Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma.
Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma.
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DOI：
10.1002/cncr.33393
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发表时间：
2021-05-15
期刊：
影响因子：
6.2
通讯作者：
Izumchenko E
中科院分区：
文献类型：
--
作者：
Shanmugam A;Hariharan AK;Hasina R;Nair JR;Katragadda S;Irusappan S;Ravichandran A;Veeramachaneni V;Bettadapura R;Bhati M;Ramaswamy V;Rao VUS;Bagadia RK;Manjunath A;Manjunath NML;Solomon MC;Maji S;Bahadur U;Bettegowda C;Papadopoulos N;Lingen MW;Hariharan R;Gupta V;Agrawal N;Izumchenko E
口腔鳞状细胞癌（Oral cavity squamous cell carcinoma，OCSCC）是头颈部最常见的恶性肿瘤。虽然晚期疾病患者的存活率仍为约20- 60%，但当在早期检测时，存活率接近80%，迫切需要一种针对具有发展OCSCC高风险的患者的经过充分验证的分析方法。唾液中的肿瘤DNA检测可以提供一个强大的生物标志物平台，克服目前诊断测试的局限性。然而，目前尚无针对口腔鳞状细胞癌患者的常规唾液筛查方法。我们设计了一个定制的下一代测序面板，其具有独特的分子标识符，覆盖了OCSCC中7个频繁突变基因的编码区，并将其应用于从121个未经治疗的OCSCC和匹配的术前唾液标本中提取的DNA。使用严格的变体调用标准，在106个肿瘤中检测到突变，与至少88%的预测检测一致。此外，在原发性恶性肿瘤中鉴定的突变也在93%的唾液样本中检测到。为了确保变体不是导致假阳性调用的错误，我们对该方法进行了多步分析验证：（i）对46个唾液样品进行重新测序，确认了88%的体细胞变体;（ii）在来自11个没有烟草和酒精历史的健康受试者的唾液样品中没有检测到功能相关的突变;（iii）在8次测序运行中使用一组7个合成基因座，我们证实了我们的平台是可重复的，并提供了与液滴数字PCR相当的灵敏度。这些数据突出了在OCSCC诊断后收集的唾液中的驱动基因中进行体细胞突变鉴定的可行性。我们设计了一种具有独特分子标识符的自定义NGS检测，其覆盖了OCSCC中7个频繁突变基因的编码区（该最小基因集预测了88%的OCSCC患者中至少一种体细胞畸变的发生率）。我们的研究结果表明，这种快速，灵敏，非侵入性的方法可用于检测低频率的肿瘤相关突变的唾液标本收集的口腔鳞状细胞癌患者。
Oral cavity squamous cell carcinoma (OCSCC) is the most common head and neck malignancy. While survival of patients with advanced stage disease remains ~20–60%, when detected at early stage, survival approaches 80%, posing a pressing need for a well-validated profiling method for patients with high risk of developing OCSCC. Tumor DNA detection in saliva may provide a robust biomarker platform that overcomes limitations of current diagnostic tests. However, there is no routine saliva-based screening method for patients with OCSCC. We have designed a custom next generation sequencing panel with unique molecular identifiers that covers coding regions of 7 frequently mutated genes in OCSCC, and applied it on DNA extracted from 121 treatment-naïve OCSCCs and matched preoperative saliva specimens. Using stringent variants calling criteria, mutations were detected in 106 tumors, consistent with a predicted detection of at least 88%. Moreover, mutations identified in primary malignancies, were also detected in 93% of saliva samples. To ensure that variants are not errors resulting in false positive calls, we performed a multistep analytical validation of this approach: (i) re-sequencing of 46 saliva samples confirmed 88% of somatic variants; (ii) no functionally relevant mutations were detected in saliva samples from 11 healthy subjects without history of tobacco and alcohol; (iii) using a panel of 7 synthetic loci across 8 sequencing runs, we confirmed that our platform is reproducible and provides sensitivity on par with droplet digital PCR. These data highlight the feasibility of somatic mutation identification in driver genes in saliva collected upon OCSCC diagnosis. We have designed a custom NGS test with unique molecular identifiers that covers the coding regions of 7 frequently mutated genes in OCSCC (this minimal gene set predicted incidence of at least one somatic aberration in 88% of OCSCC patients). Our results demonstrate that this quick, sensitive, and non-invasive method can be used for detection of low frequency tumor-associated mutations in saliva specimens collected from patients with OCSCC.
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影响因子：
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