全外显子组测序筛查食管鳞状细胞癌放疗抵抗相关基因研究

摘要/Abstract

摘要：

目的比较食管鳞状细胞癌（ESCC）术后放疗后不同预后患者的基因谱差异，筛选与放疗抵抗相关的遗传变异。方法选择2015年1月至2019年12月在南通大学附属医院接受根治性手术及术后辅助放疗的32例ESCC患者为研究对象，根据1年内是否出现照射野内复发分为复发组（放疗抵抗组，n=16）和稳定组（放疗敏感组，n=16）。分别提取患者的基因组DNA，利用全外显子组测序（WES）技术进行高通量测序。应用Trimmomatic、BWA、Picard生物信息学分析软件处理数据，通过GATK对比获得比对文件，然后利用Vardict软件从测序数据中筛选出两组的各类遗传变异。采用Kaplan-Meier法估算患者无瘤生存期（DFS）、总生存期（OS）。Cox比例风险回归模型分析影响ESCC患者DFS和OS的独立危险因素。结果经样本数据质量控制，本研究最终纳入26例患者进行后续分析，复发组和稳定组各13例。全组非沉默肿瘤突变负荷中位数为0.95个/Mb，突变碱基替换类型均以C>T的转换为主，其次为C>G的颠换；发生频率最高的遗传变异依次是单核苷酸多态性（SNP）（75.1%）、缺失突变（13.7%）、插入突变（10.5%）；复发组中肿瘤特有突变数目较稳定组稍高（中位突变数分别为36、34），且两组突变频率前十的基因谱明显不同。复发组中检测到392个特有突变基因，前5个分别为：MUC19、NPIPA5、EPPK1、FLG和FOXG1。稳定组检测到192个特有突变基因，前5个分别为TCHH、WNK1、AIM1L、COL6A5和DPCR1。复发组中位DFS和中位OS分别为15.0个月（95%CI为10.1个月~未达到）和26.2个月（95%CI为19.8个月~未达到），稳定组患者均未出现复发或转移。单因素分析发现，GRIK2（χ²=6.81，P=0.009）、MUC4（χ²=4.25，P=0.039）、MUC5B（χ²=4.03，P=0.045）、PRRG1（χ²=5.15，P=0.023）基因突变、3p缺失（χ²=4.16，P=0.041）和14q缺失（χ²=7.09，P=0.008）与DFS相关；FLG（χ²=6.41，P=0.011）、NPIPA5（χ²=4.57，P=0.033）、PKD1L2（χ²=6.41，P=0.011）、FOXG1（χ²=4.57，P=0.033）基因突变、3p缺失（χ²=3.88，P=0.049）、14q缺失（χ²=5.66，P=0.017）和18p缺失（χ²=3.85，P=0.050）与OS相关。多因素分析发现，14q缺失（HR=3.65，95%CI为1.18~11.32，P=0.025）是影响术后辅助放疗ESCC患者DFS的独立危险因素，FLG（HR=8.94，95%CI为1.52~52.74，P=0.016）、NPIPA5（HR=6.36，95%CI为1.23~33.03，P=0.028）基因突变和14q缺失（HR=3.82，95%CI为1.18~12.31，P=0.025）是影响术后辅助放疗ESCC患者OS的独立危险因素。结论WES结果提示，术后辅助放疗ESCC复发组与稳定组的基因突变类型和突变率基本一致，但两组的基因突变谱明显不同。FLG、NPIPA5基因突变和14q缺失可作为预测术后辅助放疗ESCC患者预后的分子标志物。

关键词:食道鳞癌,全外显子组测序,遗传变异,放疗抵抗

Abstract:

