| 摘要: |
| 【】 目的 探究基因组图谱中可作为非小细胞肺癌(NSCLC)放化疗(CRT)疗效以及胸部放射性毒性预测生物标志物的基因。方法 前瞻性连续纳入在2022年3月至2024年3月期间在我院单中心接受根治性CRT(dCRT)治疗的III期NSCLC患者55例,41例为鳞状细胞癌(SCCs)患者,其他为腺癌(ADCs)。所有患者均采集制备无细胞DNA(cfDNA),使用安捷伦SureSelect Human All Exome V4试剂盒和Twist Human Core Exome试剂盒完成cfDNA文库制备。使用Illumina Hiseq 4000平台进行全外显子组(WES)测序。筛选基础质量＜30、cfDNA样本覆盖深度＜20、读数＜3个且映射到双链的单核苷酸变异(SNVs)或Indels。通过将靶基因编码区的所有碱基替换和Indels相加来计算肿瘤突变负荷(TMB)。通过FACETS从cfDNA WES数据中估算肿瘤分数(TF),TF%=2/(1/次等位基因频率+1)。使用PrimePCR?ddPCR?突变检测试剂盒进行液滴数字聚合酶链反应(ddPCR)评估突变等位基因频率。所有患者均进行随访观察,根据放射治疗肿瘤组(RTOG)的毒性标准评估对放射性损伤。结果 随访期间,42例(76.36%)患者出现疾病进展,18例(32.73%)患者因癌症相关死亡。治疗期间共有21例患者(38.18%)出现≥2级毒性反应,包括17例患者出现≥2级放射性肺炎,6例患者出现放射性食管炎。共有110个cfDNA配对样本接受了WES,dCRT前后平均TF分别为36.30%和50.30%。体细胞SNVs数量与TF%和肿瘤直径相关(rho=0.257,P=0.033和rho=0.274,P=0.025)。WES与ddPCR的变异等位基因频率相关性良好(rho=0.971,P＜0.001)。经单变量及LASSO-Cox回归分析确定使用吸烟史(OR=2.265,P=0.036)、共济失调毛细血管扩张突变(ATM)基因(OR=2.074,P=0.042)、Kelch样ECH相关蛋白1(KEAP1)基因(OR=2.585,P=0.014)和人类混合谱系白血病基因2(MLL2)(OR=3.202,P=0.011)构建dCRT后无疾病进展生存的预测模型。ATM突变型、KEAP1突变型或MLL2突变型患者的中位无进展生存期分别为8.08个月(vs. 11.04个月,P=0.008,)、7.79个月(vs. 11.04个月,P=0.001)和5.95个月(vs. 9.67个月,P＜0.001)均较野生型基因患者更短。通过富集分析发现,锌指蛋白217(ZNF217)和DNA聚合酶δ1催化亚基(POLD1)编码基因与胸部放射性损伤有关,ZNF217或POLD1突变使得≥2级胸部放射性损伤发生风险分别增加5.325倍(95%CI:1.098～25.827,P=0.038)和9.756倍(95%CI:1.670～57.008,P=0.011)。结论 建议临床对携带ATM、KEAP1、MLL2、ZNF217、POLD1等这些基因突变的不可切除III期NSCLC患者进行放疗时格外谨慎。 |
| 关键词: 非小细胞肺癌 基因组图谱 放化疗 放射性毒性 疗效 |
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| 基金项目:湖北省自然科学基金面上项目(2022CFB078) |
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| Screening of new predictive biomarkers related to the efficacy and thoracic radiation toxicity of radio-chemotherapy in non-small cell lung cancer using genomic profiling |
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Liu Yuanxiang
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Hubei Cancer Hospital
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| Abstract: |
| 【】 Objective: To identify genes in genomic profiling that can serve as predictive biomarkers for chemoradiotherapy (CRT) efficacy and thoracic radiation toxicity in non-small cell lung cancer (NSCLC). Methods: A total of 55 patients with III stage NSCLC who received radical CRT (dCRT) at our single center between March 2022 and March 2024 were prospectively and consecutively enrolled, including 41 squamous cell carcinoma (SCC) patients and others with adenocarcinoma (ADC). Cell-free DNA (cfDNA) was collected and prepared from all patients. cfDNA library preparation was performed using the Agilent SureSelect Human All Exome V4 Kit and Twist Human Core Exome Kit. Whole exome sequencing (WES) was conducted on the Illumina Hiseq 4000 platform. Single-nucleotide variants (SNVs) or indels with base quality＜30, cfDNA sample coverage depth＜20, read count＜3, or mapping to both DNA strands were filtered out. Tumor mutation burden (TMB) was calculated by summing all base substitutions and indels in the coding regions of target genes. Tumor fraction (TF) was estimated from cfDNA WES data using FACETS, with TF% = 2/(1/allele frequency+1). Droplet digital polymerase chain reaction (ddPCR) was performed using the PrimePCR? ddPCR? Mutation Detection Kit to assess mutant allele frequency. All patients were followed up, and radiation-induced injuries were evaluated according to the toxicity criteria of the Radiation Therapy Oncology Group (RTOG). Results: During follow-up, 42 patients (76.36%) experienced disease progression, and 18 (32.73%) died from cancer-related causes. A total of 21 patients (38.18%) developed ≥ grade 2 toxic reactions during treatment, including 17 with ≥ grade 2 radiation pneumonitis and 6 with radiation esophagitis. A total of 110 paired cfDNA samples underwent WES, with mean TF values of 36.30% before and 50.30% after dCRT. The number of somatic SNVs was correlated with TF% (rho=0.257, P=0.033) and tumor diameter (rho=0.274, P=0.025). WES showed a strong correlation with variant allele frequency measured by ddPCR (rho=0.971, P＜0.001). Univariate and LASSO-Cox regression analyses identified smoking history (OR=2.265, P=0.036), ataxia telangiectasia mutated (ATM) gene (OR=2.074, P=0.042), Kelch like ECH associated protein 1 (KEAP1) gene (OR=2.585, P=0.014), and mixed lineage leukemia 2 (MLL2) gene (OR=3.202, P=0.011) to construct a predictive model for disease-free survival after dCRT. Patients with ATM, KEAP1, or MLL2 mutations had shorter median progression-free survival than wild-type counterparts: 8.08 months vs. 11.04 months (P=0.008), 7.79 months vs. 11.04 months (P=0.001), and 5.95 months vs. 9.67 months (P＜0.001), respectively. Enrichment analysis identified zinc-finger protein 217 (ZNF217) and DNA polymerase delta 1 catalytic subunit (POLD1) as genes associated with thoracic radiation injury. ZNF217 or POLD1 mutations increased the risk of ≥ grade 2 thoracic radiation injury by 5.325-fold (95%CI: 1.098～25.827, P=0.038) and 9.756-fold (95%CI: 1.670～57.008, P=0.011), respectively. Conclusion: Clinicians are advised to exercise extra caution when administering radiotherapy to unresectable III stage NSCLC patients with ATM, KEAP1, MLL2, ZNF217, or POLD1 gene mutations. |
| Key words: Non-small cell lung cancer Genomic profiling Chemoradiotherapy Radiation toxicity Efficacy |
