Disclosure • Nothing to disclose Molecular Diagnostics in Thyroid Cancer Jonathan George, MD, MPH Assistant Professor Current Practices & Future Trends Head and Neck Oncologic & Endocrine Surgery UCSF Medical Center November 8, 2014 Problem Overview • FNA Cytology • FNA Cytology • 525,000 FNAs annually – Bethesda Classification • Indeterminate FNA – Indications for Molecular testing • 20-30% read as indeterminate (158,000) • Molecular Diagnostics • 75% benign, 25% chance of malignancy – Gene Expression Classifier 30-40% Diagnostic thyroidectomy • – Molecular Alteration Testing • 75-80% of indeterminate FNAs taken to surgery for diagnostic thyroidectomy are benign on final path • 119,000 “unnecessary” surgeries • $6-$10K • Possible complications • Time lost from work, child care, etc • Anxiety, pain, recovery Problem Problem • Molecular Testing of FNA Samples • Molecular Testing of FNA Samples • Advances in molecular testing may increase • Advances in molecular testing may increase diagnostic accuracy of FNA diagnostic accuracy of FNA • Alexander EK et al. NEJM 2012. • Accurately predict whether a cytologically indeterminate nodule is benign in 93% of cases • Permits a more conservative approach to management • Avoid unnecessary thyroid lobectomy Alexander EK, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med , 2012.367(8):705-715. N Engl J Med , 2012.367(8):705-715.
FNA Cytology FNA Cytology Bethesda Classification Reducing Unnecessary Diagnostic Procedures • Category 1: Non-diagnostic • Improving the Indeterminate FNA • Category 2: Benign – Can indeterminate FNA samples be re- classified as benign using genetic testing? • Category 3: Indeterminate (FLUS and AUS) • Gene Expression Classifier Rule-out • Category 4: Follicular neoplasm, SFN, HN, SHN cancer • Category 5: Suspicious for malignancy • Category 6: Malignant – Can the risk of cancer in an FNA specimen be confirmed using mutational analysis? • Molecular Alteration Testing Rule-in cancer Algorithm for Evaluating Thyroid Nodules. Gene Expression Classifier Gene Expression Classifier Microarray Technology: Rule Out Cancer GEC FNA Analysis • Multigene expression (mRNA) test – Total RNA is extracted from the FNA and whole- transcript amplification is prepared for hybridization onto a custom gene expression microarray. – Developed using 167 gene expression profiles • Result • Benign • Suspicious GEC benign : 93% chance of benign histology (<6% malignant) Jameson JL. N Engl J Med 2012;367:765-767. Algorithm for Evaluating Thyroid Nodules. Gene Expression Classifier Gene Expression Classifier GEC FNA Analysis Affirma FNA Analysis GEC suspicious : 40% risk of malignancy Jameson JL. N Engl J Med 2012;367:765-767.
Gene Expression Classifier Gene Expression Classifier Affirma FNA Analysis: GEC Benign Microarray Technology: Rule Out Cancer • Multigene expression (mRNA) test – High sensitivity and NPV – NPV • 95% for FLUS • 94% for follicular neoplasm – Equivalent to a NPV of a benign FNA Gene Expression Classifier Gene Expression Classifier Multigene expression (mRNA) Pilot Study Multigene expression (mRNA) Pilot Study • Alexander EK, NEJM 2012 • Alexander EK, NEJM 2012 – Microarray chip using 167 genes • Final Pathology (not cytopath): 85/265 indeterminate nodules were malignant (32%) – 49 clinical sites • 78/85 malignant nodules identified on cytopath – N = 3789 patients GEC analysis as suspicious – 4812 FNAs • Sensitivity: 92% – Final inclusion criteria 265 indeterminate FNAs • Specificity 52%, PPV 40% • a positive test is less likely to represent cancer Alexander EK, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. Alexander EK, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med , 2012.367(8):705-715. N Engl J Med , 2012.367(8):705-715. Gene Expression Classifier Gene Expression Classifier Multigene expression (mRNA) Pilot Study Multigene expression (mRNA) Pilot Study • Alexander EK, NEJM 2012 • Alexander EK, NEJM 2012 • Negative Predictive Value (NPV) • Summary • Bethesda 3 : FLUS/AUS 95% • GEC is a multigene expression (mRNA) test used that can reliably reclassify indeterminate FNAs as • Bethesda 4 : FN, SFN 94% benign and therefore allow conservative approach (observation) • Bethesda 5 : Suspicious 85% • High sensitivity : 92% • High NPV : 94-95% for FLUS and FN • Equivalent to NPV of a benign FNA Alexander EK, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. Alexander EK, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med , 2012.367(8):705-715. N Engl J Med , 2012.367(8):705-715.
