循环血肿瘤DNA含量变化对评价兔VX2肿瘤放疗早期反应的动物实验研究
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摘要
目的观察外周血循环Shope病毒DNA含量变化比较VX2移植瘤放疗早期治疗效果,探讨液态活检技术在临床的潜在应用价值。方法采用组织块大腿外侧皮下接种法建立兔VX2肿瘤模型,随机分为实验组和对照组,实验组给予单剂量20Gy放射治疗。根据已知的Shope病毒基因库设计Shope病毒特征性探针,应用实时定量PCR法检测放疗前1天、放疗后第3天及放疗后第7天血浆中Shope病毒特征性DNA片段含量。于放疗后第7天留取肿瘤组织块行病理组织学分析肿瘤细胞坏死率,分组标准以肿瘤细胞坏死率达80%为界,≥80%者为放疗有效组,小于80%为放疗抵抗组。采用SAS 9. 1统计软件分析,循环血shope病毒DNA含量组间差别采用方差分析,以P <0. 05为差异有统计学意义。结果根据病理肿瘤坏死率分组,对照组n=4,有效组n=6,抵抗组n=6。病理分析发现放疗有效组肿瘤组织存在大量坏死灶,放疗有效组坏死率明显高于放疗抵抗组(P <0. 05);放疗后3天,有效组的循环血shope病毒DNA含量明显高于抵抗组(P <0. 05),放疗后7天,放疗有效组循环血shope病毒DNA含量明显低于抵抗组(P <0. 01)。外周血中shope病毒的动态变化与VX2肿瘤放疗早期病理改变相关。结论循环血Shope病毒DNA可以作为兔VX2肿瘤特征性肿瘤标志物,循环血肿瘤DNA变化在早期监测肿瘤放疗疗效时具应用价值。
Objective To evaluate the potential clinical value of circulating tumor DNA(ctDNA) by observing the changing of DNA levels of Shope virus in peripheral blood of VX2 tumor bearing rabbits undergoing radiotherapy. Methods VX2 tumor bearing animal models were established by tissue transplantation in left thigh of rabbits. The animals were randomly divided into the experimental group and the control group. The experimental group was given single dose of 20 Gy radiotherapy. The Shope virus specific DNA probe was designed according to the known Shope virus gene pool. The DNA fragment of Shope virus was detected by real-time quantitative PCR 1 day before,3 days after the radiotherapy and 7 days after the radiotherapy.Statistical analysis was processed by SAS 9. 1 statistical software. The differences of circulating shope virus DNA levels among three groups were calculated by analysis of variance,with P < 0. 05 as statistically significant cutoff. Results Histopathological analysis of tumor cell necrosis were performed on 7 days after the radiation treatment,with tumor necrosis ratio ≥ 80% as the radio-sensitive group(n = 6),while tumor necrosis ratio less than 80% as the radio-resistance group(n = 6),tumor bearing animal without radiation as the control group(n = 4). Pathological analysis showed that there was a large number of necrotic lesions in radiotherapy group,and the necrosis rate of radio-sensitive group was significantly higher than that of radioresistance group(P < 0. 05). 3 days after the radiotherapy,the circulating shope virus DNA level of sensitive group was significantly higher than that of resistance group(P < 0. 05). At 7 days after the radiotherapy,the circulating shope virus DNA level of radiotherapy sensitive group was significantly lower than that in resistant group(P < 0. 01). The dynamic changes of shope virus DNA in peripheral blood were related to the early pathological changes of VX2 tumor after the radiotherapy. Conclusion Circulating Shope virus DNA can be used as a specific bio-marker of VX2 tumor in rabbits,moreover,circulating tumor specific DNA is useful in the early monitoring of tumor radiotherapy response.
