American Society of Cytopathology Companion Society Symposium Uses and Misuses of Ancillary Tests in Cytopathology

Transcription

1 American Society of Cytopathology Companion Society Symposium Uses and Misuses of Ancillary Tests in Cytopathology Zubair W. Baloch. MD, PhD. Professor of Pathology, UPENN Medical Center Perelman School of Medicine Philadelphia, PA. USA

2 None to disclose Disclosures

3 Where We are Now in the Management of Thyroid Nodules? The Present

4 Fig. 1 Thyroid Nodule Management Paradigm Otolaryngologic Clinics of North America , DOI: ( /j.otc )

5 Thyroid FNA Bethesda Classification Scheme The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC): Implied Risk of Malignancy and Recommended Clinical Management Diagnostic Category Risk of Malignancy (%) Usual Management Non-diagnostic or Unsatisfactory Repeat FNA with ultrasound guidance Benign 0-3% Clinical follow-up Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance (AUS/FLUS) ~ 5-15% Repeat FNA Follicular Neoplasm or Suspicious for a Follicular Neoplasm (Specify if Hurthle type or Oncocytic) 15-30% Surgical lobectomy Suspicious for Malignancy 60-75% Near-total thyroidectomy or surgical lobectomy Malignant 97-99% Near-total thyroidectomy

6 Colloid - Watery Nuclear Atypia Susp-Malig Malignant Monotonous cells, Microfollicles, Nuclear overlapping & Crowding Benign AUS/FLUS Foll-Neop Susp-Malig Malignant Follicular Cells

7 Colloid - Watery Nuclear Atypia Benign AUS/FLUS Foll-Neop Susp-Malig Malignant Follicular Cells

8 Management of patients found to have an indeterminate or malignant result on initial FNA Published in: Henry B. Burch; Kenneth D. Burman; David S. Cooper; James V. Hennessey; Nicole O. Vietor; The Journal of Clinical Endocrinology & Metabolism 2016, 101,

9 Molecular Diagnostics Promises vs. Reality

10 What We Know

11 Papillary Thyroid Carcinoma-PTC 70-75% of PTC Tall cell and classic papillary cancer Propensity for dedifferentiation 20-40% poorly differentiated 30-40% anaplastic Classic papillary cancer Younger age at at presentation 10-20% ~20% of of sporadic PTC PTC 40-70% 40-70% of of radiation induced PTC (RET/PTC3 Frequent lymph assoc node with metastases solid variant) Frequent lymph node metastases PAX8/PPARg 1-5% Follicular variant of papillary cancer (NRAS, HRAS 61) More frequent encapsulation

12 20-40% FA Follicular Patterned Lesions RAS HRAS, NRAS codon 61 KRAS codons 12,13 40% FC ~70% Follicular CA PAX8/PPARg 30% FC 2-13% FA

13 Mutations Thyroid Tumor Prevalence (%)* Primary Signaling Functional Impact on the Protein and Tumor Type Pathways Affected BRAFV600E CPTC 45 MAPK Activating; promoting tumorigenesis, invasion, metastasis, recurrence and mortality FVPTC 15 TCPTC ATC 25 BRAFK601E FVPTC 5 MAPK Activating; probably similar to BRAFV600E HRAS, KRAS, NRAS FTA MAPK and PI3K AKT Activating; promoting tumorigenesis, invasion and metastasis of PDTC and FTC FTC FVPTC PDTC ATC PTEN (mutation) FTA 0 PI3K AKT Inactivating the gene but activating the PI3K pathway; promoting tumorigenesis and invasiveness FTC ATC PTC 1 2 PTEN (deletion) FTC 30 PI3K AKT Inactivating the gene but activating the PI3K pathway; promoting tumorigenesis and invasiveness PIK3CA FTA 0 5 PI3K AKT Activating; promoting tumorigenesis and invasiveness FTC 5 15 ATC PTC 1 2 IDH1 FTC 5 25 IDH1-associated metabolic pathways FVPTC 20 CPTC 10 ATC Inactivating; impact on tumors is unclear AKT1 Metastatic cancer 15 PI3K AKT Unclear; seems to favour metastasis CTNNB1 PDTC 25 WNT β-catenin Activating; promoting tumour progression ATC TP53 PDTC 25 p53-coupled pathways Inactivating; promoting tumour progression ATC, anaplastic thyroid cancer; CPTC, conventional PTC; CTNNB1, β-catenin; FTA, follicular thyroid adenoma; FTC, follicular thyroid cancer; FVPTC, follicular-variant PTC; IDH1, isocitrate dehydrogenase 1; PDTC, poorly differentiated thyroid cancer; PTC, papillary thyroid cancer; TCPTC, tall-cell PTC. Xing M. Nature Reviews.13, (2013).

