Validation of BRAF Mutational Analysis in Thyroid Fine Needle Aspirations: A Morphologic- Molecular Approach
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1 Validation of BRAF Mutational Analysis in Thyroid Fine Needle Aspirations: A Morphologic- Molecular Approach Kerry C. Councilman, MD Assistant Professor University of Colorado Denver
2 Goals: BRAF Mutation Validation Study To confirm that Diff Quick stained, direct cytology smears from thyroid can be used for both diagnosis and molecular assayssimilar to liquid based samples. To show that thyroid cell-specific high-quality DNA can be obtained using pathologist directed microdissection methods for enrichment. To compare mutational data from cytology material and surgical pathology sample, in order to validate cytology assay methods.
3 Risk of Thyroid Malignancy and Clinical Management by Bethesda Category Benign <1% Clinical follow-up ACUS/FLUS Follicular neoplasm Suspicious Malignant 5-10% 20-30% 50-75% 100% Repeat FNA Lobectomy Thyroidectomy Thyroidectomy
4 BRAF Mutation Schematic illustration of the MAPK pathway. Most common genetic alteration in PTC (~45%) The most frequent mutation is C.1799T>A (V600E) is basis of assay BRAF mutation activates MAPK, promoting cancer formation Figure from Xing M Endocrine Reviews 2007;28: by Endocrine Society
5 BRAF Mutation Testing of Thyroid Nodules Typically used only for indeterminate cases Approximately half of PTC have BRAF mutation BRAF mutation >95% PPV for PTC Kim (2010), Cantara (2010), Nikiforov (2009, 2011)
6 Methodological Approaches Liquid Based Advantages Ease of transfer Superior DNA Disadvantages May need extra pass Unknown cell composition Must prepare up front Cell Block Validated like surgical Few cells May need extra pass Direct Smear Visualize target cells Slide sacrifice Superior DNA Routinely prepared Cell specific DNA Enrichment
7 Processing the DQ Slide
8 Cytology Cases Tested for BRAF Mutation: Validation Study Cytologic Dx N Mutation + Total 37 5/37 PTC 9 5/9 Indeterminate 17 0/17 Negative 11 0/11
9 Validation: Concordance Data Comparison of FNA cytology and surgical pathology specimen derived BRAF data to validate methodology 100% mutation negative FNA cases were negative (on follow-up surgical when available) 1/9 PTC case was positive in FNA, negative in surgical resection Possible explanations Tumor heterogeneity: Sampled multiple areas of the final resection specimen, some areas (+), other areas (-) Sampling Multiple papillary cell clones may represent separate collision tumors
10 Summary/Conclusions Microdissection of pathologist directed cells from DQ stained direct smear slide is as effective as FFPE sample BRAF mutation analysis Advantages: FNA material can be used to determine if surgery is necessary on indeterminate lesions Uses routinely prepared and stained cytology smears Cytopathologist directed cell harvesting
11 References Ali S, Cibas E. The Bethesda System for Reporting Thyroid Cytopathology. New York: Springer, 2010 Baloch ZW, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: a synopsis of the National Cancer Institute Thyroid Fineneedle aspiration State of the Science Conference. Diagn Cytopathol. 2008;36: Cooper DS, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009;19: Nikiforova MN, Nikiforov YE. Molecular diagnostics and predictors in thyroid cancer. Thyroid. 2009;19: Cantara S, et al. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology. J Clin Endocrinol Metab. 2010;95: Kim SW, et al. BRAF V600E mutation analysis in fine needle aspiration cytology specimens for evaluation of thyroid nodules: a large series in a BRAF V600E prevalent population. J Clin Endocrinol Metab. 2010;95: Nikiforov YE, et al. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab. 2009;94: Nikiforov YE, et al. Mutational Profiling of Thyroid FNA Samples. J Clin Endocrinol Metab. 2011;96:0000
12 Acknowledgments CMOCO (Colorado Molecular Correlates) Laboratory Dara Aisner, MD, PhD Wilbur Franklin, MD Natalie Thomas Justin Bohn Patrick Chesnut Department of Pathology Ann Thor, MD Sherif Said, MD, PhD Department of Endocrinology Brian Haugen, MD Joshua Klopper, MD Photography Lisa Litzenberger
13 Indeterminates Of 17 indeterminate cytology cases tested (all BRAF negative) 8 had surgical follow-up All remained BRAF -negative on surgical follow-up Expected as BRAF -positive mutation rate is low in indeterminate nodules Nikiforov, et al (2011): Our validation study: Suspicious: 19.2% - 1 Suspicious Follicular neoplasm: ~1% - 4 Follicular neoplasm ACUS: ~2% - 12 ACUS
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