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 Thyroid 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 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

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 Cancer 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 Thyroid Cancer 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 Endocrinology 2007;148:936-941

Papillary Thyroid Carcinoma
 Mutations in Papillary Carcinoma

BRAF V600E
46-75%

N RET/PTC
~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

KRT20 – metastatic
TERT EIF1AX
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, neuroblastoma 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, thyroid adenoma associated; TP53, tumor protein 53; TSHR, thyroid-stimulating hormone 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 biopsy results

Molecular diagnostic testing and the indeterminate thyroid nodule.

2
Yip, Linwah. Current Opinion in Oncology. 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

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
Thyroid Nodule 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