DICER1 Mutations Are Frequent in Adolescent-Onset Papillary Thyroid Carcinoma

DICER1 Mutations Are Frequent in Adolescent-Onset Papillary Thyroid Carcinoma Abstract Context Papillary thyroid carcinoma (PTC) is a common malignancy in adolescence and is molecularly and clinically distinct from adult PTC. Mutations in the DICER1 gene are associated with thyroid abnormalities, including multinodular goiter and differentiated thyroid carcinoma. Objective In this study, we sought to characterize the prevalence of DICER1 variants in pediatric PTC, specifically in tumors without conventional PTC oncogenic alterations. Patients Patients (N = 40) who underwent partial or total thyroidectomy and who were <18 years of age at the time of surgery were selected. Design The 40 consecutive thyroidectomy specimens (30 malignant, 10 benign) underwent genotyping for 17 PTC-associated variants, as well as full sequencing of the exons and exon-intron boundaries of DICER1. Results Conventional alterations were found in 12 of 30 (40%) PTCs (five BRAFV600E, three RET/PTC1, four RET/PTC3). Pathogenic DICER1 variants were identified in 3 of 30 (10%) PTCs and in 2 of 10 (20%) benign nodules, all of which lacked conventional alterations and did not recur during follow-up. DICER1 alterations thus constituted 3 of 18 (16.7%) PTCs without conventional alterations. The three DICER1-mutated carcinomas each had two somatic DICER1 alterations, whereas two follicular-nodular lesions arose in those with germline DICER1 mutations and harbored characteristic second somatic RNase IIIb “hotspot” mutations. Conclusions DICER1 is a driver of pediatric thyroid nodules, and DICER1-mutated PTC may represent a distinct class of low-risk malignancies. Given the prevalence of variants in children, we advocate for inclusion of DICER1 sequencing and gene dosage determination in molecular analysis of pediatric thyroid specimens. Thyroid carcinoma is the most common malignancy in adolescent and young adult women, and incidence is increasing across all age groups (1, 2). Nodular thyroid disease is also common in adolescence, and increasing access to cervical ultrasound has exposed high rates of previously subclinical thyroid nodules. Thyroid carcinoma in children and adolescents presents with several distinct clinical features when compared with adults. First, there is a substantially higher rate of regional and distally metastatic disease. In addition, recurrence rates are higher in this age group (3). Paradoxically, despite higher initial burden of disease, outcomes in children and adolescents are excellent (4). Certain histologic variants, including the diffuse sclerosing and solid variants of papillary thyroid carcinoma (PTC) are identified more commonly in children and adolescents (5, 6). Biologic and molecular mechanisms explaining differing clinical behavior of pediatric and adult PTC have thus far been elusive. A discrete set of conventional somatic variants in BRAF, HRAS, NRAS, and KRAS or gene fusions involving RET or PAX8 are identified in up to 80% of adult thyroid carcinomas (7, 8). However, several recent studies assessing these variants in children have suggested a lower frequency of variants in these genes than in adults, resulting in a higher proportion of pediatric tumors lacking common oncogenic variants, so called “dark-matter” cases (Supplemental Table 1). The oncogenic drivers in this “dark-matter” subset remain unidentified. Aberrant regulation of microRNA (miRNA) processing has been associated with multiple human malignancies (9–11), including differentiated and anaplastic thyroid carcinomas (12–15). The DICER1 gene product, DICER1, is an endoribonuclease responsible for processing RNA into small interfering RNA and miRNA, which subsequently posttranscriptionally downregulate messenger RNAs. Pathogenic, typically truncating germline variants in DICER1 cause DICER1 syndrome [Online Mendelian Inheritance in Man (OMIM) 601200], characterized by a spectrum of benign and malignant tumors of mainly pediatric onset. These include pleuropulmonary blastoma, ovarian Sertoli-Leydig cell tumor, cystic nephroma, pituitary blastoma, and multinodular goiter. Truncating DICER1 mutations are accompanied by characteristic somatic missense mutations affecting highly specific metal ion-binding residues within the RNase IIIb domain in syndrome-related tumors (16). In this study, we sought to define the prevalence of DICER1 variants in 40 (30 malignant and 10 benign) consecutive pediatric thyroidectomy specimens (Table 1; Supplemental Table 2). Table 1. Baseline Characteristics of the Cases Studied   PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA    PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA  Abbreviations: NA, not applicable. View Large Table 1. Baseline Characteristics of the Cases Studied   PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA    PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA  Abbreviations: NA, not applicable. View Large Materials and Methods Patients and samples This study was approved by the Hospital for Sick Children Research Ethics Board (project number 1000039078) and the institutional review board of the Research Institute of the McGill University Health Centre (project number MP-37-2017-2949). Patients undergoing partial or total thyroidectomy who were <18 years of age at the time of surgery were identified, and informed consent for participation in this study was obtained. All patients were assessed for a personal or family history of syndromic cancers. Patients were excluded if