Thyroid Fine-Needle Aspiration Cytology & Molecular Testing Opinions & Criticisms

Zubair W. Baloch, MD, PhD Professor of Pathology & Laboratory Medicine UPENN Medical Center, Philadelphia, PA

Disclosure

• Nothing to disclose Lets Start with Basics Objectives of FNA

• Recognize specific diagnostic entities • Provide meaningful, management oriented diagnosis • Utilization of ancillary techniques as needed

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC)

The Bethesda System for Reporting Thyroid Cytopathology: 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 ~ 5-15% Repeat FNA Follicular Lesion of Undetermined Significance (AUS/FLUS)

Follicular or Suspicious for a 15-30% Surgical lobectomy Follicular Neoplasm (Specify if Hurthle type or Oncocytic) Suspicious for Malignancy 60-75% Near-total or surgical lobectomy

Malignant 97-99% Near-total thyroidectomy Easy Breezy Cytopathology Diagnosis Not Diagnostically Challenging

Excellent Concordance Between Clinical Features, Ultrasound and Pathology Nodular Goiter

Colloid: Generally abundant . Follicular cells Variable morphology Oncocytes, Macrophages Degeneration/regeneration: Calcification, stromal proliferation, mitoses Chronic Lymphocytic Thyroiditis

Oncocytes Lymphocytes: In the background & infiltrating the cell groups Papillary Thyroid Carcinoma

Nuclear features – Major Diagnostic Features Elongation, chromatin clearing, Nuclear membrane irregularities Intranuclear grooves Inclusions Small peripheral nucleoli

Not so easy - Head Scratching Everyday Thyroid Cytopathology

Intdeterminate Lesions Or Indeterminate Pathologist?

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC)

The Bethesda System for Reporting Thyroid Cytopathology: 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 ~ 5-15% Repeat FNA Follicular Lesion of Undetermined Significance (AUS/FLUS)

Follicular Neoplasm or Suspicious for a 15-30% Surgical lobectomy Follicular Neoplasm (Specify if Hurthle type or Oncocytic) Suspicious for Malignancy 60-75% Near-total thyroidectomy or surgical lobectomy

Malignant 97-99% Near-total thyroidectomy Diagnosis Follicular Neoplasm

80% Benign on Surgical Excision Diagnosis Follicular Lesion / Neoplasm The Atypical Category

The Dreaded AUS/FLUS 10-15% or more Risk of Malignancy Reasons for AUS/FLUS

• History – TFT’s, H/O prior FNA • Ultrasound features – Cystic vs. solid • Operator – sampling • Adequacy • Cytology Preparation • Interpretation • Surgical follow-up – ? Gold standard

Questions to Myself? How Can I Find Relevance of My Practice of Cytopathology in a Landscape That Won’t Stop Shifting? Let’s Make Sense of Present & Predict Future

In Light of Past The Timing of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC)

Growing Body of Literature Showing Inconsistencies in Surgical Pathology Diagnosis of Thyroid Among Experts – Encapsulated Follicular Variant of PTC The Cytology Gold Standard is not so Gold

TBSRTC Follow-up Clinicopathologic Studies Showing Over-diagnosis and Over-treatment of Thyroid Carcinoma – PTC. Concept of Low and High Risk Disease

TBSRTC TBSRTC

Clinical and Radiology Guidelines American & European Thyroid Association American College of Radiology American Society of Radiologist in Ultrasound TBSRTC

Molecular Profiling of Thyroid Tumors + Molecular Diagnosis of Thyroid Nodules

Diagnostic Tests with High Negative and Positive Predictive Value

Mutational Analysis Gene Expression Classifier Next Gene Sequencing Growing Body of Literature Showing Inconsistencies in Surgical Pathology Diagnosis of Among Experts – Encapsulated Follicular Variant The Cytology Gold Standard is not so Gold

Follow-up Clinicopathologic Studies Showing Over-diagnosis and Over- treatment of Thyroid Carcinoma – PTC. Concept of Low and High Risk Disease TBSRTC

Clinical and Radiology Guidelines American & European Thyroid Association American College of Radiology American Society of Radiologist in Ultrasound

Molecular Profiling of Thyroid Tumors Molecular Diagnosis of Thyroid Nodules Diagnostic Tests with high Negative and Positive Predictive Value TBSRTC

The Aftermath Literature Influx since 12/2007

• PUBMED -1635 publications mentioning • >20% focused on TBSRTC in title and AUS/FLUS Category abstract content – English Literature Thyroid FNA Bethesda Classification Scheme

