classifying your thyroid fna specimens using...

Classifying Your Thyroid FNA Specimens

Using Bethesda Terminology: Use of

Adjunct Molecular Reflex Testing

Constantine Theoharis, MD

Assistant Professor

David Chhieng, MD, MBA, MSHI

Professor

Director of Cytology

Department of Pathology

Yale University

New Haven CT

Overall Objectives

� To apply the Bethesda Thyroid FNA Classification

System in the evaluation of thyroid FNA.

� To utilize molecular testing as an adjunctive test in

thyroid FNA. thyroid FNA.

� To advise clinicians on the implications of the each

diagnostic category of the Bethesda Classification

system and the results of molecular testing.

Overview

� Usage and clinical implications of the Bethesda Thyroid

Fine Needle Aspiration Classification System

� Indications and implications of molecular testing in thyroid

FNA

3

The Utilization of the Bethesda

System & BRAF Molecular

Adjunct Testing on Thyroid

Cytologic Samples Cytologic Samples

Part I of II

4

Constantine Theoharis, MDAssistant Professor

Department of PathologyYale School of Medicine

ASCP 2011

Objectives

� Review the Bethesda Thyroid FNA Classification System

� Describe its utilization at Yale & the questions it raises

Cellularityraises

� Posit molecular testing as prequel to part 2

� Challenge our view of the modern cytopathologist/surgical pathologist

5

Architecture Cytology

The Art of Cytopathology

Background� Thyroid Cancer

� Most common endocrine malignancy

� Incidence increasing

� Problematic clinically� Nodules

� ~7% of the population

� But TFNA is Compromised

� 60% benign

� 10% malignant

� 20% equivocal

� 10% non-diagnostic/unsatisfactory

� Lack of Uniformity� ~7% of the population� Only 5% are malignant

� Thyroid FNA (TFNA) = � the keystone modality� Diagnostic test*� Screening test *

*more in a moment6

� Lack of Uniformity

� Terminology

� Criteria

� Clinical Implications

� Bethesda Classification

� Resolutions?

� Molecular Diagnostics

� Not expressly advocated in the Bethesda System

The Bethesda System

1. Nondiagnostic or

Unsatisfactory

� Cyst fluid only

� Virtually acellular specimen

� Obscuring factors

4. Follicular neoplasm or

suspicious for a follicular

neoplasm

� Specify if Hürthle cell type

5. Suspicious for malignancy

2. Benign

� Benign follicular nodule e.g. adenomatoid nodule, colloid nodule

� Lymphocytic thyroiditis

3. Atypia of undetermined

significance

5. Suspicious for malignancy

6. Positive for malignancy

� PTC

� Medullary carcinoma

� Anaplastic carcinoma

� Lymphoma

� Metastatic neoplasm

� Other

7Ali and Cibas (Ed): The Bethesda System for Reporting Thyroid Cytopathology. Springer. 2009

Audience ResponseHow many thyroid FNAs do you see at your

institution?

� Answer choice #1: 1-100 cases/year.


� Answer choice #3: 501-1000 cases/year.� Answer choice #3: 501-1000 cases/year.


� Answer choice #5: >3000 cases/year.

Yale Endocrine Cytopathology

and Surgical Pathology

� Endocrine Surgery Referral Center

�Over 3,200 Thyroid FNAs annuallyFNAs annually

� Representing 2,500 patients seen in clinic per year

� Approximately 400 operations performed annually

9

Yale Smilow Cancer Center

Audience ResponseDo you utilize the Bethesda Thyroid FNA

Classification at your institution?

� Answer choice #1: No. We don’t have a

classification system per se.

� Answer choice #2: No. We have our own

departmental system.departmental system.

� Answer choice #3: Yes. We use TBS as it has been

described in the literature.

� Answer choice #4: Yes, however, we’ve modified it

somewhat to fit our needs.

