Molecular Markers in Thyroid Disease Yeah or Meh? Walter E. Pofahl, MD, FACS Professor and Vice Chair East Carolina University Department of Surgery Resident Class 1994

Faculty Disclosure No disclosures

Case 65 year old female Incidentally noted thyroid nodule No risk factors for malignancy No suspicious US characteristics Right thyroid nodule (1.9 cm) FNAB benign Left thyroid nodule (0.9 cm Atypia of undetermined significance (Bethesda III) Gene expression classifier testing suspicious

Educational Need/Practice Gap Inappropriate use of molecular markers in thyroid disease Educational Need Due to lack of understanding of role of markers in clinical context

Objectives Upon completion of this educational activity, you will be able to: Discuss the different commercially available molecular marker products Describe clinical situations where use of molecular markers are helpful Outline treatment based on results of molecular testing

Expected Outcome Appropriate utilization of molecular markers in the management of thyroid disease

Rationale Bethesda Classification Thyroid Cytopathology Category Malignancy Risk I Non-diagnostic II Benign 0-3% III Atypia/follicular lesion of undetermined significance 5-15% IV Follicular neoplasm/suspicious for follicular neoplasm 15-30% V Suspicious for malignancy 60-75% VI Malignant 97-99% 15-30% thyroid FNAs indeterminate 15-50% subsequently found to be malignant

Progression of Thyroid Tumorigenesis From Xing M; Nature 2013;13:184-199

Molecular Biology Gene mutations Gene amplifications T1799A point mutation in BRAF Expression of BRAF-V600E mutant protein Gene amplifications Genes encoding PI3K-AKT pathway members More prevalent in anaplastic cancers Gene translocations RET-PTC Aberrant gene methylation Hypermethylation driven by BRAF-V600E

Gene Mutations in Thyroid Tumors From Xing M; Nature 2013;13:184-199

ATA Guidelines Recommendation 13 Recommendation 14 If molecular testing is being considered, patients should be counseled regarding the potential benefits and limitations of testing and about the possible uncertainties in the therapeutic and long-term clinical implications of results Recommendation 14 If intended for clinical use, molecular testing should be performed in Clinical Laboratory Improvement Amendments/College of American Pathologists (CLIA/CAP)-certified molecular laboratories, or the international equivalent, because reported quality assurance practices may be superior compared to other settings Haugen BR, et al. Thyroid 2016

ATA Guidelines Recommendation 15 Recommendation 16 For nodules with AUS/FLUS cytology, after consideration of worrisome clinical and sonographic features, investigations such as repeat FNA or molecular testing may be used to supplement malignancy risk assessment in lieu of proceeding directly with a strategy of either surveillance or diagnostic surgery. Informed patient preference and feasibility should be considered in clinical decision-making If repeat FNA cytology, molecular testing, or both are not performed or inconclusive, either surveillance or diagnostic surgical excision may be performed for and AUS/FLUS thyroid nodule, depending on clinical risk factors, sonographic pattern, and patient preference Recommendation 16 Diagnostic surgical excision is the long-established standard of care for the management of FN/SFN cytology nodules. However, after consideration of clinical and sonographic features, molecular testing may be used to supplement malignancy risk assessment in lieu of proceeding directly with surgery. Informed patient preference and feasibility should be considered in clinical decision-making If molecular testing is either not performed or inconclusive, surgical excision may be considered for removal and definitive diagnosis of an FN/SFN thyroid nodule Haugen BR, et al. Thyroid 2016

ATA Guidelines Recommendation 17 If the cytology is reported as suspicious for papillary carcinoma (SUSP), surgical management should be similar to that of malignant cytology, depending on clinical risk factors, sonographic features, patient preference, and possibly results of mutational testing (if performed) After consideration of clinical and sonographic features, mutational testing for BRAF or the seven-gene mutation marker panel (BRAF, RAS, RET/PTC, PAX8/PPAR𝛾) may be considered in nodules with SUSP cytology if such data would be expected to alter surgical decision-making Haugen BR, et al. Thyroid 2016

Commercially available products Affirma GEC Veracyte ThyGen X and ThyraMIR Interpace Diagnostics ThyroSeq CBL Path

Gene Expression Classifier Testing Affirma (Veracyte) mRNA expression profile Micro-array technology 142 genes Proprietary algorithm Reported as benign or suspicious Rule-out test

Gene Expression Classifier Testing

ThyGenX and ThyraMIR Interpace Diagnostics ThygenX ThyraMIR Mutation panel Rule-in test ThyraMIR miRNA gene expression 10 genes Rule-out test

ThyroSeq CBL Path Next-generation sequencing Simulataneous sequencing and detection >1000 hotspots of 14 thyroid cancer-related genes 42 types of gene fusions Comprehensive rule-in and rule-out test

Comparisons NPV Sensitivity PPV Specificity GEC 75-100% 90-100% 14-57% 7-52% ThygenX 85% 69% 71% 86% ThyGenx + ThyraMIR 94% 89% 74% Thyroseq 96% 90% 83% 93%

Zhang M, Lin O. Arch Pathol Lab Med doi: 10.5858/arpa.2016-0100-RA

Nishino M. Cancer Cytopathol 2016

Nishino M. Cancer Cytopathol 2016

Nishino M. Cancer Cytopathol 2016

Clinical Scenarios Where Helpful Where Not Helpful Avoiding operation Determining extent of resection Where Not Helpful Total thyroidectomy planned regardless Patient unwilling to proceed based on results

Summary

Future Treatment Algorithm