The Bethesda System for Reporting Thyroid Cytopathology: 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 ~ 5-15% Repeat FNA Follicular Lesion of Undetermined Significance (AUS/FLUS)

Follicular Neoplasm or Suspicious for a 15-30% Surgical lobectomy Follicular Neoplasm (Specify if Hurthle type or Oncocytic) Suspicious for Malignancy 60-75% Near-total thyroidectomy or surgical lobectomy

Malignant 97-99% Near-total thyroidectomy

Thyroid FNA Molecular Testing Helping with the Indeterminate Cases 2003 to Present

Mutations, Translocations & Rearrangements Further Insights into Morphology Molecular Markers for Thyroid FNA Specimens

• Gene expression (mRNA) • Gene mutations • Circulating TSHR mRNA • miRNAs • Proteomics • Combination of marker types

Schematic representation of the Mitogen Activated Protein Kinase)MAPK signalling pathway.

Ciampi R , Nikiforov Y E 2007;148:936-941 Papillary Thyroid Carcinoma Mutations in Papillary Carcinoma

BRAF V600E 46- 75%

RET/PTC N ~75% Papillary CA 15%

RAS 15-43%

PAX8-PPARγ 37% BRAF V601K Non-Papillary Follicular Patterned Lesions

Adenoma vs. Carcinoma Mutations & Translocations in Follicular Carcinoma

FA FC

~70% Follicular CA 40% N PAX8-PPARγ 30%

FC Non-Papillary Oncocytic Follicular Lesions

Adenoma vs. Carcinoma Oncocytic Lesions

Kras 12/13 75%, NRAS 61 15%, HRAS 4.5%, GRIM-19, TSHR Mutations (autonomously functioning adenomas) PTEN, P13CA & TP53 TCGA Data: Cell. 2014 What we know now..

• BRAF mutations – Most common V600E – Papillary carcinoma pathway (Classic & Tall cell) – Can occur in Anaplastic Carcinoma • Ras mutations (Nras, Hras, Kras) – Follicular patterned lesions (Foll-Adenoma, FVPTC, Foll-Carcinoma) – Kras Oncocytic follicular lesions – Poorly differentiated carcinoma – Nras (18-55%)

Very Low Frequency of Limited Panel of Mutation Markers in Follicular Neoplasm Fast forward to

Next Generation Sequencing Next-Generation Sequencing

• 5-10 ng of input DNA with successful analysis of 99.6% of samples • Simultaneous parallel sequencing of thousands to millions of short nucleic acid sequences • High sensitivity and quantitative assessment • Large scale analyses required for discovery projects • Targeted panels may be useful for molecular dx of thyroid nodules Next-Generation Sequencing UPMC group--34 primers for amplification of targeted genomic areas

Nikiforova J Clin Endocrinol Metab 2013;98:E1852-60 Next-Generation Sequencing UPMC group—WHOLE genome/exome/ transcriptome sequencing in mutation negative 26 PTC and 16 FTC

Nikiforov, JCEM ThyroSeq v.2 NGS Mutation Panel (Nikiforova et.al. JCEM 2014 & 2015) 14 genes for mutations; 42 fusion types; 16 genes for expression

Gene Mutations Gene Fusions Gene expression (DNA) (RNA) (RNA) NRAS RET RET PGK1 – pan-cell marker HRAS TSHR PPARG KRT7 NTRK1 TG Thyroid KRAS AKT1 NTRK3 TTF1 epithelial cells BRAF TP53 BRAF SLC5A5 ALK PIK3CA GNAS Calcitonin – MTC Other PTEN CTNNB1 PTH – parathyroid TERT EIF1AX KRT20 – metastatic Other Molecular Alterations Detected in Fine-Needle Aspiration Samples Diagnosed as Follicular Neoplasm / Suspicious for Follicular Neoplasmand Associated Cancer Risk

No. of Samples

Cancer Identified at Negative Because of Low Alteration Positive Unique Diagnostic Events Surgery (Cancer Risk, %) Level

Point mutations NRAS 16 13 13 (81) 2 KRAS 6 6 5 (83) 4 HRAS 2 2 2 (100) 0 TERT 4 2 4 (100) 0 TSHR 3 3 1 (33) 1 BRAF V600E 1 1 1 (100) 0 BRAF K601E 1 1 0 (0) 0 TP53 1 0 1 (100) 0 PIK3CA 1 0 1 (100) 0 Gene fusions THADA 5 5 5 (100) 0 PPARG 4 4 4 (100) 0 NTRK3 2 2 2 (100) 0