The Bethesda System (Yale Version)

1. Nondiagnostic/ Unsatisfactory� Insufficient cellularity

� Poor preservation

� Obscuring factors

2. Benign � Benign mixed macro/micro-

follicular hyperplastic nodule i.e. goiter

� Lymphocytic thyroiditis/

4. Follicular neoplasm� Microfollicular pattern

� S/O Follicular Variant of PTC

5. Hürthle cell neoplasm

6. Suspicious for malignancy� Papillary Thyroid Carcinoma

� Medullary carcinoma� Lymphocytic thyroiditis/ Hashimoto thyroiditis

� Cyst Contents or Colloid Nodule-if USG matches

3. Indeterminate [AUS/FLUS]� Low cellularity with

predominance of microfollicles and absence of colloid

� Atypical nuclear features



� Lymphoma

� Metastatic malignancy

7. Positive for malignancy� Papillary Thyroid Carcinoma



� Lymphoma

� Metastatic malignancy

11Theoharis et al. Thyroid 2009;19:1215

Is Thyroid FNA a Diagnostic or a

Screening Test?

� Diagnostic�Positive for PTC/MTC/ATC

�Suspicious for…

�Negative for Malignancy

AUS/Indeterminate?

� Screening

�Follicular Neoplasm

�Hürthle Cell Neoplasm

�AUS/Indeterminate?

12


� Architecture�Group Cell Features

�Single Cell Features

� Cytology�Nucleus v.s. Cytoplasm


� Architecture

�Group Cell Features

�Single Cell Features

� Cytology

�Nucleus v.s. Cytoplasm?

Cyto-Method Differs from Histology

� Cellularity

� Follicular Cells

� Adequacy

� 6 groups

� 10-15 cells/group

� Colloid

13

� Colloid

� Not needed

� But useful

� Lymphs /Macs/ Others?

� Architecture

� Group Cell Features

� Single Cell Features

� Cytology

� Nucleus vs. Cytoplasm

NegativeUSG: Hyperechoic nodule(s), capsular calcifications, bordering vessels…non-specific

15

Papillary Thyroid Carcinoma� Ultrasound

� Hypoechoic

� Microcalcifications

� Increased Vascularity

� Cellularity

� Follicular Cells!

16

� Follicular Cells!

� Architecture

� +/- Papillae, sheets,

caps

� Cytology

� Irregular Nuclear

Membranes & Grooves

� Intra-Nuclear

Cytoplasmic Invaginations (INCI’s)

Papillary Thyroid Carcinoma -

Papillae

17

Papillary Thyroid Carcinoma -

Inclusion (INCI)

18

Psammoma Body (Calcospherite)

19

Papillary Thyroid Carcinoma-

Papillae

20

Papillary Thyroid Carcinoma –

Inclusion (INCI)

21Does a classic positive PTC need molecular testing? BRAF? For Dx? for Pgx?

PTC Papillae & Psammoma

22

PTC with Oncocytic Features

23

Papillary Thyroid Carcinoma

to LN

24

Papillary Thyroid Carcinoma

to LN

25Often, few if any lymphocytes are present

Follicular & Hürthle Cell

Thyroid Neoplasms� Architecture

�Microfollicles

�NO Papillae

�Capsule & Vessel?

26

�Capsule & Vessel?

� Cytoplasm

�Amount v.s. Nucleus

� Nuclei�Smooth

�Enlarged

�+/- Nucleoli

�+/- Scant Colloid

�NO INCI’s

USG: Isoechoic nodule, no calcifications, no central vascularity…non-specific findings

