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：甲状腺超声（US）、细针抽吸活检（FNAB）和分子检测已广泛用于对甲状腺结节的恶性肿瘤风险进行分层。本研究的目的是结合超声特征和基因改变，研究一种细胞学不确定的甲状腺结节 (ITN) 的新诊断方法。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 都会收到 US 评分 (XUS)，根据其患甲状腺癌 (TC) 的风险，范围在 0 到 0.9 之间。从适用于所有患者的病理材料中提取 DNA 和 RNA，并进行 Oncomine 综合检测 v2 (OCAv2) 下一代测序。每个基因改变都根据其与 TC 的关联强度进行注释，其总和作为基因组分类器评分 (XGC)。总风险评分 (TRS) 是 XUS 和 XGC 的总和。生成 ROC 曲线以评估 XUS、XGC 和 TRS 的诊断准确性。结果：研究队列由 50 名患者组成（39 名女性和 11 名男性），年龄 47.5±14.8 岁。三名患者因分子检测失败而被排除。其余 47 名患者中，28 名（59.6%）被诊断为 TC。 BRAFV600E是乳头状TC中最常见的突变，NIFTP中存在PAX8-PPARG融合，Hrthle细胞TC中存在SLX4、ATM和NRAS的致病性变异，髓质TC中存在RET突变。与 XUS 相比，XGC 和 TRS 的诊断准确性显着更高（分别为 88 vs. 62.5%，p < 0.001；85.2 vs. 62.5%，p < 0.001）。然而，这种准确性的提高是由于灵敏度显着提高（分别为 80.7 vs. 34.6%，p < 0.001；84.6 vs. 34.6%，p < 0.001），而没有提高特异性（94.7 vs. 90%，p = 0.55；85.7 vs. .90%，p = 0.63）。结论：对于具有高风险 US 模式（XUS = 0.9）的 ITN 可能不需要进行分子检测，因为仅基于 Xus 的 TC 诊断特异性就足够了，并且分子检测不会提高该诊断的特异性。 OCAv2 可用于指导具有低至中度风险 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 和较低变性温度下的共扩增技术，确定了 57 名患有体细胞 BRAFV600E 突变阳性原发肿瘤的甲状腺乳头状癌 (PTC) 患者的血浆样本中是否存在含有 BRAFV600E 突变 (cfBRAFV600E) 的游离 DNA。 （冷）PCR。在 24/57 (42.1%) 的受检患者中检测到突变 cfBRAFV600E 等位基因。 cfBRAFV600E 的存在与肿瘤大小 (p = 0.03)、多灶性生长模式 (p = 0.03)、甲状腺外总体扩展 (p = 0.02) 和肺微转移 (p = 0.04) 的存在显着相关。在低、中、高风险 PTC 患者中，cfBRAFV600E 的检测率分别为 4/19 (21.0%)、8/22 (36.3%) 和 12/16 (75.0%)。与未检测到 cfBRAFV600E 的患者相比，检测到 cfBRAFV600E 的患者对治疗反应不佳的可能性高出 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 相结合，能够在 PTC 患者的液体活检中检测到 BRAFV600E，并可能有助于识别结构或生化风险增加的 PTC 患者。对治疗的不完全或不确定的反应。