the indication for thyroidectomy was Graves disease without focal lesion or if consent was declined. Forty consecutive pediatric thyroidectomy specimens were obtained (30 malignant, 10 benign) from patients without a personal or family history of syndromic cancers (Table 1). Patients 3 and 6 had prior exposure to therapeutic ionizing radiation. No other patients had a history of radiation exposure or iodine deficiency, and no patients received thyroid hormone treatment prior to thyroidectomy. Patients were followed postoperatively for a median of 22.4 months (range: 0.3 to 94.4 months). Carriers of DICER1-variant tumors were followed for a median of 21 months (6.3 to 94.4 months), whereas those with conventional-variant tumors were followed for 34.4 months (3.6 to 93.3 months) (Supplemental Table 3). The duration of follow-up was not statistically different for these two groups (P = 0.27). Patients were considered to have no evidence of disease at follow-up if there was no structural disease identified on ultrasound or radioactive iodine scan, and thyroglobulin was less than 0.5 µg/L with levothyroxine-suppressed thyroid-stimulating hormone. Elevated thyroglobulin without structural correlate was considered biochemically persistent disease, and evidence of persistent malignancy based on imaging was classified as structurally persistent disease. There were no patients who achieved no evidence of disease who subsequently demonstrated disease recurrence. Samples were snap-frozen in liquid nitrogen within 30 minutes of excision. DNA was extracted from tissue using the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Germantown, MD). Constitutional DNA was extracted from peripheral blood mononuclear cells using the Gentra Puregene Blood Kit (QIAGEN) in the Clinical Laboratory Improvement Amendments-College of American Pathologists molecular diagnostic laboratories at The Hospital for Sick Children. Pathology review Hematoxylin and eosin-stained pathology specimens were reviewed by two pathologists (O.M. and R.C.) and reported according to a standardized approach. In the case of discrepancy between reviewers, cases were discussed until consensus was achieved. One specimen (case 8) was unavailable for review, and the original pathologists’ interpretation was used for the purposes of this study. Genotyping of conventional PTC-associated alterations Genotyping for the 17 most common thyroid carcinoma associated genetic alterations in BRAF, HRAS, KRAS, NRAS, RET, and PAX8 (17) was performed by allele-specific quantitative polymerase chain reaction (PCR)/quantitative reverse transcription-PCR using the Thyroid Cancer Mutation Detection Kit and the Thyroid Cancer Fusion Detection Kit (Entrogen Inc., Woodland Hills, CA). Equivocal results were obtained for seven samples that underwent gene fusion analysis. These results were validated using a Digital Droplet PCR assay on a QX200 Digital Droplet PCR System (Bio-Rad, Hercules, CA) utilizing the same oligonucleotides used for the Thyroid Cancer Fusion Detection Kit assay (Entrogen Inc.). Variants screened for are detailed in the Supplemental Methods. DICER1 sequencing The full coding region and exon-intron boundaries of DICER1 (NM_177438.2) were sequenced using a custom-designed Fluidigm Access Array (Fluidigm, Markham, Ontario, Canada) (18). Variants were called using FreeBayes version 0.4.1 via the Galaxy toolshed (www.usegalaxy.org/), and annotation of functional consequences was performed using wANNOVAR (http://wannovar.wglab.org/). Integrative Genomics Viewer (http://software.broadinstitute.org/software/igv) was used to manually visualize variants, which were then validated by Sanger sequencing. Matched-normal DNA was used to determine whether mutations were germline or somatic in origin. Copy number variation analyses CytoScanHD Array (Affymetrix, Santa Clara, CA) analysis was performed on 36 tumor samples to screen for genome-wide copy number alterations. DNA was hybridized on a CytoScanHD Array (Affymetrix), and normalization and analysis were performed using the Chromosome Analysis Suite (version 3.2.0.1252; r10346). In addition, CytoScanHD (Affymetrix) was performed in the same manner on peripheral blood mononuclear cell-derived DNA from two patients (patients 2 and 3) whose tumors were found to harbor copy number losses involving the DICER1 locus to determine whether the identified changes were germline or somatic in origin. Results Conventional PTC-associated alterations were identified in 12 of 30 (40%) malignant lesions: five cases with BRAFV600E, three with RET/PTC1, and four with RET/PTC3 (Table 2), consistent with published results (Supplemental Table 1). Sequencing of DICER1 identified pathogenic mutations in 3 of 30 (10%) PTCs, all of which lacked conventional alterations, thus accounting for 16.7% (3 of 18) of “dark-matter” cases. Two of 10 (20%) benign lesions were also found to be DICER1 mutated (Table 3; Fig. 1; Supplemental Table 2; Supplemental Figs. 1–4). The three malignant lesions each harbored two somatically acquired DICER1 alterations, whereas the patients with DICER1-mutated benign nodules each carried a germline truncating mutation and an additional somatic RNase IIIb “hotspot” mutation in the nodule, as has been observed previously (19–22). One additional tumor (case 5) carried a synonymous DICER1 variant, and two others (cases 4 and 6) each harbored a polymorphism classified as likely benign according to ClinVar (www.ncbi.nlm.nih.gov/clinvar/; variation IDs: 133971 and 133962, respectively). Neither case showed evidence