Follicular Adenoma Capsule

27

Follicular Thyroid Carcinoma

� CAPSULAR INVASION

� VASCULAR INVASION

� Architecture�Microfollicles

28

�Microfollicles

�NO Papillae

� Nuclei�Smooth

�Enlarged

�+/- Nucleoli

�+/- Scant Colloid

�NO INCI’s

FTC Capsular Mushrooming

29

Follicular Thyroid Carcinoma

30

Follicular Neoplasm >

Follicular Adenoma

31

Follicular Neoplasm >

Follicular Carcinoma

32

Hürthle Cell Carcinoma

33Vascular Invasion

Hürthle Cell Neoplasm

34

Hürthle Cell Neoplasm >

Hürthle Cell Carcinoma

Normal sized cells

35Rarely, the degree of atypia is so great…

Massive cell with atypia

Indeterminate/FLUS/AUS

� For specimens that contain cells (follicular, lymphoid, or

other) with architectural and/or nuclear atypia

insufficient to be classified as suspicious for a follicular

neoplasm, suspicious for malignancy, or malignancy

� Criteria� Criteria

� Borderline cellularity with predominance of follicular cells

and absence/scant colloid

� Borderline cellularity with predominance of Hürthle cells

� Focal nuclear atypia s/o PTC (nuclear enlargement with

pale chromatin, nuclear grooves) particularly in patients

with lymphocytic thyroiditis or cystic changes

� Atypical lymphoid population

� Atypia with obscuring factors and/or air drying artifacts

36

AUS/FLUS/Indeterminate

(Yale Version)

� Architectural Atypia�Low Cellularity

�Microfollicles

�Absent/Scant Colloid

37

� Incipient Changes of

Neoplasia?

Indeterminate-Architectural Atypia

Follicular Adenoma with

intact capsule on resection

38

AUS/FLUS/Indeterminate

(Yale Version)

� Nuclear Atypia�Elongation/Enlargement

�Nuclear Membrane Irregularities/Grooves

�Rare possible(?)

39

�Rare possible(?) pseudoinclusions

� Incipient Changes of

Neoplasia?

Indeterminate-Nuclear Atypia

Mixed Classic and Follicular

variant PTC on resecton

40

� “At our institution, the term ‘indeterminate,’ corresponds to the NCI 2007 guidelines category “Follicular cells of undetermined significance.” Lesions designated as such my benefit from re-aspiration in the appropriate clinical context.”

Repeat FNA in 3- 6 months


� Repeat FNA in 3- 6 months

� ~20% of nodules are repeatedly diagnosed as “indeterminate”

� Surgery indicated if worrisome clinical and/or US findings

� Can we do better?

� Reflex BRAF mutational analysis?*

41*more on this coming in part 2!

Suspicious for

malignancy

� GOAL: � Maintain high PPV of

malignancy on f/u but w/o compromising sensitivity

� USUAL SUSPECTS:� Suspicious for PTC

� Most common

� Lobectomy ± frozen section (completion subsequently)subsequently)

� Suspicious MTC, ATC, NHL

� Compromised:� Quantity

� Quality

� Can we do better?

� Reflex BRAF mutational analysis?*

42*more on this coming in part 2!

Quantity Quality

The Need for Compromise

Suspicious for malignancy

Papillary group Nuclear atypia

43Overstained, obscuring blood, low cellularity, compromise dsample

What’s the difference?

Is it Atypical?

� Rule of Thumb:

� “I’m not certain it’s

negative”

Is it Suspicious

� Rule of Thumb:

� “I’m not certain it’s positive”

� You the cytopathologist

are communicating:

� “Don’t lose this patient to

follow-up”

� Malignant risk should be

low: <30%

� You the cytopathologist

are communicating:

� “Consider lobectomy

based upon this sample”

� Malignant risk should be

high: >60%

44

Medullary Thyroid Carcinoma

� Cellularity

� Cellular

� Architecture

� SINGLE Cells

� Cytology

45

� Cytology� Spindled

� Plasmacytoid

� Nuclei

� Smooth

� Salt & Pepper

� Small nucleoli

� +/- INCI’s� Ancillary Test?

