Clinical and Molecular Approach to Using Thyroid Needle Biopsy for Nodular Disease Robert L. Ferris, MD, PhD Department of Otolaryngology/Head and Neck Surgery and Yuri E. Nikiforov, MD, PhD Division of Molecular Anatomic Pathology University of Pittsburgh School of Medicine Objectives To provide an overview of molecular alterations in thyroid tumors To discuss specimen requirements for molecular testing in thyroid samples To discuss the diagnostic and prognostic utility of specific molecular markers in thyroid surgical resections and fine needle aspiration (FNA) samples
Trends in Incidence of Thyroid Cancer (1973-2002) and Papillary Tumors by Size (1988-2002) in the United States Thyroid Cancer Incidence and Mortality, 1973-2002 Davies, L. et al. JAMA 2006;295:2164-2167. Risk of Cancer in a Thyroid nodule 3 5% in a woman age 20-40 Age Sex Size Cystic Radiation as youth Standard for Evaluation Fine needle aspirate (FNA)
Benign Interpretation of FNA Malignant 1 Follicular/FLUS 2 Inadequate 2 1 ATA 2009 guidelines for central neck dissection are vague cytology may be the only information we have preoperatively 2 For certain cytologic diagnoses, lobectomy is a diagnostic procedure
Thyroid Nodules and FNA Cytology FNA cytology establishes definitive diagnosis in most cases 15-30% reported as indeterminate Category Risk of malignancy FLUS 5-10% Follicular neoplasm/lesion 20-30% Suspicious for malignancy 50-75% Baloch ZW et al. Diagn Cytopathol (2008) FNA cytology Positive for Malignancy 1-2% false positive Management of Patients with Thyroid Nodules Total Thyroidectomy I-131 Therapy Negative for Malignancy 1-3% false negative Indeterminate LUS or Follicular neoplasm Thyroid Lobectomy Completion Thyroidectomy I-131 Therapy
TSHR RTK Papillary carcinoma Follicular carcinoma β α γ RET/PTC 15% Adenylate cyclase camp PKA 15% 45% MAPK Pathway RAS BRAF MEK 45% PI3K AKT mtor PTEN PI3K/AKT Pathway ERK 30% PAX8/PPARγ Mutations in Papillary Carcinoma BRAF 45% N RET/PTC 15% ~75% Papillary CA RAS 15%
Mutations in Papillary Carcinoma BRAF 45% Tall cell and classical papillary CA Extrathyroidal extension Higher stage at presentation Higher rate of tumor recurrence Propensity for dedifferentiation N RET/PTC 15% RAS 15% Classic papillary CA Younger age at presentation Assoc. with radiation exposure Frequent l/node metastasis Lower stage at presentation Follicular variant of PTC More frequent encapsulation Less frequent l/node metastasis More frequent distant metastases Mutations in Follicular Carcinoma FA FC N 40% 30% FC ~70% Follicular CA
Mutations in Follicular Carcinoma N FA FC 40% 30% FC Larger tumor size Higher rate of distant metastasis Propensity for dedifferentiation Younger age of presentation Smaller tumor size Solid/nested growth pattern More frequent vascular invasion BRAF Mutations in Thyroid Nodules: Prevalence and Specificity Number of cases reported Benign Nodules BRAF V600E 1 (0.16%) Follicular Carcinoma Papillary Carcinoma PDC and AC 611 171 2140 125 0 45% (30-83%) 21% (0-50%) Meta-analysis of literature 2003-2007
BRAF V600E Predicts Tumor Aggressiveness Multicenter study, 107 patients Single institution, 190 patients Xing et al. JCEM (2005) Xing, M. et al. J Clin Oncol; 27:2977-2982 2009 Prevalence and Specificity of Mutant RAS Detection Goiter Follicular adenoma Follicular Carcinoma Papillary Carcinoma PDCA and AC Range 0-20% 20-45% 40-50% 0-20% 20-90% Average 5% 34% 45% 15% 30% Meta-analysis of literature 1988-2007
Prevalence and Specificity of PAX8/PPAR Detection Goiter Follicular adenoma Follicular Carcinoma Papillary Carcinoma PDCA and AC Range 0 0-20% 20-70% 0-5% 0 Average 0 7% 30% 1% 0 Meta-analysis of literature 2000-2007 Molecular Diagnostics in Thyroid FNA Samples
