The Role of Genetic Testing in the Evaluation of Thyroid Nodules. Thyroid Cancer and FNA. Thyroid Cancer. Pure Follicular Cancers.

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1 Where does Molecular Analysis of FNA Specimens fit into the evaluation of thyroid nodules? The Role of Genetic Testing in the Evaluation of Thyroid Nodules Ultrasound TSH Risk factors Jill E. Langer, MD Professor of Radiology The Perelman School of Medicine University of Pennsylvania SRU 214 Thyroid Cancer and FNA Thyroid Cancer Each thyroid nodule is either a cancer or not a cancer Thyroid FNA is the best non surgical diagnostic tool to diagnose malignancy Thyroid FNA is not always able to distinguish a cancer from a non cancerous lesion, only surgical pathology is able to make that distinction Thyroid Epithelial Tumors are derived from follicular cells (95% of all cancers) Well differentiated Papillary Cancer (8%) Pure papillary cancer Follicular Variant of Papillary cancer Well differentiated Follicular Cancer (1%) Poorly differentiated Papillary Cancer (5%) Anaplastic Cancer ( < 1%) Medullary cancers are derived from parafollicular or C cells (3 to 5%) Pure Follicular Cancers 1% of all thyroid cancers in the US; higher in iodine deficient areas female > male; peak in 6th decade 87% ten year survival (c/w 99% for PTC) Histologically similar to the benign follicular adenoma but demonstrates capsular invasion on surgical pathology Of all follicular tumors, 8% benign adenoma and 2% carcinoma Follicular cancer Capsular invasion Vascular invasion 1

2 Thyroid FNA Benign = heterogeneous population of follicular cells, no atypical nuclear features, often colloid and macrophages Malignant = papillary tissue fragments, abnormal nuclear features such as irregular contours and nuclear grooves, intranuclear cytoplasmic inclusions, usually indicates a papillary cancer Thyroid FNA Non diagnostic = insufficient number of cells to render a definite diagnosis (adequacy often defined as 6 to 8 groups of cells, each group of at least 1 cells) Indeterminate = monotonous follicular cells without abnormal nuclear features DDX: benign follicular adenoma, pure follicular cancer, follicular variant of papillary thyroid carcinoma, benign hyperplastic nodule, focal area of chronic lymphocytic thyroiditis Thyroid FNA and Risk for Cancer Benign 6 7% of FNAs 96+ % benign Non diagnostic 2 1% of FNAs Risk of cancer 1 to 1% Indeterminate 2 3% of FNAs 15 75% cancer risk Papanicolaou Society of Cytopathology Task Force on Standards of Practice, 1997 Malignant 5 15% of FNAs 99+% malignant Standard of care for Indeterminate Thyroid Lobectomy (majority of patients) With completion thyroidectomy if malignant histology, about 15 to 2% Up to 85% unnecessary surgeries for benign disease Surgical risk of permanent hypoparathyroidism and RLN injury (1 to 2 %) Total thyroidectomy if high risk history, bilateral nodules, patient preference, cytologic features FNA Benign Non diagnostic Indeterminate FLUS or AUS Neoplasm Atypia of uncertain significance Malignant Suspicious for Malignancy Baloch ZW et al, Diagnostic Cytopath 28 Malignancy risk Nondiagnostic or unsatisfactory ~1-5% Benign 2-4% Atypia or follicular lesion of 5-15% unknown significance (AUS/FLUS) Follicular neoplasm 15-3% Suspicious for malignancy 6-75% Malignant 97-1% Baloch ZW, Diag Cytopath 36:425, 28; Wang Thyroid :243 2