Objective To evaluate the potential clinical value of circulating tumor DNA(ctDNA) by observing the changing of DNA levels of Shope virus in peripheral blood of VX2 tumor bearing rabbits undergoing radiotherapy. Methods VX2 tumor bearing animal models were established by tissue transplantation in left thigh of rabbits. The animals were randomly divided into the experimental group and the control group. The experimental group was given single dose of 20 Gy radiotherapy. The Shope virus specific DNA probe was designed according to the known Shope virus gene pool. The DNA fragment of Shope virus was detected by real-time quantitative PCR 1 day before,3 days after the radiotherapy and 7 days after the radiotherapy.Statistical analysis was processed by SAS 9. 1 statistical software. The differences of circulating shope virus DNA levels among three groups were calculated by analysis of variance,with P < 0. 05 as statistically significant cutoff. Results Histopathological analysis of tumor cell necrosis were performed on 7 days after the radiation treatment,with tumor necrosis ratio ≥ 80% as the radio-sensitive group(n = 6),while tumor necrosis ratio less than 80% as the radio-resistance group(n = 6),tumor bearing animal without radiation as the control group(n = 4). Pathological analysis showed that there was a large number of necrotic lesions in radiotherapy group,and the necrosis rate of radio-sensitive group was significantly higher than that of radioresistance group(P < 0. 05). 3 days after the radiotherapy,the circulating shope virus DNA level of sensitive group was significantly higher than that of resistance group(P < 0. 05). At 7 days after the radiotherapy,the circulating shope virus DNA level of radiotherapy sensitive group was significantly lower than that in resistant group(P < 0. 01). The dynamic changes of shope virus DNA in peripheral blood were related to the early pathological changes of VX2 tumor after the radiotherapy. Conclusion Circulating Shope virus DNA can be used as a specific bio-marker of VX2 tumor in rabbits,moreover,circulating tumor specific DNA is useful in the early monitoring of tumor radiotherapy response.
引文
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[15]SHINOZAKI M,O'DAY S J,KITAGO M,et al. Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy[J]. Clin Cancer Res,2007,13(7):2068-2074.
[16]BIDARD F C,MADIC J,MARIANI P,et al. Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma[J]. Int J Cancer,2014,134(5):1207-1213.
[17]SAUSEN M,PHALLEN J,ADLEFF V,et al. Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients[J]. Nat Commun,2015,6:7686. doi:10. 1038/ncomms8686.
[18]PEREIRA E,CAMACHO-VANEGAS O, ANANd S, et al.Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers[J]. PLo S One,2015,10(12):e0145754.
[19]LIN J C,WANG W Y,CHEN K Y,et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma[J]. N Engl J Med,2004,350(24):2461-2470.
[20]LO Y M,LEUNG S F,CHAN L Y,et al. Kinetics of plasma EpsteinBarr virus DNA during radiation therapy for nasopharyngeal carcinoma[J]. Cancer Res,2000,60(9):2351-2355.
[21]WANG W Y,TWU C W,CHEN H H,et al. Long-term survival analysis of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA levels[J]. Cancer,2013,119(5):963-970.
[22]TWU C W,WANG W Y,LIANG W M,et al. Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma[J]. Int J Radiat Oncol Biol Phys,2007,67(1):130-137.
[23]Cao H,Banh A,Kwok S,et al. Quantitation of human papillomavirus DNA in plasma of oropharyngeal carcinoma patients[J]. Int J Radiat Oncol Biol Phys,2012,82(3):e351-358.
[24]AHN S M,CHAN J Y,ZHANG Z,et al. Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer[J]. JAMA Otolaryngol Head Neck Surg,2014,140(9):846-854.
[2]CARPINETTI P,DONNARD E,BETTONI F,et al. The use of personalized biomarkers and liquid biopsies to monitor treatment response and disease recurrence in locally advanced rectal cancer after neoadjuvant chemoradiation[J]. Oncotarget,2015,6(35):38360-38371.
[3]车莉萍,程超,张桉瑜,等.循环血游离Shope病毒DNA含量与兔VX2肿瘤18F-FDG PET/CT影像表现的相关性分析[J].现代生物医学进展,2014,14(15):2808-2811.