Gene Expression Classifier Gene Expression Classifier Indeterminate FNA, GEC Negative: Risk of Malignancy Study Results: Duick et al, Thyroid 2012 • 368 patients treated by 51 physicians with benign gene expression classifier results • 10-fold reduction in surgery rates for cytologically indeterminate nodules with benign GEC – From 74% (historical) to 7.6% • Patients and physicians pursuing surveillance over surgery in a large majority of cases of indeterminate FNA, GEC negative (“benign”) Indeterminate FNA, GEC negative can be managed like benign FNA per NCCN guidelines The Thyroid Pathology Uncertainty Afirma Cost Effectiveness Problem FNA indeterminate • Prospective study of 776 nodules • Cost of test - $2000 to $3200 – Cytology and surgical histology – Patient out of pocket expense $200-$300 – 90-91% pathologist concordance on histology • Cost per patient for standard care – 64-75% pathologist concordance on cytology – $12,172 – Benign vs AUS/FLUS vs. FN vs. Susp malig vs. Malig • Cost per patient with Afirma test – Experienced cytopathologists less likely to call – $10,719 indeterminate • Therefore covered by most insurance – 41 vs 55% Li H et al JCEM 2011 Cibas ESD et al Ann Intern Med 2013 FNA Cytology Molecular Alteration Testing Reducing Unnecessary Diagnostic Procedures Thyroid Cancer Genetics: MAPK Cascade Activation • Improving the Indeterminate FNA • Activation of RET protein at the cell membrane – Can indeterminate FNA samples be re- (ligand binding at the extra-cell) classified as benign using genetic testing? • Activation of RAS protein • Gene Expression Classifier Rule-out cancer • BRAF effector protein binding • Downstream – Can the risk of cancer in an FNA specimen intermediates: MEK and be confirmed using mutational analysis? ERK – Mitogen-activated protein • Molecular Alteration Testing Rule-in kinase cancer – Extracellular signal Differentiation, proliferation, and regulated kinase survival
Molecular Alteration Testing Molecular Alteration Testing DTC Mutations: MAPK Cascade DTC Mutation Prevalence • Most common and Mutation PTC FTC clinically useful molecular • RET/PTC 15% 0% markers • BRAF 45% 0% • BRAF & RAS • RAS 15% 40% – Point mutations • PAX8/PPARy 1% 40% – DNA extraction • RET/PTC & PAX8/PPARy • 75% of PTCs 3 mutations – Rearrangement mutations – BRAF – 45% – Complex RNA technology – RET/PTC – 15% – RAS – 15% Molecular Alteration Testing Molecular Alteration Testing DTC Mutations: BRAF DTC Mutations: BRAF • BRAF High Specificity & PPV • BRAF V600E: Point mutation – Thymidine to adenine transversion (chrom 7, ex 15) – High Specificity – Specific for PTC, not found in FA or FC • Nikoforov 2009: 99.3% specificity for PTC – High PPV – Positive test very likely represents presence of true disease Molecular Alteration Testing Molecular Alteration Testing DTC Mutations: BRAF DTC Mutations: RET/PTC • BRAF Low Sensitivity • Somatic intra-chromosomal rearrangement in RET gene PTC – Other mutations involved in PTC – Many PTCs (55%) are negative for BRAF mutation – Most common chromosomal – Diagnostic thyroid lobectomy is still necessary in alteration in PTC many indeterminate FNAs that are BRAF- 15% of cases Zhu Z et al 2006
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