3The Bethesda System for Reporting Thyroid Cytopathology CategoryRisk of MalignancyI. Nondiagnostic or Unsatisfactory1-4%Benign0-3%III. Atypia of undetermined significance or Follicular lesion of undetermined significance~5-15%IV. Follicular Neoplasm orSuspicious for a Follicular Neoplasm15-30%V. Suspicious for Malignancy60-75%VI. Malignant97-99%
4Use of molecular biomarkers Improve the accuracy of fine-needle aspiration cytologyProvide prognostic information
5Genetic alterations in thyroid cancer Activating and inactivating somatic mutations,Alteration in gene expression patterns,MicroRNA dysregulationAberrant gene methylation
6Thyroid cancer Follicle-derived Papillary carcinoma Well differentiated carcinomaPapillary carcinomaFollicular carcinomaPoorly differentiated carcinomaUndifferentiated (Anaplastic) carcinoma
8BRAF mutationsBRAF is a serine-threonine kinase. BRAF can be activated by point mutations, small in-frame deletions or insertions, or by chromosomal rearrangement
9BRAF Val600Glu (V600E) 98–99% of all BRAF mutations papillary carcinomapoorly differentiated carcinomaanaplastic carcinomaGTG>GAGc.1799
10Prevalence of BRAF mutations in different histologic variants of PTC Classic papillaryTall cell variantFollicular variantReview by Xing60%80%10%Seoul St. Mary’s Hospital83%100%24%Xing M. Endocr Relat Cancer 2005;12:245-62
11Review of all thyroid FNA studies using the BRAF mutation prior to 2009 No. of samplesBRAF (+)Final diagnosis in BRAF (+) samples9 prospective studies1814159 (8.7%)PTC = 159 (100%)7 retrospective studies685291 (42.5%)PTC = 291 (100%)2 FNA on thyroid specimens267131 (49.1%)PTC = 130 (99.2%) Hyperplasia* = 1 (0.8%)Total2766581 (21.0%)PTC = 580 (99.8%)* Hyperplasia = atypical nodular hyperplasiaMehta V et al. Head Neck 2012 Sep 13. Epub
12Review of all thyroid FNA studies using the BRAF mutation prior to 2009 15% to 39% of BRAF-positive FNA samples fell into the nondiagnostic or “indeterminate” categoriesSeveral patients with preoperative benign FNA results were found to be positive for BRAF mutation, and then confirmed as PTC after surgical removal of the thyroid glandThe routine use of BRAF testing would further decrease this false-negative rate.Mehta V et al. Head Neck 2012 Sep 13. Epub
17BRAF mutation test for diagnosis of malignancy in thyroid FNA AuthorMethodsSensitivitySpecificityAccuracyFNAFNA & BRAFKim SW (2010)DPO-based multiplex PCR67.5%89.6%100%99.3%*90.9%96.6%Nam SY (2010)Direct sequencing, allele specific PCR79.1%88.4%92.6%95.9%Yeo MK (2011)Pyrosequencing71.2%78.5%93.9%95.5%*Five false positive cases: 1 FA and 4 NH.Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700Nam SY et al. Thyroid 2010;20:Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560
19False positiveUltra-sensitive molecular assays with analytical sensitivity <1% should not be used.Detection of very low-level mutations can be due to the error introduced during PCR, genetic heterogeneity, and presence of mutation in a very small proportion of cells.
20RET/PTC rearrangement 10-20% of papillary thyroid carcinomasRET/PTC1 and RET/PTC3Various prevalence and specificity:Differences in specific age groups and in individuals exposed to ionizing radiation.Heterogeneous distribution within the tumorVarious sensitivities of the detection methods used.
21Review of all thyroid FNA studies using the RET/PTC mutation All RET/PTC positive FNA samples were histologically proven PTCsNo false-positive resultsHighly specific biomarker for the diagnosis of PTC
22RAS mutationsActivating point mutation in codons 12, 13, and 61 of the NRAS, HRAS, and KRAS genes
23RAS mutations Follicular thyroid neoplasms, both benign and malignant 40-50% of conventional type follicular carcinoma10-15% of oncocytic type follicular carcinoma10-20% of papillary carcinoma almost exclusively the follicular variant30% of conventional type follicular adenoma<10% of oncocytic type follicular adenomaDetection of RAS mutation indicates the presence of a tumor
24PAX8/PPARγ rearrangement 30-40% of conventional follicular carcinomas<5% of oncocytic carcinomas2-13% of follicular adenomas: may be preinvasive (in situ) follicular carcinoma, or tumors where invasion was overlooked or not sampled during examination1-5% of follicular variant of papillary carcinomas
25Molecular testing of FNA samples Which patients should be tested?Which biomarkers should be tested?What is the cost of testing?How should testing be performed?