of a second hit within the DICER1 locus. Table 2. Cases With Conventional Somatic PTC-Associated Variants Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Abbreviations: ATA, American Thyroid Association; BPD, biochemically persistent disease; Dx, diagnosis; DSV, diffuse sclerosing variant; NA, not available; NED, no evidence of disease; SPD, structurally persistent disease; Tg, thyroglobulin. a A discrete nodule was present with diffuse infiltration of the surrounding parenchyma by malignant cells. View Large Table 2. Cases With Conventional Somatic PTC-Associated Variants Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Abbreviations: ATA, American Thyroid Association; BPD, biochemically persistent disease; Dx, diagnosis; DSV, diffuse sclerosing variant; NA, not available; NED, no evidence of disease; SPD, structurally persistent disease; Tg, thyroglobulin. a A discrete nodule was present with diffuse infiltration of the surrounding parenchyma by malignant cells. View Large Table 3. Characteristics of DICER1-Mutated Cases Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Abbreviations: ALL, acute lymphoblastic leukemia; AJCC, American Joint Committee on Cancer; ATA, American Thyroid Association; Dx, diagnosis; ExAC, Exome Aggregation Consortium; FH, family history; FND, follicular nodular disease; FV, follicular variant; HSCT, hematopoietic stem cell transplantation; LOH, loss of heterozygosity; MAF, minor allele frequency; MNG, multinodular goiter; N/A, not applicable; NED, no evidence of disease; PMH, personal medical history; TBI, total body irradiation; Tg, thyroglobulin; TSH, thyroid-stimulating hormone. a The germline variant is encompassed by the deletion in the tumor and is therefore in trans with the p.D1810Y mutation. b In silico predictions for variant rs544960260 (case 4): tolerated by Sorting Intolerant From Tolerant (SIFT) (score 0.11), benign by PolyPhen-2 (score 0), Combined Annotation Dependent Depletion (CADD)-scaled C-score: 3.877 (consistent with benign), and Rare Exome Variant Ensemble Learner (REVEL) score: 0.048 (consistent with benign). c TSH was performed at a referring facility and was reported only as “normal.” It was not repeated preoperatively. d Functional analyses determined that this in-frame DICER1 variant impedes processing of miRNAs (see Supplemental Fig. 4 for details). View Large Table 3. Characteristics of DICER1-Mutated Cases Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Abbreviations: ALL, acute lymphoblastic leukemia; AJCC, American Joint Committee on Cancer; ATA, American Thyroid Association; Dx, diagnosis; ExAC, Exome Aggregation Consortium; FH, family history; FND, follicular nodular disease; FV, follicular variant; HSCT, hematopoietic stem cell transplantation; LOH, loss of heterozygosity; MAF, minor allele frequency; MNG, multinodular goiter; N/A, not applicable; NED, no evidence of disease; PMH, personal medical history; TBI, total body irradiation; Tg, thyroglobulin; TSH, thyroid-stimulating hormone. a The germline variant is encompassed by the deletion in the tumor and is therefore in trans with the p.D1810Y mutation. b In silico predictions for variant rs544960260 (case 4): tolerated by Sorting Intolerant From Tolerant (SIFT) (score 0.11), benign by PolyPhen-2 (score 0), Combined Annotation Dependent Depletion (CADD)-scaled C-score: 3.877 (consistent with benign), and Rare Exome Variant Ensemble Learner (REVEL) score: 0.048 (consistent with benign). c TSH was performed at a referring facility and was reported only as “normal.” It was not repeated preoperatively. d Functional analyses determined that this in-frame DICER1 variant impedes processing of miRNAs (see Supplemental Fig. 4 for details). View Large Figure 1. View largeDownload slide Representative DICER1-mutated thyroid neoplasms are illustrated. (A) Case 2: Encapsulated follicular-variant papillary carcinoma with minimal capsular invasion. The nontumorous thyroid parenchyma showed variable involutional changes characterized by dilated colloid containing follicles and slightly flattened epithelium. (B) Case 7: Multiple follicular nodular disease. The nonlesional thyroid parenchyma exhibited variable involutional changes. These findings are reminiscent of functional nodules despite a normal preoperative thyroid-stimulating hormone (0.64 mIU/L). Figure 1. View largeDownload slide Representative DICER1-mutated thyroid neoplasms are illustrated. (A) Case 2: Encapsulated follicular-variant papillary carcinoma with minimal capsular invasion. The nontumorous thyroid parenchyma showed variable involutional changes characterized by dilated colloid containing follicles and slightly flattened epithelium. (B) Case 7: Multiple follicular nodular disease. The nonlesional thyroid parenchyma exhibited variable involutional changes. These findings are reminiscent of functional nodules despite a normal preoperative thyroid-stimulating hormone (0.64 mIU/L). Discussion The prevalence of pathogenic DICER1 variants in this series of pediatric thyroid tumors was determined to be 12.5% (5 of 40) (Supplemental Table 3). Two large genome-wide analyses have identified low rates of pathogenic DICER1 variants in adult thyroid tumors. The Cancer Genome Atlas project identified pathogenic somatic DICER1 variants in 2 of 401 (0.5%) PTCs (Supplemental Table 4) (7), whereas a subsequent study identified no DICER1-variant tumors among 125 PTC, and two tumors with DICER1 variants in each of 30 minimally invasive follicular thyroid cancers and 25 follicular adenomas (8). DICER1-mutated PTCs are thus