ObjectiveTo compare the genetic spectrums of esophageal squamous cell carcinoma （ESCC） patients with different prognosis after postoperative radiotherapy and to screen the genetic variants associated with radiotherapy resistance.MethodsA total of 32 ESCC patients who received radical surgery and postoperative adjuvant radiotherapy in Affiliated Hospital of Nantong University from January 2015 to December 2019 were selected as the study objects. According to whether there was any recurrence in the radiation field within 1 year， they were divided into a recurrence group （radiotherapy resistance group，n=16） and a stable group （radiotherapy sensitive group，n=16）. Genomic DNA was extracted from patients and high-throughput sequencing was performed using whole exome sequencing （WES） technology. Biological information analysis software Trimmomatic， BWA and Picard were used to process the data and the alignment files were obtained by GATK comparison， then Vardict software was used to screen out various genetic variants from the sequencing data. The disease free survival （DFS） and overall survival （OS） were estimated by Kaplan-Meier method. Cox proportional hazard regression model was used to analyze the independent risk factors of DFS and OS of ESCC patients.ResultsAfter quality control of the sample data， 26 patients were finally included in this study for follow-up analysis， 13 in each of the recurrence and stable groups. The median tumor mutation burden of non-silent tumors in the whole group was 0.95 mutations/Mb. The substitution types of mutant bases were mainly C>T conversion， followed by C>G transmutation. The genetic variants with the highest frequency were single nucleotide polymorphism （SNP）（75.1%）， deletion mutation （13.7%） and insertion mutation （10.5%）. The number of tumor-specific mutations in the recurrence group was slightly higher than that in the stable group （median mutation number was 36 and 34， respectively）， and the top ten gene profiles of mutation frequency were significantly different between the two groups. In the recurrence group， 392 unique mutated genes were detected， and the top five were MUC19， NPIPA5， EPPK1， FLG and FOXG1. In the stable group， 192 unique mutation genes were detected， and the top five were TCHH， WNK1， AIM1L， COL6A5 and DPCR1. The median DFS and OS were 15.0 months （95%CI： 10.1 months-not reached） and 26.2 months （95%CI： 19.8 months-not reached） in the recurrence group respectively， and no recurrence or metastasis occurred in the stable group. Univariate analysis showed that GRIK2 （χ²=6.81，P=0.009）， MUC4 （χ²=4.25，P=0.039）， MUC5B （χ²=4.03，P=0.045）， PRRG1 （χ²=5.15，P=0.023） gene mutations， 3p deletion （χ²=4.16，P=0.041） and 14q deletion （χ²=7.09，P=0.008） were correlated with DFS. FLG （χ²=6.41，P=0.011）， NPIPA5 （χ²=4.57，P=0.033）， PKD1L2 （χ²=6.41，P=0.011）， FOXG1 （χ²=4.57，P=0.033） gene mutations， 3p deletion （χ²=3.88，P=0.049）， 14q deletion （χ²=5.66，P=0.017） and 18p deletion （χ²=3.85，P=0.050） were associated with OS. Multivariate analysis showed that 14q deletion （HR=3.65， 95%CI： 1.18-11.32，P=0.025） was an independent risk factor for DFS of ESCC patients with postoperative adjuvant radiotherapy， and FLG （HR=8.94， 95%CI： 1.52-52.74，P=0.016）， NPIPA5 （HR=6.36， 95%CI： 1.23-33.03，P=0.028） gene mutation and 14q deletion （HR=3.82， 95%CI： 1.18-12.31，P=0.025） were independent risk factors for OS of ESCC patients with postoperative adjuvant radiotherapy.ConclusionThe WES results suggest that the types and rates of gene mutations of the ESCC patients with postoperative adjuvant radiotherapy in the recurrence and stable groups are basically the same， but the mutation spectrum of the two groups is significantly different. FLG， NPIPA5 gene mutations and 14q deletion can be used as molecular markers to predict the prognosis of ESCC patients treated with postoperative adjuvant radiotherapy.

Key words:Esophageal squamous cell carcinoma,Whole exome sequencing,Genetic variation,Radiation resistance

陈志明, 陈俊杰, 李李, 丁倩, 韩钰楠, 赵洪瑜. 全外显子组测序筛查食管鳞状细胞癌放疗抵抗相关基因研究[J]. 国际肿瘤学杂志, 2023, 50(10): 592-599.

Chen Zhiming, Chen Junjie, Li Li, Ding Qian, Han Yunan, Zhao Hongyu. Identification of critical radioresistance genes in esophageal squamous cell carcinoma by whole exome sequencing[J]. Journal of International Oncology, 2023, 50(10): 592-599.