[4]车莉萍,张建,张桉瑜,等.病灶糖酵解总量变化对兔VX2肿瘤放疗反应的早期预测价值[J].中华核医学与分子影像杂志,2015,35(1):48-52.
[5]靳松,沈晋南,王晋,等.新辅助化疗后肿瘤坏死率及碱性磷酸酶同工酶变化与预后的相关性研究[J/CD].中华关节外科杂志:电子版,2012,6(3):370-375.
[6]MANDEL P,METAIS P. Les acides nucléiques du plasma sanguin chez l'homme[J]. C R Seances Soc Biol Fil,1948,142(3-4):241-243.
[7]STEINMAN C R,DEESOMCHOK U,Spiera H. Proceedings:Detection of antibody to nature DNA(N-DNA):increased specificity for active SLE by using a synthetic N-DNA antigen free of contaminating single-stranded DNA(ss-DNA). Arthritis Rheum,1975,18(3):284.
[8]STROUN M, ANKER P, MAURICE P, et al. Neoplastic characteristics of the DNA found in the plasma of cancer patients[J]. Oncology,1989,46(5):318-322.
[9]Umetani N,Giuliano A E,Hiramatsu S H,et al. Prediction of breast tumor progression by integrity of free circulating DNA in serum[J].J Clin Oncol,2006,24(26):4270-4276.
[10]PINGALI S R,JEWELL S W,HAVLAT L,et al. Limited utility of routine surveillance imaging for classical Hodgkin lymphoma patients in first complete remission[J]. Cancer,2014,120(14):2122-2129.
[11]DAWSON S J,TSUI D W,MURTAZA M,et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer[J]. N Engl J Med,2013,368(13):1199-1209.
[12]FORSHEW T,MURTAZA M,PARKINSON C,et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA[J]. Sci Transl Med,2012,4(136):136ra68. doi:10. 1126/scitranslmed. 3003726.
[13]NEWMAN A M,BRATMAN S V,To J,et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage[J]. Nat Med,2014,20(5):548-554.
[14]BETTEGOWDA C,SAUSEN M,LEARY R J,et al. Detection of circulating tumor DNA in early-and late-stage human malignancies[J]. Sci Transl Med,2014,6(224):224ra24. doi:10.1126/scitranslmed. 3007094.
[15]SHINOZAKI M,O'DAY S J,KITAGO M,et al. Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy[J]. Clin Cancer Res,2007,13(7):2068-2074.
[16]BIDARD F C,MADIC J,MARIANI P,et al. Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma[J]. Int J Cancer,2014,134(5):1207-1213.
[17]SAUSEN M,PHALLEN J,ADLEFF V,et al. Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients[J]. Nat Commun,2015,6:7686. doi:10. 1038/ncomms8686.
[18]PEREIRA E,CAMACHO-VANEGAS O, ANANd S, et al.Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers[J]. PLo S One,2015,10(12):e0145754.
[19]LIN J C,WANG W Y,CHEN K Y,et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma[J]. N Engl J Med,2004,350(24):2461-2470.
[20]LO Y M,LEUNG S F,CHAN L Y,et al. Kinetics of plasma EpsteinBarr virus DNA during radiation therapy for nasopharyngeal carcinoma[J]. Cancer Res,2000,60(9):2351-2355.
[21]WANG W Y,TWU C W,CHEN H H,et al. Long-term survival analysis of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA levels[J]. Cancer,2013,119(5):963-970.
[22]TWU C W,WANG W Y,LIANG W M,et al. Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma[J]. Int J Radiat Oncol Biol Phys,2007,67(1):130-137.
[23]Cao H,Banh A,Kwok S,et al. Quantitation of human papillomavirus DNA in plasma of oropharyngeal carcinoma patients[J]. Int J Radiat Oncol Biol Phys,2012,82(3):e351-358.
[24]AHN S M,CHAN J Y,ZHANG Z,et al. Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer[J]. JAMA Otolaryngol Head Neck Surg,2014,140(9):846-854.