more frequent in the pediatric (3 of 30) than in the adult setting (2 of 526; P = 0.0013, two-sided Fisher exact test). Although capsular or parenchymal invasion was observed among all DICER1-mutated thyroid carcinomas in our series, angioinvasion was only identified in one (case 3). None were associated with local or regional lymph node or distal metastases, and all are thus classified as low risk for persistent/recurrent disease according to the recent American Thyroid Association pediatric management guidelines (3). In contrast, four of five (80%) BRAFV600E tumors and six of seven (86%) RET/PTC tumors were associated with nodal metastases. Antithyroglobulin titers were undetectable in all individuals with DICER1 tumors and were present in 7 of 27 (26%) of the remaining individuals with PTC, a proportion concordant with recent studies (23). Patients with DICER1-mutated tumors remained disease-free at a median of 21 months follow-up (range: 9.9 to 37.4 months) (Supplemental Table 3). None of the DICER1 tumors had a chronic lymphocytic infiltrate, and four cases exhibited focal nonspecific thyroiditis. Lastly, in six of seven DICER1-mutated available specimens reviewed, the background thyroid parenchyma showed variable involutional changes similar to alterations associated with exposure to elevated thyroid hormone concentrations (Table 3; Fig. 1; Supplemental Fig. 1). DICER1 alterations impact several miRNA species in thyroid lesions (24). Although multiple studies have demonstrated alterations in miRNA sequence and expression in thyroid malignancies (7, 25–27), their mechanistic implications and role in thyroid tumorigenesis remain to be fully elucidated. It also remains to be seen whether alterations in other components of the miRNA processing machinery (DROSHA, DGCR8, AGO2, etc.) are altered in thyroid carcinoma. Conclusion DICER1 mutations in pediatric PTC are present at a frequency nearly 30 times that seen in adult PTC. DICER1 malignancies comprised 16.7% of dark-matter tumors in this pediatric series and were not associated with thyroid autoimmunity. These data establish DICER1 as a common oncogenic driver in American Thyroid Association pediatric low-risk PTC and broaden our understanding of the molecular pathogenesis of pediatric PTCs. Further delineation of the role of miRNA processing and aberrations in thyroid carcinogenesis may expand our insight into prognosis and potentially therapeutic approaches. Abbreviations: Abbreviations: miRNA microRNA PCR polymerase chain reaction PTC papillary thyroid carcinoma Acknowledgments We thank Marc Fabian, PhD, for assistance with the in vitro cleavage assay. Financial Support: This work was supported by the SickKids Foundation (to J.D.W.), the C17 Research Network, which was funded by the Childhood Cancer Canada Foundation and the Kids With Cancer Society (to J.D.W.), the Canadian Institute of Health Research Foundation Grant FDN-148390 (to W.D.F.), and the Vanier Canada Graduate Scholarship and DKG World Fellowship (to L.d.K.). Author Contributions: W.D.F. and J.D.W. designed the study. W.D.F., J.D.W., and L.d.K. wrote the manuscript. N.S. and M.K.W. performed the DICER1 genetic testing. S.F. performed bioinformatics analysis. O.M. and R.C. performed the pathology review. J.D.W., M.A., and A.S. performed oncogene genetic analysis, sample acquisition, and processing. Disclosure Summary: The authors have nothing to disclose. References 1. Pole JD, Zuk AM, Wasserman JD. Diagnostic and treatment patterns among children, adolescents, and young adults with thyroid cancer in Ontario: 1992-2010. Thyroid . 2017; 27( 8): 1025– 1033. 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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02698
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Abstract

Abstract Context Papillary thyroid carcinoma (PTC) is a common malignancy in adolescence and is molecularly and clinically distinct from adult PTC. Mutations in the DICER1 gene are associated with thyroid abnormalities, including multinodular goiter and differentiated thyroid carcinoma. Objective In this study, we sought to characterize the prevalence of DICER1 variants in pediatric PTC, specifically in tumors without conventional PTC oncogenic alterations. Patients Patients (N = 40) who underwent partial or total thyroidectomy and who were <18 years of age at the time of surgery were selected. Design The 40 consecutive thyroidectomy specimens (30 malignant, 10 benign) underwent genotyping for 17 PTC-associated variants, as well as full sequencing of the exons and exon-intron boundaries of DICER1. Results Conventional alterations were found in 12 of 30 (40%) PTCs (five BRAFV600E, three RET/PTC1, four RET/PTC3). Pathogenic DICER1 variants were identified in 3 of 30 (10%) PTCs and in 2 of 10 (20%) benign nodules, all of which lacked conventional alterations and did not recur during follow-up. DICER1 alterations thus constituted 3 of 18 (16.7%) PTCs without conventional alterations. The three DICER1-mutated carcinomas each had two somatic DICER1 alterations, whereas two follicular-nodular lesions arose in those with germline DICER1 mutations and harbored characteristic second somatic RNase IIIb “hotspot” mutations. Conclusions DICER1 is a driver of pediatric thyroid nodules, and DICER1-mutated PTC may represent a distinct class of low-risk malignancies. Given the prevalence of variants in children, we advocate for inclusion of DICER1 sequencing and gene dosage determination in molecular analysis of pediatric thyroid specimens. Thyroid carcinoma is the