Faculty Bibliography

DICER1 encodes an endoribonuclease involved in microRNA maturation and therefore has an important role in gene transcript regulation. Germline mutations scattered along DICER1 are associated with DICER1 syndrome which prominently features thyroid nodules. The tumors typically carry a second, somatic mutation in the RNase IIIb catalytic domain, referred to as "hotspot." These hotspot mutations occur in*1-2% of thyroid papillary carcinomas (PTC). The incidence of the hotspot mutations in thyroid nodules in adults, their association with malignancy and with other, germline DICER1 mutations remain largely unknown. We analyzed 6,734 consecutive clinical FNA samples from typically indeterminate cytology thyroid nodules for hotspot DICER1mutations using ThyroSeq v3 targeted next generation sequencing (NGS) assay from 11/2017-05/2018. Available follow-up was collected. A subgroup of cases underwent full DICER1 coding region and exon-intron boundaries analysis using a custom Fluidigm Access Array followed by NGS on Illumina MiSeq. Somatic DICER1 hotspot mutations were identified in 135 (2.0%) of nodules, with D1810H/V/Y and D1709G/E/N being most common. Median patient age was 37 years (range 19-79 y), 93% were females. Follow-up was available for 27 patients: 15 underwent surgery with benign diagnoses in 9 cases, NIFTP in 5 and follicular variant PTC in 1. Twelve patients were managed non-surgically, including one with a stable nodule harboring DICER1mutation at an allele frequency unchanged over 10 years between FNAs. A subset of 11 positive cases was tested for alteration in the entire DICER1 gene, which confirmed the hotspot mutations in 10 and detected additional alterations in 9 (90%), including non-hotspot mutations in 8 and LOH in 1 case. We report for the first time that likely somatic hotspot DICER1 mutations are relatively common and found in*2% of thyroid nodules in adults, who are typically mid-age women. At surgery, most of these nodules are benign, with*33% risk of NIFTP and*7% risk of follicular variant PTC. Our analysis also shows that somatic hotspot mutations are usually accompanied by a second, loss of function DICER1 mutation, which may in some cases be germline in nature

Afirma GSC utilizes RNA sequencing and machine-learning algorithms to classify cytologically indeterminate thyroid nodules into benign (B) and suspicious (S) categories. Detection of genomic variants and fusions was recently expanded beyond BRAF V600E and RET/PTC1&3 by the Xpression Atlas (XA), which identifies 761 nucleotide variants and 130 fusion gene pairs in 511 genes. Here we used XA to analyze the mutational spectrum of 190 Bethesda III/IV nodules with gold standard histologic diagnoses.190 nodules previously collected in a prospective multicenter blinded trial design were analyzed with the XA.Among the 145 histologically benign nodules, 35 (24%) contained a variant or fusion (XA+). In the 99 benign nodules with GSC-B results there were 15 (15%) with XA variants and none with a fusion. These variants were 7 TSHR, 3 SPOP, 2 EIF1AX, 1 PTEN, 1 TSHR + EZH1, and 1 GNAS. In the 46 benign nodules with GSC-S results, 18 (39%) harbored a variant and 2 (4%) a fusion. There were 9 NRAS, 6 HRAS, 2 TSHR, and 1 SPOP. Two had a PAX8/PPRARG fusion. Among the 45 histologically malignant nodules (41 GSC-S; 91% sensitivity), 22 were XA+ (49% sensitivity). In the 41 malignant nodules with GSC-S results, there were 19 variants (46%) and 2 fusions (5%). The variants were 9 NRAS, 3 HRAS, 3 BRAF V600E, 1 SPOP, 1 KRAS + EIF1AX, 1 EIF1AX, and 1 BRAF K601E. Fusions were BRAF/MKRN1 and 1 ETV6/NTRK3. In the 4 GSC-B false negative nodules (2 PTC, 1 fvPTC, 1HCC), only theHCCcontained a variant (TSHR). In 190 thyroid nodules with definitive histology, malignant nodules were twice as likely to be XA+ than benign nodules (49% vs 24%, p = 0.003 [v2]). Although GSC-S nodules were nearly 3 times more likely than GSC-B nodules to be XA+ (47% vs 16%, p < 0.0001), the PPV for malignancy did not differ among all GSC-S, GSC-S XA+, and GSC-S XA-nodules (47%, 51%, and 43%, respectively; p = 0.77). When XA+, GSC S nodules expressed mainly RAS variants, and GSC B nodules predominantly TSHR variants. Conversely, the NPV for XA was 83%. These findings support GSC as better than XA to rule-out cancer while the addition of XA to GSC-S nodules may provide additional insights into pathway activation and potential cancer treatment targets