3 BETHESDA Malignancy risk Management Nondiagnostic or ~1-5% Repeat FNA unsatisfactory Benign 2-4% Observe with US Follow up Malignant 97-1% Total Thyd Atypia or follicular lesion of unknown significance (AUS/FLUS) BETHESDA Malignancy risk 5-15% Follicular neoplasm 15-3% Suspicious for malignancy 6-75% Management Repeat FNA ATYPIA/FOLLICULAR LESION OF UNDETERMINED SIGNFICIANCE (AUS/FLUS) Frequency (%) Repeat FNA cytology in FLUS/AUS nodules 5% Benign 15% sptc AUS/FLUS on Second FNA BETHESDA Malignancy risk? Higher than 5-15% Management Surgery vs observation Follicular neoplasm 15-3%?? Hemi Thyd Suspicious for malignancy 6-75%? Total vs Hemi- Thyd Benign FLUS/AUS Foll Neo Susp PTC/PTC Baloch, Diagn Cytopathol 23; Yassa Cancer 27 Testing Beyond : Additional Testing for Indeterminate FNA Results Genetic analysis Single Oncogene (BRAF) Oncogene mutation panels (Asuragen TM ) RNA Expression Micro RNA Afirma TM gene expression classifier Testing Beyond : Additional Testing for Indeterminate FNA Results Goal is to better predict malignancy in patients having thyroid surgery and perform total thyroidectomy for indeterminate nodules with very high chance of cancer Goal is to better predict benignity to avoid surgery for the management of indeterminate nodules with a low chance of malignancy 3

4 Oncogene Analysis: Point Mutations and Chromosomal Rearrangements BRAF Mutations in 347 Thyroid Nodules: Prevalence and Specificity PAX8/PPAR Number of cases reported Benign Nodules BRAF V6E 1 (.16%) Follicular Carcinoma Papillary Carcinoma PDC and AC % (3-83%) 21% (-5%) Meta analysis of literature Nikiforov Nature Rev Endocrinol 211 7:569 Nikiforov Nature Rev Endocrinol 211 7:569 PTC with Oncogenes (7%) PAX8/PPAR 1-5% Specificity (%) Mutation panel (BRAF, RAS, RET/PTC, PPAR ) HIGH specificity Nikiforov (n=513) Cantara (n=95) Moses (n=137) Follicular neoplasm Susp for malignancy FLUS Nikiforov J Clin Endocrinol Metab :339; Cantara J Clin Endocrinol Metab 21 95:1365; Moses W J Surg 21 34:2589 Sensitivity (%) Mutation panel (BRAF, RAS, RET/PTC, PPAR ) NOT optimal sensitivity Nikiforov (n=513) Cantara (n=95) Moses (n=137) Many cancers are not positive Follicular neoplasm Susp for malignancy FLUS Nikiforov J Clin Endocrinol Metab :339; Cantara J Clin Endocrinol Metab 21 95:1365; Moses W J Surg 21 34: % 2 13% FA RAS HRAS, NRAS codon 61 KRAS codons 12,13 PAX8/PPAR FA Overlap of Follicular Adenoma and CA 4% 3% FC FC ~7% Follicular CA 4

5 BRAF RET/PTC PPAR RAS 1% CANCER False positives Proposed Algorithm by Endocrine Society 45 4 Nikiforov n=61 Cantara n=13 Moses n= RAS False positive rate Nikiforov J Clin Endocrinol Metab :339; Cantara J Clin Endocrinol Metab 21 95:1365; Moses W J Surg 21 34:2589 Frequency (%) 211 by Endocrine Society Nikiforov Y E et al. JCEM 211;96: Mutation panel (BRAF, RAS, RET/PTC, PPAR ) Should not use to AVOID surgery if the test is negative not optimal sensitivity you will miss cancers When a patient is going to surgery, you want a test to optimize the surgical procedure High specificity high positive predictive value Refer patients with cytology of suspicious for PTC who test positive for the Oncogene panel for total thyroidectomy instead of hemi thyroidectomy Malignancies with FN and FLUS less likely to be mutation positive than sptc 1 Yip J Clin Endocrinol Metab :195 When do we use Oncogene Panel testing at Penn? suspicious for malignancy Nodule with follicular neoplasm or FLUSx2 cytology when observation is NOT felt to be an option Large nodules >3cm Suspicious sonographic features? Non diagnostic cytology x 2 Cantara et al: 14/53 nodules with nondx cytology had mutations 12 cancers Cantara J Clin Endocrinol Metab 21 95:1365 Testing Beyond : Additional testing for Indeterminate FNA Results Oncogene testing BRAF Oncogene mutation panels (Asuragen TM ) RNA Expression Micro RNA Afirma TM gene expression classifier Hypothesis of GEC A novel, molecular diagnostic test could be developed with high Negative Predictive Value (NPV) for malignancy. When applied to cytologically indeterminate thyroid nodules, a benign gene expression classifier (GEC) result would accurately predict a non cancerous nodule. 5