Detection of Mutations in Thyroid FNA Samples Individual mutations/retrospective studies: RAS Sciacchitano S et al. Diagn Mol Pathol (1994) RET/PTC - Cheung C et al. JCEM (2001), Salvatore G et al. JCEM (2004) BRAF - Cohen Y et al. Clin Cancer Res (2004), Xing M et al. JCEM (2004), Salvatore G et al. JCEM (2004) Mutation panel/prospective study: BRAF, RET/PTC, RAS, PAX8/PPARg - Nikiforov et al., JCEM (2009) BRAF Mutations in Thyroid FNA Samples Samples (n) BRAF Positive Final Diagnosis in BRAF Positive Samples Thyroid Nodule FNA 9 Prospective Studies Thyroid Nodule FNA 7 Retrospective Studies 1,814 159 PTC = 159 (100%) 685 291 PTC = 291 (100%) Research FNA of Surgically Removed Thyroid, 2 studies 267 131 PTC = 130 (99.2%) HN*= 1 (0.8%) Total, 18 studies 2,766 581 PTC=580 (99.8%) HN*= 1 (0.2%) *Hyperplastic nodule reported as atypical nodular hyperplasia PTC - Papillary Thyroid Carcinoma Nikiforova MN & Nikiforov YE, Thyroid 2009 [Epub ahead of print]
Testing for Multiple Mutations in Thyroid FNA Samples Prospective study, 2003-2006, two centers (Univ. of Cincinnati, Univ. of Colorado) Thyroid nodule tested: 470 Mutation detected: 32 Risk of Cancer BRAF (18) 100% RAS (8) 87% RET/PTC (5) 100% PAX8/PPARg (1) 100% Nikiforov et al. JCEM (2009) Mutation Detection in Thyroid FNA Samples Results of Cytology and Molecular Analysis Cancer Probability in Thyroid Nodules Positive Cytology and Positive for Mutation 100% Indeterminate Cytology Alone 40.4% Indeterminate Cytology and Positive for Mutation 100% Indeterminate Cytology and Negative for Mutation 16.2% Negative cytology Alone 2.1% Negative cytology and Negative for Mutation 0.9% Nikiforov et al. JCEM (2009)
Molecular Testing of Thyroid FNA Samples with Indeterminate Cytology Cytology Diagnosis Molecular Testing (MT) HistologicDiagnosis MT Performance FLUS (21) 5-10% risk of malignancy Mutation Positive (3) BRAF (1), RAS(2) Mutation Negative(18) Malignant (3) PC (3) Benign (18) HN (15), FA (3) Sensitivity 100% Specificity 100% PPV 100% NPV 100% Accuracy 100% Follicular or Hürthle cell neoplasm (23) 20-30% risk of malignancy Mutation Positive (9) BRAF (4), RAS(3), PAX8/PPARγ(1), RET/PTC (1) Mutation Negative(18) Malignant (9) PC (7), FC (2) Malignant (3) PC (1), FC (2) Benign (11) HN (10), FA (1) Sensitivity 75% Specificity 100% PPV 100% NPV 79% Accuracy 87% Suspicious for malignancy (7) 50-75% risk of malignancy Mutation Positive (3) BRAF (4), RET/PTC (1) Mutation Negative(4) Malignant (3) PC (3) Sensitivity 60% Specificity 100% Malignant (2) PC (2) Benign (2) HN (2) PPV 100% NPV 50% Accuracy 71% Nikiforov et al. JCEM (2009) Molecular Testing of Thyroid FNA Samples at Univ. of Pittsburgh Clinical algorithm initiated in April 2007: Cytology Positive Indeterminate Negative MolecularTesting Molecular Testing for Mutations BRAF, BRAF, NRAS, NRAS, HRAS, HRAS, KRAS, KRAS, RET/PTC, RET/PTC, PTC,PAX8/PPAR? PAX8/PPARγ BRAF, RAS, RET/PTC, PAX8/PPARg Total NA Isolation Total NAIsolation Real-time PCR Real-time PCR Real-time RT-PCR Real-time RT-PCR
Results April 2007- February 2009 Thyroid nodule tested: 925 Mutation detected: 105 (11%) BRAF - 52 NRAS -34 HRAS - 10 46 KRAS - 2 RET/PTC -4 PAX8/PPARg -3 Mutation-positive nodules with surgery: 95 Nikiforov, JCEM, 2011, in press BRAF Mutations Molecular Cytology Histology BRAF+ (52) Surgery (50) MALIG (39) INDET (10) 4 FLUS 1 - FN 5 SUSP PTC (39) PTC (10) INSUF (1) PTC (1)