14 Molecular Testing of Thyroid FNA Specimens Questions and Considerations Never If there is another gold standard dx test Always If molecular testing always provides the correct answer Sometimes

15 The ACCE Model System for evaluating genetic tests

16 How to evaluate molecular testing: Can I use it in my practice? Know the test Know the studied population Know your patients These are NOT YET algorithmic

17 Analytical Validity: Accuracy and reliability to measure genotype of interest How often is it positive when a mutation is present? What range of patient specimens have been tested? Quality control program Robustness- concordance of results in multiple labs

18 Clinical Validity: Accuracy with which a test predicts the disorder of interest The sensitivity, specificity, and predictive values of a test in relation to a particular disorder Evaluation of testing in a similar population to which test is targeted Longitudinal cohort studies

19 Sensitivity-- ability of test to identify disease, so the fraction of those with disease who test positive Total cancer= TP +FN Total benign= FP + TN TP TP + FN Specificity-- ability of test to identify those without disease, so the fraction of those without disease who will test negative TN TN + FP

20 Sensitivity and specificity are characteristics of the test. The population does not affect the results. The relevant questions for the clinician and patient-- What is the chance that a person with a positive test truly has the disease? What is the chance that a person with a negative test result is disease free? Positive and negative predictive values are influenced by the prevalence of disease in the population being tested.

21 Test: 90% sensitivity and 90% specificity Example 1: 20% of population has cancer PPV TP/all positive results= 18/26 = 69% NPV TN/all negative results=72/74 = 97% Example 2: 70% of population has cancer PPV TP/all positive results= 63/66 = 95% NPV TN/all negative results= 27/34 = 79% 70 30

22 NPV (%) NPV Decreases as Cancer Prevalence Rises TEST A Sens 90% Spec 50% 100 TEST B Sens 60% Spec 95% Prevalence of Cancer (%)

23 NPV and PPV depend upon population CANCER prevalence

24 Evaluating Molecular Tests to apply to your patients: How robust is the definition of the studied population? Cytology Histopathology did all pts have surgery? Is the cancer risk of the studied population similar to your patients?

25 Molecular tests since 2011 mrna expression panel Gene expression classifier (Veracyte, 2011) Mutation (point mutations/translocation) panels mirinform Thyroid (Asuragen -> Interpace, 2011) Thyroid Cancer Mutation Panel (Quest Diagnostics, 2011) Next generation sequencing (NGS panel) ThyroSeq v.2 (CBL Path/UPMC, 2014) Mutation panel with mirna classifier ThyGenX + ThyraMIR (Interpace, 2015)

26 Veracyte Afirma Gene Expression Classifier Mutation Panels 7 Gene panel (BRAF, RAS, RET/PTC, PAX8/PPARγ) Mutation Panels Plus ThyroSeq enhanced multi-gene panel with NexGen sequencing University of Pittsburgh/CBL Pathology ThyroMIR enhanced multi-gene panel with mirna Interpace Diagnostics

27 Afirma GEC AIM: to develop a test with high sensitivity and high NPV Alexander, et al. N Engl J Med :206

28 Alexander J Clin Endocrinol Metab 2014; 99:119; McIver J Clin Endocrinol Metab 2014;99:4069; Harrell Endocrin Pract 2014;20:364 We cannot determine NPV because we don t know the truth (histopathology) about benign GEC nodules We cannot determine PPV because of selection bias Not all GEC suspicious patients had surgery

29 Patients undergoing surgery (%) Study variability in surgery rates Multicenter Harrell Mayo FLUS and Follicular neoplasm cytology % % Suspicious Afirma GEC Benign Alexander J Clin Endocrinol Metab 2014; 99:119; McIver J Clin Endocrinol Metab 2014;99:4069; Harrell Endocrin Pract 2014;20:364; Yang Cancer Cytopathol 2016;124:100

30 Frequency of cancer (%) Site variability in cancer rate with suspicious GEC NOT TRUE PPVs % 15-67% FLUS Afirma GEC Suspicious NEJM Follicular neoplasm Alexander J Clin Endocrinol Metab 2014; 99:119; McIver J Clin Endocrinol Metab 2014;99:4069; Harrell Endocrin Pract 2014;20:364; Yang Cancer Cytopathol 2016;124:100

