Markers in Thyroid Nodule Evaluation. Yuri E. Nikiforov, MD, PhD Division of Molecular & Genomic Pathology University of Pittsburgh Medical Center

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1 Markers in Thyroid Nodule Evaluation Yuri E. Nikiforov, MD, PhD Division of Molecular & Genomic Pathology University of Pittsburgh Medical Center

2 Disclosures Quest Diagnostics (consultant) UPMC/CBLPath contract to offer ThyroSeq test; owner of intellectual property related to ThyroSeq

3 Outline Clinical need Progress on genomics of thyroid cancer Molecular tests for cancer diagnosis Molecular markers for cancer prognostication

4 The Thyroid Nodule Dilemma: Benign or malignant? Thyroid nodule fine-needle aspiration (FNA) biopsy Fine-needle aspiration biopsy of the thyroid: an appraisal 526,700 thyroid FNA in U.S. in % annual growth rate 19.8% of thyroidectomies result in cancer diagnosis Sosa, JA et al. AAES Meeting Abstract 2013 Sosa, JA et al. Surgery, 2013; 154:L Gharib, H. et al. Ann Intern Med 1993;118:28

5 The Thyroid Nodule Dilemma: Benign or malignant? The Bethesda System for Reporting Thyroid Cytopathology Diagnostic Category Risk Usual of cancer management I. Nondiagnostic or Unsatisfactory 1-4% Repeat FNA with US II. Benign 0-3% Clinical follow-up III. Atypia of Undetermined Significance 5-15% Repeat FNA or Follicular Lesion of Undetermined Significance (AUS/FLUS) IV. Follicular Neoplasm or Suspicious 15-30% Surgical lobectomy for a Follicular Neoplasm (FN/SFN) V. Suspicious for malignancy 60-75% Total or lobectomy VI. Malignant 97-99% Total thyroidectomy Baloch ZW et al. Diagn Cytopathol 2008;36:425 Cibas ES & Ali SZ. Am J Clin Pathol 2009;132:658

6 Thyroid Cancer Incidence and Mortality Thyroid Cancer Incidence and Mortality in the U.S. PTC incidence rates by tumor size ( ) <1 cm cm cm >5 cm Davies L &Welch HG. JAMA (2006) Data age-adjusted to the 2000 U.S. Census with 95% CI. Cramer JD et al. Surgery. 2010;148:1147

7 Thyroid cancer risk stratification Risk of Structural Disease Recurrence (In patients without structurally identifiable disease after initial therapy) High Risk Gross extrathyroidal extension, incomplete tumor resection, distant metastases, or lymph node >3cm Intermediate Risk Aggressive histology, minor extrathyroidal extension, vascular invasion, or > 5 involved lymph nodes (0.2-3 cm) Low Risk Intrathyroidal DTC 5 LN micrometastases (< 0.2 cm) FTC, extensive vascular invasion ( 30-55%) pt4a gross ETE ( 30-40%) pn1 with extranodal extension, >3 LN involved ( 40%) PTC, >1 cm, TERT mutated ± BRAF mutated* (>40%) pn1, any LN > 3 cm ( 30%) PTC, extrathyroidal, BRAF mutated* ( 10-40%) PTC, vascular invasion ( 15-30%) Clinical N1 ( 20%) pn1, > 5 LN involved ( 20%) Intrathyroidal PTC, < 4 cm, BRAF mutated* ( 10%) pt3 minor ETE ( 3-8%) pn1, all LN < 0.2 cm ( 5%) pn1, 5 LN involved ( 5%) Intrathyroidal PTC, 2-4 cm ( 5%) Multifocal PMC ( 4-6%) pn1 without extranodal extension, 3 LN involved (2%) Minimally invasive FTC ( 2-3%) Intrathyroidal, < 4 cm, BRAF wild type* ( 1-2%) Intrathyroidal unifocal PTMC, BRAF mutated*, ( 1-2%) Intrathyroidal, encapsulated, FV-PTC ( 1-2%) Unifocal PMC ( 1-2%) Haugen BR, et al. Thyroid 2016, 26:1-133

8 Clinical need: 1. provide accurate cancer diagnosis in nodules with indeterminate cytology 2. predict cancer risk pre-operatively (to allow most optimal surgery and post-surgical management)

9 56 YO Male With a 4 x 2 x 3 cm Thyroid Nodule Left lobe long Final Cytology Diagnosis ATYPIA OF UNDETERMINED SIGNIFICANCE (BETHESDA CATEGORY III) Right lobe trans No abnormal cervical LNs Observation? Diagnostic lobectomy? Total Thyroidectomy? Would molecular testing help in this case?