most common malignancy in adolescent and young adult women, and incidence is increasing across all age groups (1, 2). Nodular thyroid disease is also common in adolescence, and increasing access to cervical ultrasound has exposed high rates of previously subclinical thyroid nodules. Thyroid carcinoma in children and adolescents presents with several distinct clinical features when compared with adults. First, there is a substantially higher rate of regional and distally metastatic disease. In addition, recurrence rates are higher in this age group (3). Paradoxically, despite higher initial burden of disease, outcomes in children and adolescents are excellent (4). Certain histologic variants, including the diffuse sclerosing and solid variants of papillary thyroid carcinoma (PTC) are identified more commonly in children and adolescents (5, 6). Biologic and molecular mechanisms explaining differing clinical behavior of pediatric and adult PTC have thus far been elusive. A discrete set of conventional somatic variants in BRAF, HRAS, NRAS, and KRAS or gene fusions involving RET or PAX8 are identified in up to 80% of adult thyroid carcinomas (7, 8). However, several recent studies assessing these variants in children have suggested a lower frequency of variants in these genes than in adults, resulting in a higher proportion of pediatric tumors lacking common oncogenic variants, so called “dark-matter” cases (Supplemental Table 1). The oncogenic drivers in this “dark-matter” subset remain unidentified. Aberrant regulation of microRNA (miRNA) processing has been associated with multiple human malignancies (9–11), including differentiated and anaplastic thyroid carcinomas (12–15). The DICER1 gene product, DICER1, is an endoribonuclease responsible for processing RNA into small interfering RNA and miRNA, which subsequently posttranscriptionally downregulate messenger RNAs. Pathogenic, typically truncating germline variants in DICER1 cause DICER1 syndrome [Online Mendelian Inheritance in Man (OMIM) 601200], characterized by a spectrum of benign and malignant tumors of mainly pediatric onset. These include pleuropulmonary blastoma, ovarian Sertoli-Leydig cell tumor, cystic nephroma, pituitary blastoma, and multinodular goiter. Truncating DICER1 mutations are accompanied by characteristic somatic missense mutations affecting highly specific metal ion-binding residues within the RNase IIIb domain in syndrome-related tumors (16). In this study, we sought to define the prevalence of DICER1 variants in 40 (30 malignant and 10 benign) consecutive pediatric thyroidectomy specimens (Table 1; Supplemental Table 2). Table 1. Baseline Characteristics of the Cases Studied   PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA    PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA  Abbreviations: NA, not applicable. View Large Table 1. Baseline Characteristics of the Cases Studied   PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA    PTC Classic Variant  PTC Follicular Variant  PTC Diffuse Sclerosing Variant  PTC Solid Variant  Benign Lesions  Number of cases (N = 40)  17  6  6  1  10  Sex     Female  8  5  4  1  10   Male  9  1  2  0  0  Mean age, y (range)  12.9 (5–17)  15.1 (11–17)  10.6 (8–13)  15  14.4 (11–18)  Size of dominant nodule, mm (range)  28.5 (9–60)  29 (7–40)  Nodal metastases present, n (%)  10 (59%)  0 (0%)  6 (100%)  0 (0%)  NA  Persistent/recurrent disease, n (%)  7 (41%)  0 (0%)  2 (33%)  0 (0%)  NA  Abbreviations: NA, not applicable. View Large Materials and Methods Patients and samples This study was approved by the Hospital for Sick Children Research Ethics Board (project number 1000039078) and the institutional review board of the Research Institute of the McGill University Health Centre (project number MP-37-2017-2949). Patients undergoing partial or total thyroidectomy who were <18 years of age at the time of surgery were identified, and informed consent for participation in this study was obtained. All patients were assessed for a personal or family history of syndromic cancers. Patients were excluded if the indication for thyroidectomy was Graves disease without focal lesion or if consent was declined. Forty consecutive pediatric thyroidectomy specimens were obtained (30 malignant, 10 benign) from patients without a personal or family history of syndromic cancers (Table 1). Patients 3 and 6 had prior exposure to therapeutic ionizing radiation. No other patients had a history of radiation exposure or iodine deficiency, and no patients received thyroid hormone treatment prior to thyroidectomy. Patients were followed postoperatively for a median of 22.4 months (range: 0.3 to 94.4 months). Carriers of DICER1-variant tumors were followed for a median of 21 months (6.3 to 94.4 months), whereas those with conventional-variant tumors were followed for 34.4 months (3.6 to 93.3 months) (Supplemental Table 3). The duration of follow-up was not statistically different for these two groups (P = 0.27). Patients were considered to have no evidence of disease at follow-up if there was no structural disease identified on ultrasound or radioactive iodine scan, and thyroglobulin was less than 0.5 µg/L with levothyroxine-suppressed thyroid-stimulating hormone. Elevated thyroglobulin without structural correlate was considered biochemically persistent disease, and evidence of persistent malignancy based on imaging was classified as structurally persistent disease. There were no patients who achieved no evidence of disease who subsequently demonstrated disease recurrence. Samples were