Case: 42 yo woman with right thyroid nodule Cytology Molecular Histology V600E WT FINAL DIAGNOSIS: FNA RIGHT THYROID: SATISFACTORY FOR INTERPRETATION. SUSPICIOUS FOR MALIGNANT CELLS. CLUSTERS OF ATYPICAL FOLLICULAR CELLS PRESENT SUSPICIOUS FOR PAPILALRY CARCINOMA. Addendum Molecular Anatomic Pathology Testing: THYROID, RIGHT, FINE NEEDLE ASPIRATION- A. BRAF T1799A mutation IS IDENTIFIED. B. Mutations in HRAS61, in NRAS61, HRAS61, and RET/PTC1, RET/PTC3 and PAX8/PPARg rearrangements NOT IDENTIFIED. FINAL DIAGNOSIS: THYROID, TOTAL THYROIDECTOMY (17.8 GRAMS) - A. PAPILLARY THYROID CARCINOMA, CONVENTIONAL TYPE, 1.1 CM B. NO EXTRATHYROIDAL EXTENSION. C. RESECTION MARGINS ARE NEGATIVE RAS Mutations Molecular Cytology Histology RAS+ (46) Surgery (39) INDET (39) FLUS (15) Foll. Neoplasm (18) SUSP (6) PTC (30) FTC (2) ATC (1) FA (4) HN (2) 85% 10% 5%
Case 2: 37 yo woman with right thyroid nodule Cytology Molecular Histology MUT WT FINAL DIAGNOSIS: FNA RIGHT THYROID: LESION OF UNDETERMINED SIGNIFICANCE. MACROPHAGES AND FEW FOLLICULAR GROUPS (SEE COMMENT). Addendum Molecular Anatomic Pathology Testing: THYROID, RIGHT, FINE NEEDLE ASPIRATION- A. NRAS61 mutation IS IDENTIFIED. B. Mutations in BRAF, HRAS61, KRAS12/13, and RET/PTC1, RET/PTC3 and PAX8/PPARg rearrangements NOT IDENTIFIED. FINAL DIAGNOSIS: THYROID, TOTAL THYROIDECTOMY (36 GRAMS) - A. PAPILLARY THYROID CARCINOMA, FOLLICULAR VARIANT, PARTIALLY ENCAPSULATED, 4.0 CM. B. NO ANGIOLYMPHATIC INVASION OR EXTRATHYROIDAL EXTENSION. C. RESECTION MARGINS ARE NEGATIVE. Molecular Testing of FNA Samples Diagnostic Impact Thyroid nodule tested: 925 Mutation detected - 105 (11%) Surgery performed - 95 Malignant - 89 (94%) Neoplastic - 93 (98%) Cancer diagnosis confirmed (cytology malignant) 43 patients Cancer diagnosis established (cytology indeterminate) 52 patients 20 - FLUS cytology 21 - FL cytology
BRAF and Aggressiveness of Papillary MicroCA Table 1 Analysis of BRAF V600E mutation in PTMCs BRAF mutation Positive (n = 24) Negative (n = 40) p Age (yr) 49.1 ± 10.4 45.1 ± 11.2 0.533 Male:female ratio 2:22 6:34 0.358 Nodular goiter 6 (25%) 8 (20%) 0.432 Hashimoto thyroiditis 0 8 (20%) >0.99 Extrathyroidal extension 12 (50%) 4 (10%) 0.001* Nodes metastasis 12 (50%) 6 (15%) 0.003* Tall cell variant 2 (8.3%) 2 (5%) 0.483 T 1 :T 3/4 ratio 12:12 36:4 0.001* * Statistically significant Lee X et al. Ann Surg Oncol (2009) BRAF and Aggressiveness of Papillary Micro-carcinoma AGGRESSIVE GROUP N= 29 (26 PTS) NON-AGGRESSIVE GROUP N= 30 (28 PTS) P VALUE BRAF 20/26 (77%) 8/25 (32%) 0.0016 ANATOMIC LOCATION - LEFT LOBE/ RIGHT LOBE 15 (52%) / 14 (48%) 14 (47%) / 14 (47%) 0.4495 TUMOR LOCATION - SUBCAPSULAR 27 (93%) 9(30%) < 0.0001 POSITIVE MARGIN 2 (7%) 1 (3%) 0.4876 COMPLETE CAPSULE 2 (7%) 8 (27%) 0.0469 INFILTRATIVE BORDER 29 (100%) 24 (80%) 0.0174 MICROSCOPIC VARIANT - CLASSIC PAPILLARY 13 (45%) 11 (37%) 0.3546 - FOLLICULAR VARIANT 8 (28.0%) 18 (60%) 0.0124 - TALL CELL FEAT/VAR 8 (28%) 1 (3%) 0.0129 INTRATHYROIDAL SPREAD 22 (76%) 6 (20%) <0.0001 FIBROSIS - NO 2 (7%) 9 (30%) 0.026 - YES, 1+ 3 (10%) 9 (30%) 0.0603 - YES, 2+ 24 (83%) 12 (40%) 0.001 EXTRATHYROIDAL EXTENSION 19 (66%) 4 (13%) <0.0001 MULTI-CENTRIC 6 (21%) 3 (10%) 0.0739 Niemeier L et al. Cancer, 2011, in press
Molecular-Pathological Score Defines Papillary MicroCA Aggressiveness Scoring Criteria BRAF Mutation + 2 Subcapsular (superficial) location 1 Extrathyroidal extension 1 Tumor fibrosis, 2+ 1 Intrathyoidal spread 1 60 50 40 % 30 20 10 0 5 4 3 2 1 0 MP Score, BRAF + 4 features aggressive control Score 4 3 0-2 High risk Interm risk Low risk Niemeier L et al. Cancer, 2011, in press Revised Management Guidelines for Patients with Thyroid Nodules and Differentiated Thyroid Cancer American Thyroid Association 2009
SUMMARY Molecular Analysis of Thyroid Neoplasms Known gene mutations occur in 75% of well differentiated thyroid carcinomas - can be detected in thyroid cytology and surgical samples In FNA samples, BRAF, RET/PTC, and PAX8/PPARg have a 100% PPV for cancer In FNA samples, RAS mutations have 85-87% PPV for cancer BRAF can be used as a molecular marker of aggressiveness in papillary carcinomas