紫杉醇相关外周神经病变的单核苷酸多态性及黑色素瘤不同转移灶免疫谱的差异研究

发布时间：2018-01-15 13:10

本文关键词：紫杉醇相关外周神经病变的单核苷酸多态性及黑色素瘤不同转移灶免疫谱的差异研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：[研究背景]化疗介导的外周神经病变是常见的剂量限制性药物不良反应,影响患者生活质量。紫杉醇为常用的化疗药物,寻找与其介导的外周感觉神经病变(peripheral sensory neuropathy,PSN)相关的标记物具有重要意义。过去研究多集中在临床病理因素上,结论不一。近年来,随着药物基因组学的发展,一些可能与2/3级紫杉醇介导PSN相关的单核苷酸基因多态性(single nucleotide polymorphism,SNP)位点在其他人种中得到证实。然而目前缺乏在中国汉族(Han Chinese in Beijing,CHB)人群中的相关研究数据。本研究旨在探讨在CHB人群中,与2/3级紫杉醇介导PSN发生风险相关的SNP位点。[研究方法]在2014年5月至2016年12月期间,入组北京协和医院216名接受紫杉醇方案化疗的中国汉族患者,提取外周血DNA。根据NCI-CTCAE标准,由受训的临床医生完成患者随访及PSN分级。查阅文献,筛选所有全基因组关联分析(genome wide association study,GWAS)研究或单基因研究报道的,在其他人种中,与2级及以上紫杉醇介导PSN可能相关,且在NCBI-dbSNP数据库的CHB人群中,最小等位基因频率(Minor allele frequency,MAF)0.05的候选SNP位点。使用Sequenom MassARRARY iPLEX平台测定患者DNA的候选SNP基因型。分析各SNP位点以及临床病理因素与2/3级紫杉醇介导PSN发生的相关性。[研究结果]最终209名患者纳入分析,在34个通过质控的候选SNP位点中,仅 rs4141404:CA(LIMK2 与 2/3 级 PSN 显著相关(OR 4.32,95%CI 2.37-7.89,校正 p0.0001),rs501461:TG(GLIS3)(OR 1.77,95%CI 1.07-2.92,p=0.027,校正p=0.75)的Bonferroni校正p值未达统计学意义。临床病理单因素logistic回归中,既往铂类用药史与发生2/3级PSN显著相关(OR 1.99,95%CI 1.07-3.69,p=0.03)。在多因素logistic回归中,rs4141404:CA(LIMK2)多态性以及既往铂类用药史与2/3级PSN发生风险相关。[研究结论]本研究是首个在最大队列的中国汉族人群中,独立开展的紫杉醇介导PSN的药物基因组学研究。Rs4141404:CA(LIMK2)多态性可能与发生紫杉醇介导的2/3级PSN相关。既往有铂类用药史这一临床病理因素可能增加紫杉醇介导的2/3级PSN的发生风险;这有助于指导紫杉醇化疗方案的个体化选择。[研究背景]黑色素瘤是最具有侵袭性的皮肤癌症之一,随着免疫治疗的发展,晚期患者的总生存得到了大大提高,但针对治疗反应的临床研究发现,具有肝转移的患者比具有肺转移的患者治疗反应更差。长期以来,研究显示免疫系统对肿瘤的生发和调节起重要作用。研究不同转移病灶的免疫谱可以帮助人们更好的理解病灶之间的差异特点,为临床治疗策略提供一定的启示。[研究方法]利用澳大利亚墨尔本Peter MacCallum癌症中心募集的10名患者的尸检癌症转移灶标本,对福尔马林固定石蜡包埋的标本(FFPE)进行肿瘤灶的显微解剖,使用nCounter Nanostring(?)技术进行免疫谱mRNA的测定,对所得数据进行肝脏转移灶对比非肝脏转移灶的差异表达分析,使用ClueGO进行差异表达基因构成分析。使用逆转录实时定量PCR(qPCR)对差异表达明显的CRP和C5基因进行测定,验证Nanostring的结果。针对差异表达最显著的基因,在手术活检标本中进行免疫组化进一步验证。对所有病灶进行CD3和PD-L1免疫组化染色,使用Vectra自动数字定量系统进行定量,比较组间差异。[研究结果]由于1个肝脏转移灶的基因表达数据标准分数(z-score)＞3.0而被排除,分析共纳入9个肝脏转移灶、21个非肝转移灶、5个正常组织。肝脏转移灶和非肝转移灶的免疫系统基因谱中,差异表达最大的基因是C反应蛋白(CRP),显著差异的基因主要涉及补体激活途径。针对同一标本,qPCR对CRP和C5基因验证的结果与Nanostring高度一致。进一步在3个手术肝转移标本、7个非肝手术转移标本进行CRP免疫组化检查,其结果与Nanostring数据结果一致。此外,在肝脏和非肝脏转移灶的肿瘤中心和浸润边缘,CD3和PD-L1表达水平均较低,组间无显著差异。[研究结论]黑色素瘤患者肝转移病灶和非肝转移病灶中,CRP、补体等表达差异是构成其免疫基因表达的主要差别,该研究结果可能为临床免疫治疗提供一定的靶向治疗策略。
[Abstract]:[background] peripheral neuropathy chemotherapy mediated adverse reactions of drugs is a common dose limiting, affect life quality of patients. Paclitaxel chemotherapy drug used, find its mediated peripheral sensory neuropathy (peripheral sensory, neuropathy, PSN) has important significance related to mark the previous research. Focus on the clinical pathological factors, not a conclusion. In recent years, with the development of pharmacogenomics, some may be mediated by paclitaxel and 2/3 single nucleotide polymorphisms related to PSN (single nucleotide polymorphism, SNP) who is confirmed in other ethnic groups. However, the lack of China Han (Han Chinese in in Beijing CHB), relevant research data in the crowd. This study aims to investigate among CHB, 2/3 and paclitaxel mediated PSN risk related SNP locus. Research methods in May 2014 to 2016 12 During the month, the group of 216 Peking Union Medical College Hospital received paclitaxel chemotherapy China Han patients, extracted from peripheral blood of DNA. according to the NCI-CTCAE standard, by trained clinicians and patients completed follow-up PSN grading. The literature, all screening analysis of genome-wide association (genome wide association study, GWAS) or of single gene reported in. In other races, and 2 and above of paclitaxel mediated by PSN may be related, and in the NCBI-dbSNP database of the CHB population, the minimum allele frequency (Minor, allele frequency, MAF) SNP 0.05. The candidate candidate loci SNP genotype patients were determined using Sequenom DNA MassARRARY iPLEX platform. The SNP locus and clinical analysis pathological factors and 2/3 Kisugi Suke correlation between the results of the occurrence of PSN. Of 209 patients included in the final analysis], in 34 through the quality control of candidate