snap-frozen in liquid nitrogen within 30 minutes of excision. DNA was extracted from tissue using the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Germantown, MD). Constitutional DNA was extracted from peripheral blood mononuclear cells using the Gentra Puregene Blood Kit (QIAGEN) in the Clinical Laboratory Improvement Amendments-College of American Pathologists molecular diagnostic laboratories at The Hospital for Sick Children. Pathology review Hematoxylin and eosin-stained pathology specimens were reviewed by two pathologists (O.M. and R.C.) and reported according to a standardized approach. In the case of discrepancy between reviewers, cases were discussed until consensus was achieved. One specimen (case 8) was unavailable for review, and the original pathologists’ interpretation was used for the purposes of this study. Genotyping of conventional PTC-associated alterations Genotyping for the 17 most common thyroid carcinoma associated genetic alterations in BRAF, HRAS, KRAS, NRAS, RET, and PAX8 (17) was performed by allele-specific quantitative polymerase chain reaction (PCR)/quantitative reverse transcription-PCR using the Thyroid Cancer Mutation Detection Kit and the Thyroid Cancer Fusion Detection Kit (Entrogen Inc., Woodland Hills, CA). Equivocal results were obtained for seven samples that underwent gene fusion analysis. These results were validated using a Digital Droplet PCR assay on a QX200 Digital Droplet PCR System (Bio-Rad, Hercules, CA) utilizing the same oligonucleotides used for the Thyroid Cancer Fusion Detection Kit assay (Entrogen Inc.). Variants screened for are detailed in the Supplemental Methods. DICER1 sequencing The full coding region and exon-intron boundaries of DICER1 (NM_177438.2) were sequenced using a custom-designed Fluidigm Access Array (Fluidigm, Markham, Ontario, Canada) (18). Variants were called using FreeBayes version 0.4.1 via the Galaxy toolshed (www.usegalaxy.org/), and annotation of functional consequences was performed using wANNOVAR (http://wannovar.wglab.org/). Integrative Genomics Viewer (http://software.broadinstitute.org/software/igv) was used to manually visualize variants, which were then validated by Sanger sequencing. Matched-normal DNA was used to determine whether mutations were germline or somatic in origin. Copy number variation analyses CytoScanHD Array (Affymetrix, Santa Clara, CA) analysis was performed on 36 tumor samples to screen for genome-wide copy number alterations. DNA was hybridized on a CytoScanHD Array (Affymetrix), and normalization and analysis were performed using the Chromosome Analysis Suite (version 3.2.0.1252; r10346). In addition, CytoScanHD (Affymetrix) was performed in the same manner on peripheral blood mononuclear cell-derived DNA from two patients (patients 2 and 3) whose tumors were found to harbor copy number losses involving the DICER1 locus to determine whether the identified changes were germline or somatic in origin. Results Conventional PTC-associated alterations were identified in 12 of 30 (40%) malignant lesions: five cases with BRAFV600E, three with RET/PTC1, and four with RET/PTC3 (Table 2), consistent with published results (Supplemental Table 1). Sequencing of DICER1 identified pathogenic mutations in 3 of 30 (10%) PTCs, all of which lacked conventional alterations, thus accounting for 16.7% (3 of 18) of “dark-matter” cases. Two of 10 (20%) benign lesions were also found to be DICER1 mutated (Table 3; Fig. 1; Supplemental Table 2; Supplemental Figs. 1–4). The three malignant lesions each harbored two somatically acquired DICER1 alterations, whereas the patients with DICER1-mutated benign nodules each carried a germline truncating mutation and an additional somatic RNase IIIb “hotspot” mutation in the nodule, as has been observed previously (19–22). One additional tumor (case 5) carried a synonymous DICER1 variant, and two others (cases 4 and 6) each harbored a polymorphism classified as likely benign according to ClinVar (www.ncbi.nlm.nih.gov/clinvar/; variation IDs: 133971 and 133962, respectively). Neither case showed evidence of a second hit within the DICER1 locus. Table 2. Cases With Conventional Somatic PTC-Associated Variants Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Abbreviations: ATA, American Thyroid Association; BPD, biochemically persistent disease; Dx, diagnosis; DSV, diffuse sclerosing variant; NA, not available; NED, no evidence of disease; SPD, structurally persistent disease; Tg, thyroglobulin. a A discrete nodule was present with diffuse infiltration of the surrounding parenchyma by malignant cells. View Large Table 2. Cases With Conventional Somatic PTC-Associated Variants Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Case  Age at Dx (y)  Sex  Gene  Variant  Histologic Variant  Tumor Classification  Anti-Tg (kIU/L)  Size of Dominant Nodule (mm)  ATA Risk  Current Status [Follow-Up Duration (mo)]  9  15  Female  BRAF  p.V600E  Classic  Malignant  <20  21  Low  BPD (36)  10  17  Male  BRAF  p.V600E  Classic  Malignant  <20  47  Intermediate  NED (19)  11  10  Male  BRAF  p.V600E  Classic  Malignant  54  Diffuse  High  SPD (90)  12  16  Male  BRAF  p.V600E  Classic  Malignant  <20  11  Intermediate  NED (11)  13  14  Male  BRAF  p.V600E  Classic  Malignant  <20  32  High  NED (6)  14  15  Female  RET/PTC1  Rearrangement  Classic  Malignant  120  50  High  SPD (32)  15  17  Female  RET/PTC1  Rearrangement  Follicular  Malignant  NA  9  Low  NED (5)  16  10  Male  RET/PTC1  Rearrangement  Classic  Malignant  2233  35  High  SPD (62)  17  12  Female  RET/PTC3  