31 NPV PPV NPV & PPV for Afirma GEC for FLUS, FN based upon N Engl J Med : NPV PPV 94% 38% NEJM paper Prevalence of malignancy Courtesy of Bryan McIver

32 ndeterminate cytology (AUS/FLUS and follicular neoplasm) at Mt. Sinai and Memorial Sloan Indeterminate cyto nodules Mt. Sinai 71 nodules Memorial Sloan 91 nodules GEC Benign GEC Suspicious 52% 48% 26% 74% Marti Ann Surg Oncol 2015; 22: Memorial Sloan Mt. Sinai Alexander, NEJM

33 Increase rate of Suspicious GEC Afirma Results in Benign Oncocytic Nodules Suspicious nodules w surgery Benign Malignant Harell et al. Endo Pract (43%) - 9 (69%) oncocytic lesions McIver et al. JCEM (84%) - 12 (44%) oncocytic lesions 17 (57%) 5 (16%) Brauner et al. Thyroid * 37 (84%) 6(14%) Lastra et al. Cancer Cytopath (54%) - 15 (58%) oncocytic lesions 22(46%) Wu et al. Thyroid (43%) (74.2% oncocytic lesions)? 59(57%) Samulski et al. Diag Cytopathol [Epub] (65%) 25 (29%) oncocytic lesions 45 (35%) Total (67%) -73 (71%) oncocytic lesions 50 (33%)

34 Limitations of ALL GEC STUDIES Retrospective selection bias Not all indeterminate cyto nodules had GEC Not all GEC susp nodules had surgery, NO PPV Limited nonsurgical FU for benign GEC nodules, NO NPV Variability in GEC performance across sites Difference in malignancy rates greatest in FLUS nodules cyto classification with highest interobserver variability Alexander J Clin Endocrinol Metab 2014; 99:119; McIver J Clin Endocrinol Metab epub May 2014; Harrell Endocrin Pract 2014;20:364

35 Nikiforov J Clin Endocrinol Metab :3390; Beaudenon Thyroid 2014;24:1479 Mutation Panel Studies canonical Point mutations tested Nikiforov 2011 NRAS codon 61 HRAS codon 61 KRAS codons 12,14 BRAF V600E Gene Rearrangements RET/PTC1 RET/PTC3 PAX8/PPARg Beaudenon 2014 NRAS codon 61 HRAS codon 12, 61 KRAS codons 12,14 BRAF V600E Asuragen lab RET/PTC1 RET/PTC3 PAX8/PPARg

36 Nikiforov J Clin Endocrinol Metab :3390; Beaudenon Thyroid 2014;24:1479 Mutation panel (BRAF, RAS, RET/PTC, PAX8/PPARg) Only 1 in 6 FLUS or follicular neoplasm nodule harbor mutations 76-89% of mutations found in FLUS and FN nodules were RAS With RAS mutation ~80% cancer risk If cancer, histology is either follicular cancer or PTC FV generally encapsulated For encapsulated cancers <4cm, lobectomy may be sufficient (ATA 2015) Does a mutational panel alter a lobectomy?

37 NexGen additions Using NexGen sequencing UPMC developed custom panel ThyroSeq ver2 TM Point mutations in 14 genes (BRAF, NRAS, KRAS, HRAS, AKT1, PTEN, TP53, TSHR, GNAS, CTNNB1, RET, PIK3CA, TERT, EIF1AX) 38 gene fusions (RET, PPARg, NTRK1, NTRK3k, ALK, THADA,BRAF) Therefore >60 genetic markers Nikiforov Cancer 2014;120:3627; Nikiforov Thyroid epub Sept

38 UPMC ThyroSeq ver2 TM Single institution, pathologists not blinded Not all indeterminate cyto pts had surgery SENSITIVITY increased to ~90% ~25% of cancers with unique fusions/mutations SPECIFICITY decreased to ~92% Nikiforov Cancer 2014;120:3627; Nikiforov Thyroid epub Sept

39 mirna additions FLUS and FN mirnas small noncoding mrnas that regulate gene expression Evaluated expression of 10miRNA genes in addition to HRAS, KRAS, NRAS, BRAF, RET/PTC, PAX8-PPARg Applied to 282 FLUS or Follicular neoplasm nodules sent to Asuragen from 12 sites 40% had traceable surgical path report 109 cases evaluable Labourier J Clin Endocrinol Metab epub May

