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Chan Kwon Jung, MD, PhD Department of Pathology THE CATHOLIC UNIVERSITY OF KOREA Seoul St. Marys Hospital October 22, 2012.

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Presentation on theme: "Chan Kwon Jung, MD, PhD Department of Pathology THE CATHOLIC UNIVERSITY OF KOREA Seoul St. Marys Hospital October 22, 2012."— Presentation transcript:

1 Chan Kwon Jung, MD, PhD Department of Pathology THE CATHOLIC UNIVERSITY OF KOREA Seoul St. Marys Hospital October 22, 2012

2 Thyroid FNA results Indeterminate 15-30%

3 Category Risk of Malignancy I. Nondiagnostic or Unsatisfactory 1-4% II. Benign 0-3% III. Atypia of undetermined significance or Follicular lesion of undetermined significance ~5-15% IV. Follicular Neoplasm or Suspicious for a Follicular Neoplasm 15-30% V. Suspicious for Malignancy 60-75% VI. Malignant 97-99% The Bethesda System for Reporting Thyroid Cytopathology

4 Use of molecular biomarkers Improve the accuracy of fine-needle aspiration cytology Provide prognostic information

5 Genetic alterations in thyroid cancer Activating and inactivating somatic mutations, Alteration in gene expression patterns, MicroRNA dysregulation Aberrant gene methylation

6 Follicle- derived Well differentiated carcinoma Papillary carcinoma Follicular carcinoma Poorly differentiated carcinoma Undifferentiated (Anaplastic) carcinoma

7 AC PAX8-PPAR γ BRAF RAS TP53 β-catenin PI3KCA PTEN Follicular cell RAS Follicular Carcinoma Papillary Carcinoma RET/PTC PDC Follicular Carcinoma Papillary Carcinoma Follicular adenoma TP53 β-catenin PI3KCA PTEN TP53 β-catenin PI3KCA PTEN TP53 TSHR Gsα

8 BRAF mutations BRAF is a serine- threonine kinase. BRAF can be activated by point mutations, small in-frame deletions or insertions, or by chromosomal rearrangement

9 c.1799 BRAF Val600Glu (V600E) 98–99% of all BRAF mutations papillary carcinoma poorly differentiated carcinoma anaplastic carcinoma GTG>GAG

10 Prevalence of BRAF mutations in different histologic variants of PTC Classic papillaryTall cell variantFollicular variant Review by Xing 60%80%10% Seoul St. Marys Hospital 83%100%24% Xing M. Endocr Relat Cancer 2005;12:245-62

11 Thyroid FNA studies No. of samples BRAF (+)Final diagnosis in BRAF (+) samples 9 prospective studies (8.7%) PTC = 159 (100%) 7 retrospective studies (42.5%) PTC = 291 (100%) 2 FNA on thyroid specimens (49.1%) PTC = 130 (99.2%) Hyperplasia* = 1 (0.8%) Total (21.0%) PTC = 580 (99.8%) * Hyperplasia = atypical nodular hyperplasia Review of all thyroid FNA studies using the BRAF mutation prior to 2009 Mehta V et al. Head Neck 2012 Sep 13. Epub

12 Review of all thyroid FNA studies using the BRAF mutation prior to 2009 Mehta V et al. Head Neck 2012 Sep 13. Epub 15% to 39% of BRAF-positive FNA samples fell into the nondiagnostic or indeterminate categories Several 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 gland The routine use of BRAF testing would further decrease this false-negative rate.

13 ThinPrep

14

15 Cell block using ThinPrep

16 Forward Reverse

17 BRAF mutation test for diagnosis of malignancy in thyroid FNA AuthorMethodsSensitivitySpecificityAccuracy FNAFNA & BRAF FNAFNA & BRAF FNAFNA & BRAF Kim SW (2010) DPO-based multiplex PCR 67.5%89.6%100%99.3%*90.9%96.6% Nam SY (2010) Direct sequencing, allele specific PCR 79.1%88.4%100% 92.6%95.9% Yeo MK (2011) Pyrosequencing 71.2%78.5%100% 93.9%95.5% *Five false positive cases: 1 FA and 4 NH. Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700 Nam SY et al. Thyroid 2010;20: Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560

18 False positive 50 DPO-PCR false positive cases: false positive rate 1.4%; specificity 98.6% 3 MEMO-sequencing false positive cases: false positive rate 0.08%; specificity 99.9% Lee ST et al. J Clin Endocrinol Metab ;97:

19 False positive Ultra-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.

20 RET/PTC rearrangement 10-20% of papillary thyroid carcinomas RET/PTC1 and RET/PTC3 Various prevalence and specificity: 1.Differences in specific age groups and in individuals exposed to ionizing radiation. 2.Heterogeneous distribution within the tumor 3.Various sensitivities of the detection methods used.