10 Evolution of Molecular Tests for Thyroid Nodules and Cancer GENOMIC TESTS Proof of principle SINGLE GENE TESTS Progress in molecular tests for thyroid nodules/cancer % 30% 70% >90% Characterization of molecular landscape of thyroid cancer

11 Genomic Landscape of PTC 402 papillary thyroid cancers BRAF Point mutations 75% RAS Gene fusions 15% Copy number variations 7% The Cancer Genome Atlas Research Network. Cell 159: (2014)

12 Genomic Landscape of Other Types of Thyroid Cancer

13 BRAF-like and RAS-like PTC 402 papillary thyroid cancers BRAF V600E BRAF V600E fusions fusions RAS RAS The Cancer Genome Atlas Research Network. Cell 159: (2014)

14 Molecular Markers for Cancer Diagnosis Mutational markers (gene panels, ThyroSeq) Gene expression (mrna) markers (Afirma) mirna markers (ThyGenX; Rosetta GX Reveal) Circulating TSHR mrna Proteomics

15 Mutational Markers Single genes and small panels PAX8/ PPARG RET/PTC 1 RET/PTC 3 NRAS HRAS KRAS BRAF BRAF 60-65% 461 FNA samples with Bethesda III-IV cytology and surgical outcome Sensitivity 57-63% Specificity 97-99% NPV 86-94% PPV 87-88% High specificity/ppv good rule in test J Clin Endocrinol Metab. 2011;96:3390-7

16 Afirma Gene Expression Classifier N Engl J Med 2012; 367:

17 Afirma Gene Expression Classifier Multi-institutional double-blind prospective study of indeterminate cytology FNA samples Sample size 265 FNAs Cytologic diagnosis n Sensitivity Specificity NPV PPV AUS/FLUS % 53% 95% 38% FN/SFN 81 90% 49% 94% 37% SUSP 55 94% 52% 85% 76% High sensitivity/npv good rule out test Alexander E et al. N Engl J Med (2012)

18 Afirma Gene Expression Classifier NPV range %; PPV range % Nishino M. Cancer Cytopathol ; 124:14-27

19 ThyroSeq Mutation-Based Genomic Classifier ThyroSeq v.0 7-gene panel ThyroSeq v.1 15-gene panel ThyroSeq v.2 56-gene panel ThyroSeq v.3 >100-gene panel 65% 78% 90% >95% Pre-NGS Next Generation Sequencing Approaches

20 Gene Mutations (DNA) BRAF NRAS HRAS KRAS PIK3CA PTEN TERT RET TSHR AKT1 TP53 GNAS CTNNB1 EIF1AX ThyroSeq v2 56-genes: 14 genes for mutations; 42 fusion types; 16 genes for expression Next generation sequencing assay Gene Fusions (mrna) RET PPARG NTRK1 NTRK3 BRAF ALK Other Primary diagnostic and prognostic markers Gene expression (mrna) PGK1 KRT7 TG TTF1 NIS Calcitonin PTH KRT20 Other pan-cell marker Thyroid epithelial cells MTC parathyroid metastatic Specimen QA/Cell lineage markers Nikiforov et al. Cancer 2014,120:3627; Nikiforov et al. Thyroid 2015;25:1217

21 ThyroSeq performance in Bethesda IV nodules Patients with FN/SFN (Bethesda IV) cytology and known surgical outcome 143 consecutive FNA samples Retrospective and prospective arms Cancer prevalence 27.3% Sensitivity 90% (CI: 80-99%) Specificity 93% (CI: 88-98%) NPV 96% (CI: 92-95%) PPV 83% (CI: 72-95%) Accuracy 92% (CI: 88-97%) Nikiforov et al. Cancer 2014,120:

22 ThyroSeq performance in Bethesda III nodules 0.6%- PT 465 consecutive FNA samples with AUS/FLUS (Bethesda III) cytology 96 patients with surgical outcome Prospectively evaluated Cancer prevalence 22.5% Sensitivity 91% (CI: %) Specificity 92% (CI: 86-98%) NPV 97% (CI: %) PPV 77% (CI: 61-93%) Accuracy 92% (CI: 86-97%) Nikiforov et al. Thyroid 2015;25:

23 ThyroSeq v2: Independent Studies 148 Bethesda III and IV nodules Boston Medical Center Disease prevalence - 44% Sensitivity 95% Specificity 60% NPV 94% PPV 66% 102 Bethesda III and IV nodules Moffitt Cancer center Disease prevalence - 20% Sensitivity 70% Specificity 77% NPV 91% PPV 42% Toraldo G et al. ATA abstract 2016 Valderrabano P et al. ERC, 2017

24 ThyraMIR+ThyGenX Test 7 gene panel + 10 mirna classifier Validation: 109 non consecutive cross-sectional specimens from 12 sites Bethesda III and IV cytology only, with corresponding histology Cytology (Bethesda) n Sensitivity Specificity NPV PPV III 58 94% 80% 97% 68% IV 51 82% 91% 91% 82% Total 109 Labourier E J Clin Endocrinol Metab 2015; 100: 2743

25 Rosetta GX Reveal mirna classifier 24 mirna Classifier Training set of 375 FNA specimens Validation with 201 of which 189 stained microscope slide specimens met RNA control No Hurtle cell cancers in validation set Cytology (Bethesda) n Sensitivity Specificity NPV PPV Entire Data Set Agreement Data III+IV % 74% 92% 43% III+IV % 80% 100% 41% Lithwick-Yanai G J Clin Pathol published online 10/2016

26 Reported Diagnostic Performance of Available Molecular Tests Afirma Rosetta ALL Rosetta Agreement ThyGenX + ThyraMir ThyroSeq Cytology (Bethesda) Cases (N) Cancers (N) NPV (%) PPV (%) III IV Total III+IV III+IV III IV Total III IV Total Alexander E. et al, N Engl J Med 2012; 367: ; Lithwick-Yanai G J Clin Pathol published online 10/2016; Labourier E J Clin Endocrinol Metab 2015; 100: 2743; Nikiforov et al. Cancer 2014,120:3627; Nikiforov et al. Thyroid 2015;25:1217

27 Cancer Probability Associated with Individual Mutations Cancer probability BRAF ALK NTRK1,3 PPARG RET/PTC TERT; P53 RAS PTEN EIF1AX TSHR GNAS >95% 80% 5-10% Very low* *Unless AF>30% Nikiforov et al. Cancer 2014,120: Nikiforov et al. Thyroid 2015;25: Karunamurthy et al. Endocr Relat Cancer. 2016;23:295

28 Cancer Probability in Nodules with RAS Mutations Medicine 2015;94:1-6 Total FNA samples 198 Total RAS+ 31 RAS+ Cancer 7 (23%) RAS+ Benign 24 (77%) BioMed Res Int 2015;2015: Total FNA samples 132 Total RAS+ 27 RAS+ Cancer 26 (96%) RAS+ Benign 1 (4%) Nuclear features of Papillary Carcinoma Clearly absent Clearly present

29 Multi-step cancer progression Colon cancer Normal epithelium APC Adenoma RAS BRAF SMAD4 Carcinoma in situ TP53 TGFB BAX Invasive carcinoma Breast cancer Normal breast epithelium Initiated cells Carcinoma in situ Invasive carcinoma Metastasis Craene B. & Berx G. (2013); Brisken C. (2013)

30 Multi-step cancer progression BENIGN MALIGNANT BENIGN PREMALIGNANT IN SITU MALIGNANT

31 NIFTP Non-Invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features Completely encapsulated or clearly demarcated follicular-pattern tumors Nuclear features of PTC No invasion No aggressive histology Oncogene-driven (RAS and RASlike gene mutations) Previously known as encapsulated follicular variant of papillary carcinoma Nikiforov et al. JAMA Oncology 2016; 2:

32 No RAI! Nikiforov et al. JAMA Oncology 2016; 2:

33 Nikiforov et al. JAMA Oncology 2016; 2:

34 Not all benign thyroid nodules are created equal: Variability in malignant potential Benign hyperplastic nodule Do not progress to cancer! Non-clonal Benign follicular adenoma Atypical FA FTC Clonal NIFTP PTC

35 Not all benign thyroid nodules are created equal: Variability in malignant potential Benign hyperplastic nodule Do not progress to cancer! No Non-clonal mutations Benign follicular adenoma Atypical FA FTC Mutation Clonal + (eg. RAS) NIFTP PTC

36 Molecular Changes Precede Histological Changes C A NGS Point mutations Indels NRAS c.181c>a B Point mutations Indels Copy number alteration Gene fusions Point Gene mutations expression Indels NRAS c.181c>a NRAS c.181c>a Adopted from Simon et al. Nature 2013 Gupta M et al. JCEM (2013)