SNP loci, only rs4141404: CA LIMK2 was significantly correlated with 2/3 level (PSN (OR 4.32,95%CI 2.37-7.89, P0.0001, rs501461:TG correction) (GLIS3) (OR 1.77,95%CI 1.07-2.92, p=0.027, adjusted p=0.75) Bonferroni correction p value did not reach statistical significance. The clinical pathology of single factor Logistic regression, 2/3 PSN level was significantly related to previous platinum drug history and occurrence (OR 1.99,95%CI 1.07-3.69, p=0.03). In logistic regression, rs4141404:CA (LIMK2) polymorphism and previous platinum drug history and 2/3 PSN risk related research conclusion. This study is the first in the largest cohort of China Han population, drug paclitaxel mediated genome independently by PSN research.Rs4141404:CA (LIMK2) polymorphism may be associated with the occurrence of paclitaxel mediated 2/3 PSN. With a history of platinum drug history the clinical pathological factors may increase the risk of developing paclitaxel mediated by 2/3 PSN; this will help Individual choice. Background melanoma in paclitaxel chemotherapy is one of the most aggressive skin cancer, along with the development of the immune therapy, the overall survival of patients with advanced has been greatly improved, but the clinical study of the treatment response, patients with liver metastasis than patients with pulmonary reaction metastasis is poor. For a long time, research shows that the immune system of the tumor development and plays an important role in the regulation. The difference between features can help people better understand the immune lesion of different metastatic lesions spectrum, autopsy of 10 patients of cancer metastasis specimens provide inspiration. Some research methods using Australia Melbourne Peter MacCallum raise the cancer center for clinical treatment strategies, on formalin fixed paraffin embedded specimens (FFPE) of microanatomy of tumors, the use of nCounter (Nanostring ?) determination of immune spectrum mRNA technology, the data were compared between non hepatic metastasis of liver metastasis using ClueGO expression analysis, analyses the differences between the composition of gene expression. Using quantitative reverse transcription PCR (qPCR) expression of CRP gene and C5 gene were determined for obvious differences, confirm the results of Nanostring. According to the differences of expression the most significant gene, immunohistochemistry was performed in surgical biopsy specimens verified. All lesions were stained for CD3 and PD-L1 immune group, the use of Vectra digital automatic quantitative system quantitatively, the difference between groups was compared. Results due to 1 of liver metastasis gene expression data standard score (Z-score) > 3 were excluded from the analysis included a total of 9 liver metastases, 21 non liver metastases and 5 normal tissues. Liver metastasis genes in tumor and non liver metastasis of the immune system, the biggest difference expression The gene is C reactive protein (CRP), significant differences of genes mainly involved in complement activation pathways. For the same specimen, qPCR of CRP and C5 gene results are highly consistent with Nanostring. The transfer samples were further in 3 liver surgery, 7 non metastatic liver surgery were tested by CRP immunohistochemical examination, the result with the data of Nanostring results. In addition, in the liver and non liver metastasis and infiltration of tumor center edge, the expression level of CD3 and PD-L1 were low, no significant difference between groups. Conclusion] patients with liver metastasis of melanoma lesions and non metastatic liver lesions, CRP, the difference is the main difference between the expression of complement structure the immune gene expression, the results of this study may provide a target to treatment strategies for clinical immunotherapy.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.5

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