26Single marker test vs Multimarker panels KoreaWesternPTC Prevalence % %BRAF (+) rate >80% of PTC % of PTCMolecular testBRAFBRAF, RAS, RET/PTC, PAX8-PPARγ
27Korean Studies BRAF mutation test for diagnosis of malignancy in thyroid FNA SensitivityAccuracyKim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700Nam SY et al. Thyroid 2010;20:Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560
28A study with a panel of mutation analyses First two passesCytologic evaluation3~4 FNA passesIndeterminate:AUS/FLUSFN/SFNSMCResidual materialMolecular analysis:BRAF, HRAS, NRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPARγ400 μL nucleic acid preservative solutionIsolation of total nucleic acidsNikiforov YE, et al. J Clin Endocrinol Metab 2011;96: 3390–7
29AUS/FLUS (n=212) 14% Thyroid mutation panel Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNAAUS/FLUS (n=212)Cancer risk based on cytology only14%Thyroid mutation panel(BRAF, RAS, RET/PTC, PAX8/PPARγ )Sensitivity 63%Specificity 99%PPV 88%NPV 94%Accuracy 94%PositiveNegativeCancer risk88% %Clinical managementTotal thyroidectomyLobectomy vs. observationNikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:
30FN/SFN (n=214) 27% Thyroid mutation panel Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNAFN/SFN (n=214)Cancer risk based on cytology only27%Thyroid mutation panel(BRAF, RAS, RET/PTC, PAX8/PPARγ )Sensitivity 57%Specificity 97%PPV 87%NPV 86%Accuracy 86%PositiveNegativeCancer risk87% %Clinical managementTotal thyroidectomyLobectomyNikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:
31SMC (n=52) 54% Thyroid mutation panel Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNASMC (n=52)Cancer risk based on cytology only54%Thyroid mutation panel(BRAF, RAS, RET/PTC, PAX8/PPARγ )Sensitivity 68%Specificity 96%PPV 95%NPV 72%Accuracy 81%PositiveNegativeCancer risk95% %Clinical managementTotal thyroidectomyLobectomyNikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:
32Application of tumor specific mRNA/miRNA expression patterns in FNAC diagnosis
33mRNA expressionMicroarray studies revealed very distinct changes in the expression of certain genesNo single markerThe aim of current approaches is to identify the minimal number of discriminating genesAfirma Gene Expression Classifier (Veracyte, South San Francisco, CA) evaluates mRNA expression levels for 142 genes.
34Gene Expression Classifier A prospective, multicenter validation study involving 49 clinical centers in the USA: 4,812 FNAs from 3789 patients with thyroid nodules ≥1 cm in diameter over a 19-month periodA gene-expression classifier was used totest 265 indeterminate nodulesSensitivity %Specificity 52%Negative predictive values AUS % Follicular neoplasm 94% Suspicious 85%N Engl J Med 2012;367:705-15
35Gene Expression Classifier Patients with an indeterminate cytology, but benign gene expression classifier test results have a very low risk of cancer.The test requires two additional needle insertions during FNA biopsy and it is costly.
36How much does the molecular test cost? In the USAMolecular panel testing (BRAF, RET/PTC, and RAS): $650Afirma Gene Expression Classifier: $4,200Thyroid surgery: $10,00 to $15,000
37MicroRNAsmall RNA sequences (19–25 nucleotides) that function to regulate the expression of genesregulate around 30% of the human genomedevelopment, apoptosis, cell proliferation, immune response, and hematopoiesistumor suppressor genes and oncogenes
38miRs aberrantly expressed in human thyroid carcinomas of follicular cell origin Endocrine-Related Cancer (2010) 17 F91–F104
39- + Summary Bethesda system BRAF or RET/PTC: PTC PAX8/PPARγ: FTC NondiagnosticBenignAUS/FLUSFollicular neoplasmSuspicious for MalignancyMalignantBenign-Somatic mutation+BRAF or RET/PTC: PTCPAX8/PPARγ: FTCRAS: FTC, FA, fvPTC
40Summary Bethesda system Nondiagnostic Benign AUS/FLUS Benign Follicular neoplasmSuspicious for MalignancyMalignantBenigngene expressionSuspicious