Rearrangement  Classic  Malignant  3348  55  High  NED (24)  18  8  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  Diffuse  High  SPD (25)  19  10  Female  RET/PTC3  Rearrangement  DSV  Malignant  <20  15a  High  NED (33)  20  11  Male  RET/PTC3  Rearrangement  DSV  Malignant  <10  Diffuse  High  SPD (19)  Abbreviations: ATA, American Thyroid Association; BPD, biochemically persistent disease; Dx, diagnosis; DSV, diffuse sclerosing variant; NA, not available; NED, no evidence of disease; SPD, structurally persistent disease; Tg, thyroglobulin. a A discrete nodule was present with diffuse infiltration of the surrounding parenchyma by malignant cells. View Large Table 3. Characteristics of DICER1-Mutated Cases Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Abbreviations: ALL, acute lymphoblastic leukemia; AJCC, American Joint Committee on Cancer; ATA, American Thyroid Association; Dx, diagnosis; ExAC, Exome Aggregation Consortium; FH, family history; FND, follicular nodular disease; FV, follicular variant; HSCT, hematopoietic stem cell transplantation; LOH, loss of heterozygosity; MAF, minor allele frequency; MNG, multinodular goiter; N/A, not applicable; NED, no evidence of disease; PMH, personal medical history; TBI, total body irradiation; Tg, thyroglobulin; TSH, thyroid-stimulating hormone. a The germline variant is encompassed by the deletion in the tumor and is therefore in trans with the p.D1810Y mutation. b In silico predictions for variant rs544960260 (case 4): tolerated by Sorting Intolerant From Tolerant (SIFT) (score 0.11), benign by PolyPhen-2 (score 0), Combined Annotation Dependent Depletion (CADD)-scaled C-score: 3.877 (consistent with benign), and Rare Exome Variant Ensemble Learner (REVEL) score: 0.048 (consistent with benign). c TSH was performed at a referring facility and was reported only as “normal.” It was not repeated preoperatively. d Functional analyses determined that this in-frame DICER1 variant impedes processing of miRNAs (see Supplemental Fig. 4 for details). View Large Table 3. Characteristics of DICER1-Mutated Cases Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Case  Age at Dx (y)  Sex  DICER1 Screening Result  PMH  FH  Pre-Op TSH (mIU/L)/Free T4 (pmol/L)  Anti-Tg (kIU/L)  Tumor Pathology, Staging, and Risk  Current Status [Duration (mo)]  Variant(s)  Predicted Consequence  Germline vs Somatic Origin  ExAC MAF  Histologic Variant  Tumor Classification  Size of Dominant Nodule (mm)  AJCC
Stage  ATA
Risk  1  16.5  Female  c.2875A>T, p.K959*  Deleterious  Somatic  N/A  0  0  0.81/13.8  <20  Encapsulated classical PTC  Malignant  60  T3N0M0  Low  NED (62)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  2  14  Female  c.5428G>T, p.D1810Y  Deleterious  Somatic  N/A  0  0  1.84/16.0  <20  Minimally invasive, encapsulated FV PTC  Malignant  37  T2N0Mx  Low  NED (41)  LOH of DICER1 (del chr14:94,043,795-104,822,229)  Deleterious  Somatic  N/A  c.1124C>G, p.P375R (rs148758903)  Likely benign  Germlinea  0.0003545  3  11.7  Female  c.5439G>C, p.E1813D  Deleterious  Somatic  N/A  ALL,TBI, HSCT  0  1.41/11.0  <10  Infiltrative classical PTC  Malignant  25  T2N0Mx  Low  NED (22)  LOH of DICER1 (del chr14:78,529,021-100,616,514)  Deleterious  Somatic  N/A  4  10  Male  c.4260_4262delGGA, p.E1420del (rs544960260)b  Likely benign  Not known  0.001588  0  0  Normalc  <10  Classical PTC with focal hobnail and tall cell change  Malignant  35  T2N0M0  Low  NED (94)  5  15  Female  c.2997T>G, p.L999L (rs12018992)  Silent  Germline  0.005387  0  MNG  0.96/13.0  <20  Minimally invasive solid-variant PTC  Malignant  13  T1bNxMx  Low  NED (38)  6  17.4  Female  c.20A>G, p.Q7R (rs117358479)  Likely benign  Not known  0.00181  ALL, TBI  0  2.05/10.8  Not done  (1) Minimally invasive FV PTC  Malignant  12  T1bNxMx  Low  NED (9)  (2) Classical-variant papillary microcarcinoma  3  7  12  Female  c.2535_2539delinsAATCAACTTCAAGCATT, p.T847delinsNFKHSd  Deleterious  Germline  Not in ExAC  0  MNG  0.64/11.9  Not done  Multifocal FND with papillary growth  Benign  28  N/A  —  NED (64)  c.5438A>G, p.E1813G  Deleterious  Somatic  N/A  8  16  Female  c.84dupT, p.G29Wfs*11  Deleterious  Germline  Not in ExAc  0  0  1.28/21  Not done  FND  Benign  20  N/A  —  NED (61)  c.5125G>A, p.D1709N  Deleterious  Somatic  N/A  Abbreviations: ALL, acute lymphoblastic leukemia; AJCC, American Joint Committee on Cancer; ATA, American Thyroid Association; Dx, diagnosis; ExAC, Exome Aggregation Consortium; FH, family history; FND, follicular nodular disease; FV, follicular variant; HSCT, hematopoietic stem cell transplantation; LOH, loss of heterozygosity; MAF, minor allele frequency; MNG, multinodular goiter; N/A, not applicable; NED, no evidence of disease; PMH, personal medical history; TBI, total body irradiation; Tg, thyroglobulin; TSH, thyroid-stimulating hormone. a The germline variant is encompassed by the deletion in the tumor and is therefore in trans with the p.D1810Y mutation. b In silico predictions for variant rs544960260 (case 4): tolerated by Sorting Intolerant From Tolerant (SIFT) (score 0.11), benign by PolyPhen-2 (score 0), Combined Annotation Dependent Depletion (CADD)-scaled C-score: 3.877 (consistent with benign), and Rare Exome Variant Ensemble Learner (REVEL) score: 0.048 (consistent with benign). c TSH was performed at a referring facility and was reported only as “normal.” It was not repeated preoperatively. d Functional analyses determined that this in-frame DICER1 variant impedes processing of miRNAs (see Supplemental Fig. 4 for details). View Large Figure 1. View