40 Cancer in 35/109 (32%) of FLUS and FN nodules 31/35 identified by molecular panel 88% SENSITIVITY 24/35 (68%) with canonical mutations 7/35 (20%) with NEW mirna Benign 74/109 85% SPECIFICITY Molecular analysis positive in 11(1 with new mirna) Labourier J Clin Endocrinol Metab epub May mirna additions FLUS and FN

41 The ACCE Model System for evaluating genetic tests

42 New Terminology Recommendation Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) *Adequate sampling of entire tumor capsule is required to establish this diagnosis Changes in the Implied Risk of Malignancy for TBSRTC Categories AUS/FLUS Suspicious for Follicular Neoplasm Suspicious for Malignancy 50% decrease (Strickland et al. Thyroid 2015 & Faquin et al. Cancer Cytopathology 2015)

43 Molecular alterations in NIFTP ~80% cases classified as NIFTP (by mean score 2 or more) show clonal alterations 12/26 RAS +/- EIF1AX 7/26 PAX8/PPARG 5/26 THADA 1/26 ALK 1/26 BRAF K601E No BRAF V600E, RET/PTC in NIFTP Cases not considered NIFTP because of insufficient nuclear score were negative for mutation NIFTP Integrated genomic characterization of papillary thyroid carcinoma. Cancer Genome Atlas Research Network. Cell Oct 23;159(3):676-90

44 Rate of Malignancy How does this affect rate of malignancy (ROM)? 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 28% 48% 18% 45% 46% 45% FLUS FN Susp for malig Malig Cytology Diagnoses Faquin Faquin wo NIFTP Strickland Strickland wo NIFTP Faquin Cancer Cytopathol 2016;124:181; Strickland Thyroid 2015:9:987

45 Decrease in ROM varies depending upon institutional rate NIFT-P Dx s Faquin Cancer Cytopathol 2016;124:181

46 Afirma and NIFTP Follow-up Study Suspicious by Afirma Nodules w surgery Benign Malignant (NIFTP) Wong et al (Thyroid 2016) 63 41(65%) 22(35%) (14 cases-88%) Samulski et al (Diag Cytopath 2016) (65%) 45(35%) (11 cases-24%)

47 Cytologic Features and Molecular Alterations in a Cohort of 39 NFVPTCs and cptcs. Brooke E. Howitt et al. Am J Clin Pathol 2015;144: Copyright by the American Society for Clinical Pathology

48 ATA Strong rec Low-quality evidence If molecular testing is being considered, patients should be counseled regarding the potential benefits and limitations of testing, and about the possible uncertainties in the therapeutic and long-term clinical implications of results. 14 Strong rec Low-quality evidence If intended for clinical use, molecular testing should be performed in CLIA/CAP certified molecular laboratories, as reported quality assurance practices may be superior compared to other settings.

49 ATA 2015, continued 15A Weak rec Moderate-quality evidence: For nodules with AUS/FLUS cytology, after consideration of clinical and sonographic features, investigation such as repeat FNA or molecular testing may be used to supplement malignancy risk assessment in lieu of proceeding directly with a strategy of either surveillance or diagnostic surgery. Informed patient preference and feasibility should be considered in clinical decision-making.

50 ATA 2015, continued 16A Weak rec Moderate Quality evidence: Diagnostic surgical excision is the long-established standard of care for the management of follicular neoplasm/suspicious for follicular neoplasm (FN) cytology nodules. However, after consideration of clinical and sonographic features, molecular testing may be used to supplement malignancy risk assessment data, in lieu of proceeding directly with surgery. Informed patient preference and feasibility should be considered in clinical decisionmaking.

51 Basis for any molecular test is high quality cytology Molecular test assay methods may differ, e.g. BRAF Molecular testing for most common point mutations (BRAF, RAS) and rearrangements is specific, but not sensitive New technologies (NexGen, mirna) improve sensitivity LIMITED clinical studies of commercially available tests have SELECTION BIAS without concordant results Focus on likelihood of cancer with POSITIVE TEST NO FU study addresses likelihood of benignity with NEGATIVE GEC or newer NexGen or mirna panels NEGATIVE predictive values cannot be accurately determined without knowing disease prevalence

52 Uncertainty about performance of molecular tests in real-world clinical settings

53