� Calcitonin


46


47

Anaplastic Thyroid Carcinoma� History

� Older patient

� Rapid, recent growth

� Gross� Hemorrhage

� Necrosis

48

� Necrosis

� Architecture� SINGLE/Cellular

� Giant/Spindled

� Pleomorphic

� PMN’s� Nuclei

� Dark/Irregular

� Mitoses

� INCI’s

ATC Immunostains

49

Thyroglobulin TTF-1

Calcitonin CK-7

Anaplastic Thyroid

Carcinoma

50

Anaplastic Thyroid

Carcinoma

51

Non-Hodgkin Lymphoma

� Large B-Cell

� Hashimoto association

� Architecture�Monotypic/Cellular

52

�Monotypic/Cellular

�Cytoplasmic vacuoles

� Nuclei�Immature chromatin

�Multi-Nucleoli

Thyroid NHL

53Liquid Monolayer pseudo-groups

Thyroid NHL +CD45

54

Distribution of cytologic categories

Cytologic Category By Nodules

2008

By Patients

2008

Expected frequency

Unsatisfactory 357 (11.7%) 230 (9.0%) 10% to 15%

Benign/Negative for

Malignancy

2368 (78.0%) 1799 (72.8%) 70% to 80%

Indeterminate/Atypia of

Undetermined

Significance

95 (3.0%) 89 (3.6%) 3% to 18%

55

Significance

Follicular /Hürthle Cell

Neoplasm

176 (5.8%) 166 (6.7%) 5% to 8%

Suspicious for

Malignancy*

43 (1.4%) 39 (1.6%) 2.5% to 8%

Malignancy* 168 (5.5%) 145 (5.9%) 4% to 8%

Total 3207 2468

* Majority of them were PTC Modified from Theoharis et al. Thyroid 2009;19:1215

Comparison before and after TBS

Cytologic Category By Nodules

2008

By Patients

2008

By Nodules

2007

By Patients

2007

Unsatisfactory* 357 (11.7%) 230 (9.0%) 293 (14.4%) 197 (12%)

Benign/Negative for

Malignancy*

2368 (78.0%) 1799 (72.8%) 1361 (66.9%) 1053 (65.9%)

Indeterminate/AUS 95 (3.0%) 89 (3.6%) Susp FN 48

(2.3%)

Susp FN 45

(2.8%)

56

Follicular /Hürthle Cell

Neoplasm*

176 (5.8%) 166 (6.7%) 174 (8.3%) 156 (9.8%)

Suspicious for

Malignancy

43 (1.4%) 39 (1.6%) 29 (1.4%) 26 (2%)

Malignancy 168 (5.5%) 145 (5.9%) 119 (6%) 112 (7%)

Total 3207 2468 2035 1596

* Differences were statistically significant Theoharis et al. USCAP 2010

Cytologic-Histologic Correlation

Cytologic category

(% surgery)

MNG/HT FA CA Total

Unsatisfactory (11%) 9 8 8 25

Benign/Negative for

Malignancy (0.3%)

61 13 8* 82

Indeterminate (30%) 7 7 13 27

Follicular / Hürthle Cell

Neoplasm (61%)

33 34 35** 102

Suspicious for

Malignancy (77%)

2 2 26 30

Malignancy (77%) 0 0 112 112

Total 112 64 202 378

57

*The false negatives were micro PTC (≤ 1cm), not initially sampled by FNA** included both follicular CA and FV PTC

Operating Characteristic

As a Screening test

for NEOPLASM

As a Diagnostic test

for MALIGANCY

Sensitivity NA NA

Specificity 68% 93%

58

Specificity 68% 93%

Positive predictive

value

NA NA

Negative predictive

value

83% 91%

Theoharis et al. Thyroid 2009;19:1215

Risk of malignancy per Dx

Diagnostic Category Incidence of

malignancy at Yale

NCI recommended

rate of malignancy

Benign/Negative for

Malignancy10%* (0.3%) 0%-3%

Indeterminate 30%* (14%) 5%-15%

Follicular /Hürthle Cell 33% 20%-30%Follicular /Hürthle Cell

Neoplasm33% 20%-30%

Suspicious for Malignancy 87% 60%-75%

Malignancy 100% 97%-99%

59

* Only a selected subset of patients underwent surgery

Modified from Theoharis et al. Thyroid 2009;19:1215

� 171 nodules diagnosed as indeterminate/FLUS/AUS

between Jan 2008 to Jun 2009;

� Accounting for 2.8% of all cases


Category Number of

cases

Case with Follow-Up

(Surgery/Repeat FNA)

Malignant

Follow-Up

60

cases (Surgery/Repeat FNA) Follow-Up

Low cellularity/

microfollicular

pattern

104 (61%) 59(59%/41%) 7%

Nuclear atypia 67 (39%) 45(73%/27%) 56%

Total 171 104 (65%/35%) 20%

Adeniran et al USCAP 2010

Problems with equivocation� On Re-FNA

� <10% of Indeterminates were re-dx’d as Indeterminate

� Majority are PTC; most classified as having “nuclear atypia” cytologically

Re-FNA benign?� Re-FNA benign?

