Application of molecular diagnostics in thyroid cancer

背景：甲状腺超声（US）、细针穿刺活检（FNAB）和分子检测已被广泛应用于甲状腺结节恶性风险的分层。本研究的目的是基于美国特征和遗传改变的结合，研究一种新的细胞学上不确定的甲状腺结节（ITN）的诊断方法。方法：我们对ITN （Bethesda III/IV）患者进行了一项先导队列研究，这些患者接受了手术治疗。根据美国甲状腺协会（ATA）建立的标准化超声模式，每个ITN根据其甲状腺癌（TC）的风险获得US评分（XUS），范围在0到0.9之间。从病理材料中提取DNA和RNA，供所有患者使用，并进行Oncomine Comprehensive Assay v2 （OCAv2）下一代测序。每个遗传改变根据其与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）为XUS与XGC之和。生成ROC曲线，评估XUS、XGC和TRS的诊断准确性。结果：研究队列包括50例患者（女性39例，男性11例），年龄47.5 ~ 14.8岁。3例患者因分子检测失败而被排除。其余47例患者中，28例（59.6%）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的致病变异，髓质TC中存在RET突变。XGC和TRS的诊断准确率显著高于XUS（分别为88比62.5%,p < 0.001； 85.2比62.5%,p < 0.001）。然而，准确度的提高是由于灵敏度的显著提高（80.7 vs. 34.6%, p < 0.001; 84.6 vs. 34.6%, p < 0.001），而特异性没有提高（94.7 vs. 90%, p = 0.55; 85.7 vs. 90%, p = 0.63）。结论：对于高危US型ITN (XUS = 0.9)，可能不需要进行分子检测，单独基于XUS诊断TC的特异性是足够的，分子检测不能提高TC的特异性。OCAv2有助于指导具有中低风险US特征（XUS < 0.9）的ITN的管理，因为它提高了TC诊断的准确性。

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.