21 Review of all thyroid FNA studies using the RET/PTC mutation All RET/PTC positive FNA samples were histologically proven PTCs No false-positive results Highly specific biomarker for the diagnosis of PTC

22 RAS mutations Activating point mutation in codons 12, 13, and 61 of the NRAS, HRAS, and KRAS genes

23 Follicular thyroid neoplasms, both benign and malignant 40-50% of conventional type follicular carcinoma 10-15% of oncocytic type follicular carcinoma 10-20% of papillary carcinoma almost exclusively the follicular variant 30% of conventional type follicular adenoma <10% of oncocytic type follicular adenoma Detection of RAS mutation indicates the presence of a tumor RAS mutations

24 PAX8/PPARγ rearrangement 30-40% of conventional follicular carcinomas <5% of oncocytic carcinomas 2-13% of follicular adenomas: may be preinvasive (in situ) follicular carcinoma, or tumors where invasion was overlooked or not sampled during examination 1-5% of follicular variant of papillary carcinomas

25 Molecular 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?

26 Single marker test vs Multimarker panels KoreaWestern PTC Prevalence 95% 80-90% BRAF (+) rate >80% of PTC 30-50% of PTC Molecular testBRAF BRAF, RAS, RET/PTC, PAX8- PPARγ

27 Korean Studies BRAF mutation test for diagnosis of malignancy in thyroid FNA Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700 Nam SY et al. Thyroid 2010;20: Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560 SensitivityAccuracy

28 First two passes 3~4 FNA passes 400 μ L nucleic acid preservative solution Cytologic evaluation Indeterminate: AUS/FLUS FN/SFN SMC Isolation of total nucleic acids Molecular analysis: BRAF, HRAS, NRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPAR γ Residual material Nikiforov YE, et al. J Clin Endocrinol Metab 2011;96: 3390–7 A study with a panel of mutation analyses

29 Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA Cancer risk based on cytology only 14% Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPAR γ ) Positive Negative Cancer risk 88% 5.9% AUS/FLUS (n=212) Clinical management Total thyroidectomy Lobectomy vs. observation Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: Sensitivity 63% Specificity 99% PPV 88% NPV 94% Accuracy 94%

30 Cancer risk based on cytology only 27% Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPAR γ ) Positive Negative Cancer risk 87% 14% FN/SFN (n=214) Clinical management Total thyroidectomy Lobectomy Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: Sensitivity 57% Specificity 97% PPV 87% NPV 86% Accuracy 86% Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA

31 Cancer risk based on cytology only 54% Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPAR γ ) Positive Negative Cancer risk 95% 28% SMC (n=52) Clinical management Total thyroidectomy Lobectomy Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: Sensitivity 68% Specificity 96% PPV 95% NPV 72% Accuracy 81% Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA

32 Application of tumor specific mRNA/miRNA expression patterns in FNAC diagnosis

33 mRNA expression Microarray studies revealed very distinct changes in the expression of certain genes No single marker The aim of current approaches is to identify the minimal number of discriminating genes Afirma Gene Expression Classifier (Veracyte, South San Francisco, CA) evaluates mRNA expression levels for 142 genes.

34 Gene Expression Classifier A gene-expression classifier was used to test 265 indeterminate nodules Sensitivity 92% Specificity 52% Negative predictive values AUS 95% Follicular neoplasm94% Suspicious 85% N Engl J Med 2012;367: 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 period

35 Gene 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.

36 How much does the molecular test cost? In the USA Molecular panel testing (BRAF, RET/PTC, and RAS): $650 Afirma Gene Expression Classifier: $4,200 Thyroid surgery: $10,00 to $15,000

37 MicroRNA small RNA sequences (19–25 nucleotides) that function to regulate the expression of genes regulate around 30% of the human genome development, apoptosis, cell proliferation, immune response, and hematopoiesis tumor suppressor genes and oncogenes

38 miRs aberrantly expressed in human thyroid carcinomas of follicular cell origin Endocrine-Related Cancer (2010) 17 F91–F104

39 Bethesda system Nondiagnostic Benign AUS/FLUS Follicular neoplasm Suspicious for Malignancy Malignant Summary Somatic mutation Benign - + BRAF or RET/PTC: PTC PAX8/PPARγ: FTC RAS: FTC, FA, fvPTC

40 Bethesda system Nondiagnostic Benign AUS/FLUS Follicular neoplasm Suspicious for Malignancy Malignant Summary gene expression Benign Suspicious

41 Thank you for your attention


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