largeDownload slide Representative DICER1-mutated thyroid neoplasms are illustrated. (A) Case 2: Encapsulated follicular-variant papillary carcinoma with minimal capsular invasion. The nontumorous thyroid parenchyma showed variable involutional changes characterized by dilated colloid containing follicles and slightly flattened epithelium. (B) Case 7: Multiple follicular nodular disease. The nonlesional thyroid parenchyma exhibited variable involutional changes. These findings are reminiscent of functional nodules despite a normal preoperative thyroid-stimulating hormone (0.64 mIU/L). Figure 1. View largeDownload slide Representative DICER1-mutated thyroid neoplasms are illustrated. (A) Case 2: Encapsulated follicular-variant papillary carcinoma with minimal capsular invasion. The nontumorous thyroid parenchyma showed variable involutional changes characterized by dilated colloid containing follicles and slightly flattened epithelium. (B) Case 7: Multiple follicular nodular disease. The nonlesional thyroid parenchyma exhibited variable involutional changes. These findings are reminiscent of functional nodules despite a normal preoperative thyroid-stimulating hormone (0.64 mIU/L). Discussion The prevalence of pathogenic DICER1 variants in this series of pediatric thyroid tumors was determined to be 12.5% (5 of 40) (Supplemental Table 3). Two large genome-wide analyses have identified low rates of pathogenic DICER1 variants in adult thyroid tumors. The Cancer Genome Atlas project identified pathogenic somatic DICER1 variants in 2 of 401 (0.5%) PTCs (Supplemental Table 4) (7), whereas a subsequent study identified no DICER1-variant tumors among 125 PTC, and two tumors with DICER1 variants in each of 30 minimally invasive follicular thyroid cancers and 25 follicular adenomas (8). DICER1-mutated PTCs are thus more frequent in the pediatric (3 of 30) than in the adult setting (2 of 526; P = 0.0013, two-sided Fisher exact test). Although capsular or parenchymal invasion was observed among all DICER1-mutated thyroid carcinomas in our series, angioinvasion was only identified in one (case 3). None were associated with local or regional lymph node or distal metastases, and all are thus classified as low risk for persistent/recurrent disease according to the recent American Thyroid Association pediatric management guidelines (3). In contrast, four of five (80%) BRAFV600E tumors and six of seven (86%) RET/PTC tumors were associated with nodal metastases. Antithyroglobulin titers were undetectable in all individuals with DICER1 tumors and were present in 7 of 27 (26%) of the remaining individuals with PTC, a proportion concordant with recent studies (23). Patients with DICER1-mutated tumors remained disease-free at a median of 21 months follow-up (range: 9.9 to 37.4 months) (Supplemental Table 3). None of the DICER1 tumors had a chronic lymphocytic infiltrate, and four cases exhibited focal nonspecific thyroiditis. Lastly, in six of seven DICER1-mutated available specimens reviewed, the background thyroid parenchyma showed variable involutional changes similar to alterations associated with exposure to elevated thyroid hormone concentrations (Table 3; Fig. 1; Supplemental Fig. 1). DICER1 alterations impact several miRNA species in thyroid lesions (24). Although multiple studies have demonstrated alterations in miRNA sequence and expression in thyroid malignancies (7, 25–27), their mechanistic implications and role in thyroid tumorigenesis remain to be fully elucidated. It also remains to be seen whether alterations in other components of the miRNA processing machinery (DROSHA, DGCR8, AGO2, etc.) are altered in thyroid carcinoma. Conclusion DICER1 mutations in pediatric PTC are present at a frequency nearly 30 times that seen in adult PTC. DICER1 malignancies comprised 16.7% of dark-matter tumors in this pediatric series and were not associated with thyroid autoimmunity. These data establish DICER1 as a common oncogenic driver in American Thyroid Association pediatric low-risk PTC and broaden our understanding of the molecular pathogenesis of pediatric PTCs. Further delineation of the role of miRNA processing and aberrations in thyroid carcinogenesis may expand our insight into prognosis and potentially therapeutic approaches. Abbreviations: Abbreviations: miRNA microRNA PCR polymerase chain reaction PTC papillary thyroid carcinoma Acknowledgments We thank Marc Fabian, PhD, for assistance with the in vitro cleavage assay. Financial Support: This work was supported by the SickKids Foundation (to J.D.W.), the C17 Research Network, which was funded by the Childhood Cancer Canada Foundation and the Kids With Cancer Society (to J.D.W.), the Canadian Institute of Health Research Foundation Grant FDN-148390 (to W.D.F.), and the Vanier Canada Graduate Scholarship and DKG World Fellowship (to L.d.K.). Author Contributions: W.D.F. and J.D.W. designed the study. W.D.F., J.D.W., and L.d.K. wrote the manuscript. N.S. and M.K.W. performed the DICER1 genetic testing. S.F. performed bioinformatics analysis. O.M. and R.C. performed the pathology review. J.D.W., M.A., and A.S. performed oncogene genetic analysis, sample acquisition, and processing. Disclosure Summary: The authors have nothing to disclose. References 1. Pole JD, Zuk AM, Wasserman JD. Diagnostic and treatment patterns among children, adolescents, and young adults with thyroid cancer in Ontario: 1992-2010. Thyroid . 2017; 27( 8): 1025– 1033. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 21, 2018

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