress or T cells with cyclophospha-mide in mice[J].Nature, 1976,26(2):77-78.
22. Ghiringhelli F.Menard C,Puiq PE,etal.Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+regulatory T cells and restores T and NK effector functions in end stage cancer patients[J].Cancer Immunol Immunother,2007,56(5):641-648.
23. Hermans IF,Chong TW,PalmowskiMJ,et al.Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model[J].Cancer Res,2003,63:8408-8413.
24. Orlando L,Cardillo A,Ghisini R,et al.Trastuzumab in combination with metro nomic cyclophosphamide and methotrexate in patients with HER-2 positive met astatic breast cancer[J].BMC Cancer,2006,6(1):225-233.
25. Coleman M,Martin P,Ruan J,et al.Prednisone etoposide,pro-carbazine,and cyclophosphamide(PEP-C)oral combination chemotherapy regimen for recurring refractory lymphoma:low-dose metronomic,multidrng therapy[J].Cancer, 2008,112(10):2228-2232.
26.26. Santosh Kesari,David Schiff,Lisa Doherty,et al.Phasell study of metronomic chemotherapy for recurrent malignant gliomas inadults[J].NeuroOncology,2007,9:354-363.
27. Nelius T,Klatte T,de Riese W,Haynes A,Filleur S.Clinical outcome of patients with docetaxel-resistant hormone-refractory prostate cancer treated with second-line cyclophosphamide-based metronomic chemotherapy [J].Medical Oncology,2009,4.
28. Klement G,Baruchel S,Park J,et al.Continuous low-dose therapy with vinblastine and VEGF receptors-2 antibody induces sustained tumor regression without overt toxidty[J].Clin Invest,2000,105:R15-R24.
29. Grothry A,Future directions in vascular endothelial growth factor-targeted therapy for metastatic colorectal cancer[J].Sem in Oncol,2006,33(5 suppl10): 41-49.
30. Klement G,Huang P,Mayer B,et al,Differences in therapeutic indexes of combination metronomic chemotherapy and an anti-VEGFR-2 antibody in multidrug-resistant human breast cancer xenografts[J].Clin Cancer Res,2002,8 (1):221-232.
31. Rui Z Bai,Yang Wu,Quan Liu,et al,Suppression of lung cancer in murine model: treated by combination of recombinant human endostsatin adenovirus with low-dose cisplatin,Journal of Experimental& Clinical Cancer Research 2009,28:31 doi:10.1186/1756-9966-28-31.
32. Garcia AA,Hirte H,Fleming G,Yang D,et al,Phase Ⅱ clinical trial of bevacizumab and low-dose metronomic oral cyciophosphamide in recurrent ovarian cancer:a trial of the California,Chicago,and Princess Margaret Hospital phasell consortia[J].Clin Oncol.2008 Jan,26(1):76-82.
33. Garcia-Saenz JA,Martin M.Calles A,et al.Bevacizumab in combination with metronomic chemotherapy in patients with anthracycline and taxane-refractory breast cancer[J].Chemother.2008 Oct,20(5):632-639.
34. Buckstein R,Kerbel RS,Shaked Y,et al.High-Dose celecoxib and metronomic "low-dose"cyclophosphamide is an effective and safe therapy in patients with relapsed and refractory aggressive histology non-Hodgkin's lymphoma[J]. ClinCancer Res,200612(17):5190-519.
1. Parkin DM,Bray F,Ferlay J,et al.Estimating the world cancer burden[J].CA Cancer Clin,2005,55:74.
2. Daveau M,Scotte M,Francois A,et al.Hepatocyte growth factor,transforming growth factor alpha,and their receptors as combined markers of prognosis in hepatocellular carcinoma[J].Mol Car,2003,36(3):130.
3. Llovet JM,Bruix J.Systematic review of randomizedtrials for unresctable hepatocellularcareinoma:Chemoembolization improves survival [J].Hepatology, 2003,37(2):429.
4.晚期肝癌治疗进展-2008年ASCO会议总结,南方医科大学南方医院肿瘤中心,罗荣城,李爱民.