6 Development of a gene expression classifier (GEC) to accurately identify benign thyroid nodules in patients with indeterminate FNA cytology AFIRMA DEVELOPMENT Whole Transcriptome approach using microarray technology Molecular Classifier Trained and Validated to Distinguish Benign vs. Suspicious Nodules Feasibility Development Validation On Whole Genome Exon Array, went from 22K to 3K genes relevant to thyroid neoplasia From 3K set, selected final 142 genes and locked final algorithm Tested final 167 gene expression classifier on independent samples A multidimensional algorithm required to separate complex data sets Benign Number of Genes 22K genes 3K genes 167 genes Suspicious Chudova D, et al. J Clin Endocrinol Metab Results by Cytopathology Diagnosis N/A N (sample size) = N Engl J Med :26 Alexander EK, et al. NEJM 212. Non-diagnostic or unsatisfactory Malignancy risk Management ~1-5% Repeat FNA Benign 2-4% Observe with US Follow up Afirma in action 51 endocrinologists surveyed 9/11-3/ pts with indeterminate cytology and benign GEC Pattern of surgical referral evaluated FLUS x 2 GEC Negative FN in low risk patient 5-6% Observe with US Follow up % recommended for surgery p<.1 8 With benign GEC 74 Traditional paradigm Duick Thyroid :996; Wang Thyroid :243 6

7 Afirma TM in action Mayo clinic US FNA of 1112 nodules Nondx 9.7% Benign 73% Malignant/suspicious for malignancy 9.3% FLUS/AUS or follicular neoplasm 8%--89 pts and offered GEC testing Afirma TM in action Mayo clinic 53 nodules with GEC results 13 (24%) BENIGN 4 (76%) SUSPICIOUS McIver 212 ATA abstract 89 AUS/FLUS/FN 53 with GEC results 3 not submitted (pt declined, MD choice) 6 NO result (insuff RNA) McIver ATA abstract surgery 13 no surgery 23 (85%) BENIGN 4 (15%) CANCER Afirma TM in action Mayo clinic Positive predictive value Mayo 14% vs. NEJM 38% Different cancer prevalence in FLUS/FN NEJM 25% vs. Mayo 15% Different proportion of GEC suspicious results in FLUS/FN NEJM 57-6% vs Mayo 78% Real world application may vary... NPV NPV and PPV for Afirma TM GEC based upon N Engl J Med : % 38% 9% Sensitivity and 52% Specificity Prevalence of malignancy PPV Courtesy of Bryan McIver Negative Predictive Value falls at higher disease prevalence NPV % Sensitivity 9% Sensitivity 8% Sensitivity When do we use GEC testing at Penn? FLUS/AUS Follicular/Hürthle cell neoplasm Cancer risk <25% Gene expression classifier HIGH NEGATIVE PREDICTIVE VALUE BENIGN SUSPICIOUS Prevalence of malignancy OBSERVE LOBECTOMY Courtesy of Bryan McIver 7

8 Molecular Analysis Oncogene testing identifies indeterminate nodules with a high risk of malignancy Used for patients undergoing surgery Suspicious for PTC, suspicious nodules with FN or non dx cytology and suspicious sonographic appearance m RNA Gene expression classifier has a high NPV Used when observation rather than surgery is the goal FLUS x 2, small FN, no suspicious US features Benign Malignant Nondx* Result reported Evaluation of thyroid nodules 212 History, physical examination, TSH Thyroid US with risk stratification for FNA FNA cytology sample Follicular lesion of undetermined significance Follicular/Hürthle cell neoplasm Suspicious for malignancy Molecular analysis Result reported To reflect back 5 years... New England Journal of Medicine 1964 Thank you 214 Ultrasound FNA Molecular analysis 8

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