图/表7

表1

两组行外科根治性手术及术后辅助放疗ESCC患者临床资料比较 [例（%）]"

本指标	稳定组（n=13）	复发组（n=13）	P值
性别
男	8（61.54）	11（84.62）	0.378
女	5（38.46）	2（15.38）	0.378
年龄（岁）
≤65	9（69.23）	7（53.85）	0.688
>65	4（30.77）	6（46.15）	0.688
病理分级
G1/G1~2	2（15.38）	5（38.46）	0.378
G2/G2~3/G3	11（84.62）	8（61.54）	0.378
T分期
T_1-2	2（15.38）	3（23.08）	<0.999
T₃	11（84.62）	10（76.92）	<0.999
N分期
N₀	5（38.46）	4（30.77）	<0.999
N_1-3	8（61.54）	9（69.23）	<0.999
临床分期
Ⅰ~Ⅱ	5（38.46）	5（38.46）	<0.999
Ⅲ	8（61.54）	8（61.54）	<0.999
放疗剂量（Gy）
45~50	8（61.54）	9（69.23）	<0.999
>50	5（38.46）	4（30.77）	<0.999
辅助化疗
是	7（53.85）	9（69.23）	0.688
否	6（46.15）	4（30.77）	0.688

表1

图1

图2

表2

26例行外科根治性手术及术后辅助放疗ESCC患者基因突变对预后影响的单因素分析"

基因变异因素	例数	中位DFS （月）	χ²值	P值	中位OS （月）	χ²值	P值	基因变异因素	例数	中位DFS （月）	χ²值	P值	中位OS （月）	χ²值	P值
FLG									NPIPA5
野生型	24	NR	2.85	0.091	40.87	6.41	0.011	野生型	24	NR	1.62	0.204	40.87	4.57	0.033
突变型	2	13.17	2.85	0.091	19.73	6.41	0.011	突变型	2	10.10			10.10
GRIK2								PKD1L2
野生型	24	NR	6.81	0.009	36.07	1.94	0.163	野生型	24	NR	2.85	0.091	40.87	6.41	0.011
突变型	2	5.67	6.81	0.009	8.20	1.94	0.163	突变型	2	13.17			19.83
MUC4								FOXG1
野生型	19	24.77	4.25	0.039	33.63	3.75	0.053	野生型	24	NR	1.62	0.204	40.87	4.57	0.033
突变型	7	NR	4.25	0.039	NR	3.75	0.053	突变型	2	10.10			10.10
MUC5B								PRRG1
野生型	24	NR	4.03	0.045	40.87	3.22	0.073	野生型	24	NR	5.15	0.023	36.07	1.19	0.275
突变型	2	8.90	4.03	0.045	17.97	3.22	0.073	突变型	2	8.93			19.93

表2

表3

26例行外科根治性手术及术后辅助放疗ESCC患者基因突变对预后影响的多因素分析"

变异基因	DFS			OS
变异基因	HR值	95%CI	P值	HR值	95%CI	P值
FLG				8.94	1.52~52.74	0.016
GRIK2	4.57	0.08~260.74	0.462	-	-	-
MUC4	0.20	0.02~1.60	0.128	-	-	-
MUC5B	4.00	0.79~20.23	0.093	-	-	-
NPIPA5				6.36	1.23~33.03	0.028
PKD1L2				-	-	-
FOXG1				-	-	-
PRRG1	1.32	0.02~74.67	0.894	-	-	-

表3

表4

26例行外科根治性手术及术后辅助放疗ESCC患者的染色体臂基因拷贝数变异对预后影响的单因素分析"

染色体臂拷贝数因素	例数	中位DFS （月）	χ²值	P值	中位OS （月）	χ²值	P值
3p缺失
野生型	7	NR	4.16	0.041	NR	3.88	0.049
突变型	19	24.37	4.16	0.041	33.80	3.88	0.049
1q扩增
野生型	17	NR	2.73	0.099	53.17	2.67	0.102
突变型	9	14.97	2.73	0.099	26.23	2.67	0.102
14q缺失
野生型	19	NR	7.09	0.008	NR	5.66	0.017
突变型	7	14.97	7.09	0.008	26.23	5.66	0.017
18q缺失
野生型	11	NR	2.30	0.130	NR	2.96	0.085
突变型	15	24.77	2.30	0.130	33.80	2.96	0.085
18p缺失
野生型	14	NR	1.53	0.216	NR	3.85	0.050
突变型	12	24.77	1.53	0.216	33.40	3.85	0.050

表4

染色体臂拷贝数因素	DFS		OS
3p缺失	5.63	0.72~43.90	0.099	3.97	0.51~30.90	0.188
14q缺失	3.65	1.18~11.32	0.025	3.82	1.18~12.31	0.025
18p缺失				2.94	0.89~9.73	0.078

表5

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