5. Prete SD,Montella L,Caraglia M,et al.Sorafenib plus octreotide is an effective and safe treatment in advanced hepatocellular carcinoma:multicenter phase II So.LAR.study[J].Cancer Chemother Pharmacol,2009.12(30).
6. Lee JM,Sarosy GA,Annunziata CM,et al,Combination therapy:intermittent sorafenib with bevacizumab yields activity and decreased toxicity[J].Br Cancer, 2010,(5).
7. Amaravadi RK,Schuchter LM,McDermott DF,et al,Phase Ⅱ Trial of Temozolomide and Sorafenib in Advanced Melanoma Patients with or without Brain Metastases [J].Clin Cancer Res,2009,15(24):7711-7718.
8. Stempak D,Gammon J,Hahon J,et al.A pilot pharmaco kinetic and anti ang iogenic biomarker study of celecoxib and low-dose metronomic vinblastine or cycloph osphamide in pediatric recurrent solid tumors[J].Pediatr Hematol Oncol,2006,28 (11):720-728.
9. Mitsuoka A,BabaM,Morikawa S.Enhancement of delayed hyper-sensitivity by depletion of suppress or T cells with cyclophospha-mide in mice[J].Nature,1976, 262:77-78.
10. Ghiringhelli F,Menard C,Puiq PE,et al.Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+regulatory T cells and restores T and NK effector functions in end stage cancer patients[J].Cancer Immunol Immunother, 2007,56(5):641-648.
11. Hermans IF,Chong TW,PalmowskiMJ,et al.Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model[J].Cancer Res,2003,63:8408-8413.
12. Orlando L,Cardillo A,Ghisini R,et al.Trastuzumab in combination with metro nomic cyclophosphamide and methotrexate in patients with HER-2 positive met astatic breast cancer[J].BMC Cancer,2006,6(1):225-233.
13.Coleman M,Martin P,Ruan J,et al.Prednisone etoposide, pro-carbazine,and cyclophosphamide(PEP-C)oral combination chemotherapy regimen for recurring or refractorylymphoma:low-dose metronomic,multidrng therapy[J].Cancer 2008, 112(10):2228-2232.
14.Nelius T,Klatte T,de Riese W,Haynes A,Filleur S.Clinical outcome of patients with docetaxel-resistant hormone-refractory prostate cancer treated with second-line cyclophosphamide-based metronomic chemotherapy[J].Medical Oncology,2009,4.
15. Folkman J.Angiogenesis[J].Annu Rev Med,2006,57:1-18.
16. Garcia AA,Hirte H,Flemin Q,et al.Phase Ⅱclinical trial of bevacizumab an d low-dose metronomic oral cyclophospbamide in recurrent ovarian cancer:a trial of the California,Chicago,and Princess Margaret Hospital phase Ⅱ consortia [J]. Clin Oneol,2008,26(1):76-82.
17. Bellmunt J, Trigo JM,Calvo E,et al.Activity of a multitargeted chemo-switch regimen (sorafenib,gemcitabine,and metronomic capecitabine) in metastatic renal-cell carcinoma:a phase Ⅱ study[J].Lancet Oncol.2010 Feb 15.
18.文卫,杨连粤,黄耿文,等.TSP-1和VEGF在肝细胞癌新生血管生成中的作用[J].中华肝胆外科杂,2005,11(1)：17-19.
19.Zhu AX,Blaszkowsky L,Enzinger PC,et al.Phase II study of cetuximab in patients with unresectable or metastatic hepatocellular carcinoma[J].American Society of Clinical On-cology Gastrointestinal Cancers Symposium,SanFrancisco, CA,2006,January 26-28.
20. Suzuki H,Seto K,Shinoda Y,Mori M,Ishimura Y,Suemats M,Ishih Paracrineu pregulationof VEGF receptor mRNA inendothelialcells by hypoxia-xposed Hepgz cells[J].JAM Physiol,1999,276:92-97.
21.Holash J,Maisopiene PC,Compton D,et al.Vesel cooperation,regression and growth in tumors mediated by giopoietlns and VEGF[J].Science,1999,24(22): 1994-1998.