Abbreviations: BRAF, B-Raf proto-oncogene, serine/threonine kinase; FNAs, fine-needle aspiration samples; HRAS, Harvey rat sarcoma viral oncogene homolog; K601E, lysine to glutamic acid substitution at position 601 in BRAF; KRAS, Kirsten rat sarcoma viral oncogene homolog; NRAS, RAS viral (v-ras) oncogene homolog; NTRK3, neurotrophic tyrosine kinase, receptor, type 3; PIK3CA, phosphatidylinositol4,5-bisphosphate 3-kinase, catalytic subunit α; PPARG, peroxisome proliferator-activated receptor γ; TERT, telomerase reverse transcriptase; THADA, associated; TP53, tumor protein 53; TSHR, thyroid-stimulating receptor; V600E, valine to glutamic acid substitution at position 600 in BRAF.

Nikiforova Y, et.al. Cancer 2014; 120:3627-34

Highly accurate diagnosis of cancer in thyroid nodules with follicular neoplasm/suspicious for a follicular neoplasm cytology by ThyroSeq v2 next‐generation sequencing assay

Nikiforova Y, et.al. Cancer 2014; 120:3627-34

Gene expression classifier (GEC) accurately identifies benign thyroid nodules in patients with indeterminate FNA cytology Alexander et al. NEJM 2012

Afirma Gene Expression Classifier (GEC)

Now 167 Gene Steward & Kloos - Otolarygol Clin N Am 2014

Bethesda Categories III–V (n = 265)

GEC result Malignant reference standard (n = 85) Benign reference standard (n = 180) Sensitivity, 92% [84–97] Suspicious 78 87 Specificity, 52% [44–59] PPV, 47% [40–55] NPV, 93% [86–97] Benign 7 93 %FN results, 2.6% ROM, 32% Bethesda Category III: Atypia of undetermined significance/Follicular lesion of undetermined significance (n = 129) Sensitivity, 90% [74–98] GEC result Malignant reference standard (n = 31) Benign reference standard (n = 98) Specificity, 53% [43–63] Suspicious 28 46 PPV, 38% [27–50] NPV, 95% [85–99] Benign 3 52 %FN results, 2.3% ROM, 24% Bethesda Category IV: Follicular or Hürthle cell neoplasm/Suspicious for follicular neoplasm (FN/SFN) (n = 81) Sensitivity, 90% [68–99] GEC result Malignant reference standard (n = 20) Benign reference standard (n = 61) Specificity, 49% [36–62] Suspicious 18 31 PPV, 37% [23–52] NPV, 94% [79–99] Benign 2 30 %FN results, 2.5% ROM, 25% Bethesda Category V: Suspicious for malignancy (n = 55) Sensitivity, 94% [80–99] GEC result Malignant reference standard (n = 34) Benign reference standard (n = 21) Specificity, 52% [30–74] Suspicious 32 10 PPV, 76% [61–88] NPV, 85% [55–98] Benign 2 11 %FN results, 3.6% ROM, 62% Bethesda Category II: Cytopathology benign (n = 47) Sensitivity, 100% [29–100] GEC result Malignant reference standard (n = 3) Benign reference standard (n = 44) Specificity, 70% [55–83] Suspicious 3 13 PPV, 19% [5–46] NPV, 100% [86–100] Benign 0 31 %FN results, 0% ROM, 6% Test performance for commonly used molecular tests in indeterminate fine needle aspiration results

Molecular diagnostic testing and the indeterminate thyroid . 2 Yip, Linwah. Current Opinion in . 26(1):8-13, January 2014.

Molecular Tests

vs. Clinical Application & Practice Increase rate of Suspicious GEC - Afirma Results in Oncocytic Nodules

Suspicious Benign Malignant nodules w surgery

Harell et al. Endo Pract 2014 30 13 (43%) 17 (57%) - 9 (69%) oncocytic lesions

McIver et al. JCEM 2014 32 27 (84%) 5 (16%) - 12 (44%) oncocytic lesions

Brauner et al. Thyroid 2015 43* 37 (84%) 6(14%)

Lastra et al. Cancer Cytopath 48 26 (54%) 22(46%) 2014 - 15 (58%) oncocytic lesions

Total 153 103 (67%) 50 (33%) -73 (71%) oncocytic lesions Changes in Surgical Pathology Diagnosis / Classification of “Low Risk Tumor(s)” The Endocrine Society Working Group for Re-evaluation of the Encapsulated Follicular Variant of Papillary Thyroid Carcinoma