37 Eszlinger M et al. Thyroid Mar;27(3):

38 Wang N, et al. Cancer Oct 1;120(19):

39 Cancer Diagnosis: Challenging the Gold Standard The current update breaks with the century-old principle of diagnosis based entirely on microscopy by incorporating molecular parameters into the classification of CNS tumors

40 Prognostic Utility of Molecular Testing

41 Use of Molecular Markers for Cancer Risk Assessment 1510 patients, 97% with PTC Excised tumors tested for 7 common mutations 70% of tumors found mutation-positive Mean follow-up 33 ± 21.2 months with PTC Low risk Intermediate risk Yip et al. Ann Surg 262: (2015)

42 Cum Survival Molecular markers of high-risk cancer TERT gene, multiple mutations DTC, n=469 DTC, n=551 TERT wt ATA high risk, TERT(-) TERT mut ATA high risk, TERT(+) Follow-up (years) Melo M et al. JCEM (2014) Song YS et al. Cancer (2016) PTC, n=507 DTC, n=551 TERT RAS BRAF BRAF+TERT RAS+TERT BRAF+TERT Xing M et al. JCO (2014) Song YS et al. Cancer (2016)

43 Cancer risk assessment using ThyroSeq v2 0.6 cm nodule AUS cytology BRAF+/TERT+ AKT1+/PIK3CA+ mptc with extrathyroid ext 35 (55%) BRAF + Another HR mutation 18 (29%) RAS + Another HR mutation 3 (5%) Other Multiple HR mutations 55 (98%) Thyroid Cancer 51 (93%) Cancers with Aggressive Features: Extrathyroidal extension (55%) Vascular invasion (53%) Lymph node macrometastasis (47%) Poorly differentiated/anaplastic ca (14%) Distant metastasis (8%) Nikiforova M et al. ATA abstract 2016

44 Molecular markers to predict cancer risk Risk of Structural Disease Recurrence (In patients without structurally identifiable disease after initial therapy) Molecular Signature High Risk Gross extrathyroidal extension, incomplete tumor resection, distant metastases, or lymph node >3cm Intermediate Risk Aggressive histology, minor extrathyroidal extension, vascular invasion, or > 5 involved lymph nodes (0.2-3 cm) Low Risk Intrathyroidal DTC 5 LN micrometastases (< 0.2 cm) FTC, extensive vascular invasion ( 30-55%) pt4a gross ETE ( 30-40%) pn1 with extranodal extension, >3 LN involved ( 40%) PTC, >1 cm, TERT mutated ± BRAF mutated* (>40%) pn1, any LN > 3 cm ( 30%) PTC, extrathyroidal, BRAF mutated* ( 10-40%) PTC, vascular invasion ( 15-30%) Clinical N1 ( 20%) pn1, > 5 LN involved ( 20%) Intrathyroidal PTC, < 4 cm, BRAF mutated* ( 10%) pt3 minor ETE ( 3-8%) pn1, all LN < 0.2 cm ( 5%) pn1, 5 LN involved ( 5%) Intrathyroidal PTC, 2-4 cm ( 5%) Multifocal PMC ( 4-6%) pn1 without extranodal extension, 3 LN involved (2%) Minimally invasive FTC ( 2-3%) Intrathyroidal, < 4 cm, BRAF wild type* ( 1-2%) Intrathyroidal unifocal PTMC, BRAF mutated*, ( 1-2%) Intrathyroidal, encapsulated, FV-PTC ( 1-2%) Unifocal PMC ( 1-2%) NIFTP (<1%) BRAF+TERT, RAS+TERT Multiple driver mutations (eg. NRAS and PIK3CA or TP53) TERT ALK fusions NTRK1 fusions NTRK3 fusions BRAF V600E RET/PTC RAS BRAF K601E PAX8/PPARG BRAF V600Elike mutations RAS-like mutations Haugen BR et al. Thyroid. 2016, 26:1-133

45 Summary: Potential clinical management informed by molecular profiling Bethesda III-IV Cytology ThyroSeq v2 Test result Negative: no mutations Currently Negative: low level or LR mutations Positive: RAS-like mutation Positive: BRAF-like mutation Positive: multiple HR mutation Probability of Cancer or NIFTP 3-4% <10% 80-90% 95-99% % Tumor type, risk of recurrence N/A NIFTP or low-risk cancer NIFTP or lowrisk cancer Intermediaterisk cancer High-risk cancer Patient management Observation Active surveillance Lobectomy Total thyroidectomy or lobectomy Total thyroidectomy +/- LND