22.李江,王文亮,刘斌.人类肝细胞肝癌的细胞凋亡和癌控[J].世界华人消化杂志,1998,6(12)：1057-1060.
23. Gasparini G,Harris AL.Clinical importance of the determination of tumor angiogenesis in breast cancer much more than a new Prognostic tool[J].Clin Oncol,1995,13:765.
24.周静,邵春奎,冯智英.P53及MVD与肝细胞性肝癌肝移植术后肿瘤复发的关系[J].中国肝胆外科杂志,2008,14(6)：391-394.
25. Maeda K,ChungYS,Takatsuks S,et al.Tumor angiogermesis and tumor cell prolife ration as prognostic indicator in gastric careinoma[J].Br J Cancer,1995,72:319.
26. ScottM,Wilhelm,christopher Carter,Li Ya Tang,et al.BAY43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK Pathway and receptor tyrosine kinase involved in tumor progression and angiogenesis[J]. Cancer Research,2004,64:7099-7109.
27. Kim CK,Choi YK,Lee H,et al.The Farnesyltransferase Inhibitor LB42708 Suppresses VEGF-induced Angiogenesis by Inhibiting Ras-dependent MAPK and PI3K/Akt Signal Pathways[J].2010 (20):Epub ahead of print.
28. Chen YJ,Wang T,Liu DS,et al.Effect of Budesonide on TGF-betal enhanced VEGF Production by Lung Fibroblasts[J].Cell Biol Int.2010(1):Epub ahead of print.
29.陈涛,褚忠华,刘建平,等.体外化疗药物敏感实验对指导原发性肝癌个体化治疗的临床意义[J].癌症,2005,1018-1022.
30.刘芳,张阳,邵淑娟,等.伊立替康及联合5-氟尿嘧啶,顺铂对人肝癌细胞株的细胞毒作用实验研究[J].大连医科大学学报,2008,30(2)：127-140.
31.Gornet JM.Azoulay D,Goldwasser F,et al.Complete remission of unresectable hepatocellular carcinoma on healthy liver by the combination of aggressive surgery and high-dose-intensity chemotherapy by CPT-11[J].Anticancer Drugs, 2000,11(8):649-52.
32. G Bocci,A Falcone,A Fioravanti,et al.Antiangiogenic and anticolorectal cancer effects of metronomic irinotecan chemotherapy alone and incombination with semaxinib[J].British Journal of cancer (2008)98,1619-1629.
33. Vincent P,Zhang X,Chen C,et al.Chemotherapy with the RAF kinase inhibitor BAY43-9006 in combination with irinotecan, vinorelbine,or gemcitabine is well tolerated and efficacious in preclinical xenograft models[J].Clin Oncol,2002, 21:23b.
34. Klement G,Baruchel S,Park J,et al.Continuous low-dose therapy with vinblastine and VEGF receptors-2 antibody induces sustained tumor regression without overt toxidty [J].J Clin Invest,2000,105:R15-R24.
35. Grothry A,Future directions in vascular endothelial growth factor-targeted therapy for metastatic colorectal cancer[J].Sem in Oncol,2006,33 (5 supp 110):S41-49.
36. Rui Z Bai,Yang Wu,Quan Liul,et al.Suppression of lung cancer in murine model: treated by combination of recombinant human endostsatin adenovirus with low-dose cisplatin[J].Journal of Experimental&Clinical Cancer Research 2009,28:31 doi:10.1186/1756-9966-28-31.
37. Garcia AA,Hirte H,Fleming G,Yang D,et al,Phase Ⅱ clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer:a trial of the California,Chicago,and Princess Margaret Hospital phaseⅡ consortia[J].J Clin Oncol.2008 Jan 1;26(1):76-82.
38. Garcia-Saenz JA,Martin M,Calles A,et al.Bevacizumab in combination with metronomic chemotherapy in patients with anthracycline-and taxane-refractory breast cancer[J].J Chemother.2008 Oct;20(5):632-639.