Application of molecular diagnostics in thyroid cancer

分子诊断在甲状腺癌中的应用

• 批准号: 10255253
• 负责人: Joanna Klubo-Gwiezdzinska
• 金额: $ 46.76万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10255253
• 关键词: Age; Alleles; American; Assessment tool; Biochemical; Biological Assay; Biopsy Specimen; Cells; Characteristics; Cohort Studies; Confidence Intervals; Cytology; DNA; Detection; Diagnosis; Diagnostic; Failure; Female; Fine needle aspiration biopsy; General Population; Goals; Growth; Lung; Malignant Neoplasms; Malignant neoplasm of thyroid; Methods; Microfluidics; Micrometastasis; Molecular; Molecular Profiling; Mutation; Odds Ratio; Operative Surgical Procedures; PPARG gene; Papillary thyroid carcinoma; Pathogenicity; Pathologic; Patients; Pattern; Plasma; Polymerase Chain Reaction; Primary Neoplasm; RNA; ROC Curve; Risk; Risk stratification; Sampling; Specificity; Standardization; Structure; Sum; Temperature; Testing; Thyroid Gland; Thyroid Nodule; Time; Ultrasonography; Unnecessary Surgery; Variant; Woman; aged; base; cancer diagnosis; cancer risk; cell free DNA; diagnostic accuracy; digital; genomic signature; high risk; improved; liquid biopsy; male; medullary thyroid carcinoma; molecular diagnostics; mutant; next generation sequencing; novel diagnostics; response; treatment response; tumor

Background: Thyroid ultrasound (US), fine needle aspiration biopsy (FNAB), and molecular testing have been widely used to stratify the risk of malignancy in thyroid nodules. The goal of this study was to investigate a novel diagnostic approach for cytologically indeterminate thyroid nodules (ITN) based upon a combination of US features and genetic alterations. Methods: We performed a pilot cohort study of patients with ITN (Bethesda III/IV), who underwent surgical treatment. Based on standardized sonographic patterns established by the American Thyroid Association (ATA), each ITN received an US score (XUS), ranging between 0 and 0.9 according to its risk of thyroid cancer (TC). DNA and RNA were extracted from pathologic material, available for all patients, and subjected to Oncomine Comprehensive Assay v2 (OCAv2) next-generation sequencing. Each genetic alteration was annotated based on its strength of association with TC and its sum served as the genomic classifier score (XGC). The total risk score (TRS) was the sum of XUS and XGC. ROC curves were generated to assess the diagnostic accuracy of XUS, XGC, and TRS. Results: The study cohort consisted of 50 patients (39 females and 11 males), aged 47.5 14.8 years. Three patients were excluded due to molecular testing failure. Among the remaining 47 patients, 28 (59.6%) were diagnosed with TC. BRAFV600E was the most common mutation in papillary TC, PAX8-PPARG fusion was present in NIFTP, pathogenic variants of SLX4, ATM, and NRAS were found in Hrthle cell TC and RET mutations in medullary TC. The diagnostic accuracy of XGC and TRS was significantly higher compared with XUS (88 vs. 62.5%, p < 0.001; 85.2 vs. 62.5%, p < 0.001, respectively). However, this increased accuracy was due to significantly better sensitivity (80.7 vs. 34.6%, p < 0.001; 84.6 vs. 34.6%, p < 0.001, respectively) without improved specificity (94.7 vs. 90%, p = 0.55; 85.7 vs. 90%, p = 0.63, respectively). Conclusion: Molecular testing might not be necessary in ITN with high-risk US pattern (XUS = 0.9), as specificity of TC diagnosis based on Xus alone is sufficient and not improved with molecular testing. OCAv2 is useful in guiding the management of ITN with low-to-intermediate risk US features (XUS < 0.9), as it increases the accuracy of TC diagnosis. The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cfBRAFV600E) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cfBRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cfBRAFV600E was significantly associated with tumor size (p = 0.03), multifocal patterns of growth (p = 0.03), the presence of extrathyroidal gross extension (p = 0.02) and the presence of pulmonary micrometastases (p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cfBRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterized by a 4.68 times higher likelihood of non-excellent response to therapy, as compared to patients without detectable cfBRAFV600E (OR (odds ratios), 4.68; 95% CI (confidence intervals)) 1.26-17.32; p = 0.02). In summary, the combination of digital polymerase chain reaction (dPCR) with COLD-PCR enables the detection of BRAFV600E in the liquid biopsy from patients with PTCs and could prove useful for the identification of patients with PTC at an increased risk for a structurally or biochemically incomplete or indeterminate response to treatment.