Importance/UNASSIGNED:Use of next-generation sequencing of RNA and machine learning algorithms can classify the risk of malignancy in cytologically indeterminate thyroid nodules to limit unnecessary diagnostic surgery. Objective/UNASSIGNED:To measure the performance of a genomic sequencing classifier for cytologically indeterminate thyroid nodules. Design, Setting, and Participants/UNASSIGNED:A blinded validation study was conducted on a set of cytologically indeterminate thyroid nodules collected by fine-needle aspiration biopsy between June 2009 and December 2010 from 49 academic and community centers in the United States. All patients underwent surgery without genomic information and were assigned a histopathology diagnosis by an expert panel blinded to all genomic information. There were 210 potentially eligible thyroid biopsy samples with Bethesda III or IV indeterminate cytopathology that constituted a cohort previously used to validate the gene expression classifier. Of these, 191 samples (91.0%) had adequate residual RNA for validation of the genomic sequencing classifier. Algorithm development and independent validation occurred between August 2016 and May 2017. Exposures/UNASSIGNED:Thyroid nodule surgical histopathology diagnosis by an expert panel blinded to all genomic data. Main Outcomes and Measures/UNASSIGNED:The primary end point was measurement of genomic sequencing classifier sensitivity, specificity, and negative and positive predictive values in biopsies from Bethesda III and IV nodules. The secondary end point was measurement of classifier performance in biopsies from Bethesda II, V, and VI nodules. Results/UNASSIGNED:Of the 183 included patients, 142 (77.6%) were women, and the mean (range) age was 51.7 (22.0-85.0) years. The genomic sequencing classifier had a sensitivity of 91% (95% CI, 79-98) and a specificity of 68% (95% CI, 60-76). At 24% cancer prevalence, the negative predictive value was 96% (95% CI, 90-99) and the positive predictive value was 47% (95% CI, 36-58). Conclusions and Relevance/UNASSIGNED:The genomic sequencing classifier demonstrates high sensitivity and accuracy for identifying benign nodules. Its 36% increase in specificity compared with the gene expression classifier potentially increases the number of patients with benign nodules who can safely avoid unnecessary diagnostic surgery.

The molecular foundations of Hürthle cell carcinoma (HCC) are poorly understood. Here we describe a comprehensive genomic characterization of 56 primary HCC tumors that span the spectrum of tumor behavior. We elucidate the mutational profile and driver mutations and show that these tumors exhibit a wide range of recurrent mutations. Notably, we report a high number of disruptive mutations to both protein-coding and tRNA-encoding regions of the mitochondrial genome. We reveal unique chromosomal landscapes that involve whole-chromosomal duplications of chromosomes 5 and 7 and widespread loss of heterozygosity arising from haploidization and copy-number-neutral uniparental disomy. We also identify fusion genes and disrupted signaling pathways that may drive disease pathogenesis.

BACKGROUND:RAS mutations are common in the available mutational analysis of cytologically indeterminate (Cyto-I) thyroid nodules. However, their reported positive predictive value (PPV) for cancer is widely variable. The reason for this variability is unknown, and it causes clinical management uncertainty. A systematic review was performed, evaluating the PPV for cancer in RAS mutation positive Cyto-I nodules, and variables that might affect residual heterogeneity across the different studies were considered. METHODS:PubMed was searched through February 22, 2017, including studies that evaluated at least one type of RAS mutation in Cyto-I nodules, including any (or all) of the Bethesda III/IV/V categories or their equivalents and where the histological diagnosis was available. The PPV residual heterogeneity was investigated after accounting for Bethesda classification, blindedness of the histopathologist to the RAS mutational status, Bethesda category-specific cancer prevalence for each study, and which RAS genes and codons were tested. This was studied using five meta-regression models fit to different sets of Bethesda classification categories: Bethesda III, IV, or V (III/IV/V); Bethesda III or IV (III/IV); Bethesda III only; Bethesda IV only; and Bethesda V only. RESULTS: = 34.4%), with significant Cochran's Q-test for Bethesda III (p < 0.001) and IV (p = 0.04). CONCLUSION/CONCLUSIONS:The PPV of RAS mutations in Bethesda III and IV categories is quite heterogeneous across different studies, creating low confidence in the accuracy of a single estimate of PPV. Clinicians must appreciate this wide variability when managing a RAS-mutated Cyto-I nodule. Future studies should seek to resolve this unexplained variability.