Project Goals • Review a cohort of cases by experts in the field of endocrine pathology • Establish a consensus on diagnostic histologic criteria • Define the risk of adverse events based on long follow-up • Recommend new terminology that reflects tumor biology and patient outcome Naming Non-Invasive Follicular Variant of PTC as anything but “Not Carcinoma”

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

• Molecular profile - RAS and RAS-like mutations • Non-invasive FVPTC– Negligible risk of recurrence • Invasive EFVPTC - Increased risk of distant metastases Integrated Genomic Characterization of Papillary Thyroid Carcinoma. Cell (2014)

Classic PTC Encapsulated-FVPTC Foll Thyr CA Poorly Diff Thy Anapl Thyr CA Foll CA Adenoma

MUTATIONS BRAF V600E +++ + + BRAF K601E +++ + + NRAS +++ ++ + + ++ HRAS ++ + + KRAS + ++ + ++ PTEN + ++ TSHR + ++ GNAS ++ GENE FUSIONS RET/PTC +++ PAX8/PPARG ++ +++ ALK fusions + ++ ++ BRAF fusions + ETV6/NTRK3 ++ NTRK1 fusion ++ 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) Combined Institutional Data Showing TBSRTC Diagnostic Categories, Surgical Follow-Up, Risk Of Malignancy With and Without Cases of Non-Invasive Follicular Variant of Papillary Thyroid Carcinoma (NI-FVPTC) Faquin et al. Cancer Cytopathology 2015

TBSRTC Diagnostic Categories

ND Benign AUS/FLUS FN/SFN SM Malignant

Total number of FNABs, n=6943 406 (5.8%) 4221 (60.8%) 1028 (14.8%) 463 (6.6%) 238 (3.4%) 587 (8.4%)

Surgical FU

Benign Surgical FU, n=949 52 386 273 203 31 4

Malignant Surgical FU, n=877 18 40 124 101 148 446

Total PTC, n=756

Total NI-FVPTC, n=173 1 15 54 46 42 15

Risk of Malignancy

ROM 25.3% 9.3% 31.2% 33.2% 82.6% 99.1%

OROM 4.4% 0.9% 12.0% 21.8% 62.1% 75.9%

ROM excluding NI-FVPTC Cases 23.9% 5.8% 17.6% 18.0% 59.2% 95.7% **p-value 0.19 0.04§ 0.03§ 0.03§ 0.01§ 0.1 OROM excluding NI-FVPTC Cases 4.1% 0.5% 6.8% 11.8% 44.5% 73.4% **p-Value 0.18 0.05 0.02§ 0.04§ 0.02§ 0.1

% Decrease in Risk of Malignancy

ROM excluding NI-FVPTC Cases 1.4% 3.5% 13.6% 15.1% 23.4% 3.3%

OROM excluding NI-FVPTC Cases 0.2% 0.3% 5.2% 9.9% 17.6% 2.5%

Where Are We Heading to? Thyroid nodules are Common & Good Majority Will Undergo Biopsy

Thyroid nodules are Common & Good Majority Will Undergo Biopsy

Palpation Autopsy & US Ann Intern Med 1968 69:537; N Engl J Med 1993 328:553 • The Data from future thyroid FNA studies based on changes in surgical pathology diagnoses will be important for recommending potential changes in TBSRTC

• The Adjunct Molecular tests are here to stay • Never going to replace thyroid FNA cytology • Play a role in the current management paradigm of thyroid nodules Management Paradigms Aka Personalized Approach

Clinical Presentation + Ultrasound + FNA Diagnosis + Molecular Testing My View About These Tests?

• Marketing different than reality – Testing all or reflex – Local cytopathologists in the driver seat vs. a centralized lab reviewing all specimens – Cost My View About These Tests? Beyond Diagnosis • Over-simplification (as advertised) – A tissue sample “always means management oriented diagnosis” – Stick the needle get a biopsy and/or FNA - Gene analysis – Genetic profile – Predicting therapy and prognosis. – What about: Tumor heterogeneity • Majority of the mutations in the resected tumors are not fully shared in the limited tissue samples?

What I Struggle with Everyday? When My Roots are Basic Cytomorphology & My Practice is Facing Many so Called “Practice Changers”

What I Struggle with? How to avoid loosing thyroid FNA specimens?

• Good relationship with the clinicians – History – Results discussion – All matters • Good relationship with radiologist and knowledge of ultrasound • Empowering the workforce of cytopathology