背景资料：甲状腺超声（US）、细针穿刺活检（FNAB）和分子检测已被广泛用于对甲状腺结节的恶性风险进行分层。本研究的目的是探讨一种新的诊断方法，细胞学不确定的甲状腺结节（ITN）的基础上，美国的特点和遗传变异的组合。方法：我们对接受手术治疗的ITN（Bethesda III/IV）患者进行了一项试点队列研究。根据美国甲状腺协会（ATA）建立的标准化超声模式，每个ITN都接受了US评分（XUS），根据其甲状腺癌（TC）的风险，评分范围在0到0.9之间。从所有患者的病理材料中提取DNA和RNA，并进行Oncomine Comprehensive Assay v2（OCAv 2）下一代测序。每个遗传改变基于其与TC的关联强度进行注释，并且其总和用作基因组分类器得分（XGC）。总风险评分（TRS）是XUS和XGC的总和。绘制ROC曲线以评估XUS、XGC和TRS的诊断准确性。结果：研究队列包括50例患者（39名女性和11名男性），年龄47.5 - 14.8岁。3例患者因分子检测失败而被排除。在其余47例患者中，28例（59.6%）被诊断为TC。BRAFV 600 E是乳头状TC中最常见的突变，PAX 8-PPARG融合存在于NIFTP中，SLX 4、ATM和NRAS的致病性变体存在于Hrthle细胞TC中，RET突变存在于髓质TC中。XGC和TRS的诊断准确性明显高于XUS（分别为88%和62.5%，p < 0.001; 85.2%和62.5%，p < 0.001）。然而，这种准确性的提高是由于灵敏度显著提高（分别为80.7 vs. 34.6%，p < 0.001; 84.6 vs. 34.6%，p < 0.001），而特异性没有提高（分别为94.7 vs. 90%，p = 0.55; 85.7 vs. 90%，p = 0.63）。结论：在具有高风险US模式（XUS = 0.9）的ITN中可能不需要进行分子检测，因为仅基于XUS的TC诊断特异性已经足够，并且分子检测不会改善。OCAv 2可用于指导具有低至中等风险US特征（XUS < 0.9）的ITN的管理，因为它提高了TC诊断的准确性。 血浆中罕见突变靶点的检测（液体活检）已成为评估癌症患者的一种有前途的工具。我们使用微流控数字PCR和在较低变性温度（COLD）PCR下的共扩增来确定来自57名患有甲状腺乳头状癌（PTC）的患者的血浆样品中含有BRAFV 600 E突变（cfBRAFV 600 E）的无细胞DNA的存在，所述患者具有体细胞BRAFV 600 E突变阳性的原发性肿瘤。在24/57例（42.1%）受检患者中检测到突变cfBRAFV 600 E等位基因。cfBRAFV 600 E的存在与肿瘤大小（p = 0.03）、多灶性生长模式（p = 0.03）、甲状腺外大体延伸（p = 0.02）和肺微转移（p = 0.04）显著相关。在低、中和高风险PTC患者中，分别在4/19（21.0%）、8/22（36.3%）和12/16（75.0%）例病例中检测到cfBRAFV 600 E。具有可检测的cfBRAFV 600 E的患者的特征在于，与没有可检测的cfBRAFV 600 E的患者相比，对治疗的非优异反应的可能性高4.68倍（OR（比值比），4.68; 95%CI（置信区间））1.26-17.32; p = 0.02）。总之，数字聚合酶链反应（dPCR）与COLD-PCR的结合能够检测PTC患者液体活检中的BRAFV 600 E，并且可以证明对于识别结构或生物化学不完整或不确定的风险增加的PTC患者是有用的治疗反应。