� Hyperplastic nodules in both groups with Hashimoto thyroiditis more prevalent in the 2nd group

� Suspicious Category less problematic (87% CA risk)

� Adjunct testing?� Immunostaining?

� Molecular testing?61

Molecular Diagnostics?

primum non nocere*

62MAPK SIGNALING PATHWAY

* From the Greek: ὠφελέειν, ἢ µὴ βλάπτειν

Audience ResponseDo you utilize molecular testing on thyroid FNAs at

your institution?

� Answer choice #1: No.

� Answer choice #2: BRAF only.

Answer choice #3: BRAF, RET only.� Answer choice #3: BRAF, RET only.

� Answer choice #4: BRAF, RAS only.

� Answer choice #5: Panel of BRAF, RET, RAS, PAX

etc.

A Possible Guideline to Molecular

Thyroid FNA Testing

64Theoharis C, Hui P. Surgery of the Thyroid and Parathyroid Glands 2nd Ed. (in press)

Pathologist

OUTCOME

Proteomics

Novel Tech

Electron

Microscopy

Immuno &

Chem Stains

Gross

Pathology

Practice

Management

Epidemiology

Somatic

Genomics

Gene

Arrays

Pharmaco-

genomics

Image

Analysis

Molecular

Diagnostics

Knowledge

Databases

Histology

Cytology

Flow

Cytometry

Electronic Data Layer

Decision Support

Pathologist/Cytopathologist as

Diagnostic Specialist

• Pathology develops and

adopts tools to leverage

data from emerging

technologies

• We become diagnostic

65

Dx

Diagnostic

ImagingMedical

Record

Medical

Literature

Medical Rx

Options

Surgical

Techniques

Social

Environment

Patient

Health

Patient

Rx

Clinical

Laboratory

TreatingClinician(s)

• We become diagnostic

consultants, providing

outcomes based treatment

recommendations to

treating clinicians

• We become the

Department of Diagnostic

Medicine

Sinard JH, Practical Pathology Informatics, used with permission

Summary� The Bethesda 6-tier classification system

� Conveys different levels of risk of malignancy

� Excellent screening test for follicular/Hürthle cell

neoplasm

� Superb diagnostic test for identifying PTC with a

specificity of 93%

� Sub-classifying indeterminate category into 2

descriptive groups conveys different levels of risk

� Molecular Testing may have a role especially in

equivocal cases

� Part 2 follows shortly…

� Thank you!

66

Acknowledgements�Cytopathologists

�David Chhieng

�Adebowale Adeniran

�Diane Kowalski

�Malini Harigopal

�Guoping Cai

�Angelique Levi

Surgical Pathologist�Surgical Pathologist

�Manju Prasad

�Molecular Pathologist

�Pei Hui

�Surgeons

�Robert Udelsman

�Sanziana Roman

�Julie Ann Sosa

�Tobias Carling

�Radiologist

�Lynwood Hammers67

Molecular Testing in Thyroid FNA

David Chhieng, MD, MBA, MSHI

Professor

Director of Cytology

Department of Pathology

Yale University

New Haven CT

Objectives� To apply the Bethesda Thyroid FNA Classification

System in the evaluation of thyroid FNA.

� To utilize molecular testing as an adjunctive test in

thyroid FNA.

� To advise clinicians on the implications of the each

diagnostic category of the Bethesda Classification

system and the results of molecular testing.

Overview� Introduction

� BRAF testing as a diagnostic marker

� RAS mutational analysis

� Use of a molecular panel

� BRAF testing as a prognostic marker

3

Adjunctive Testing� Immunocytochemistry

� Flow cytometry

� Lymphocytic thyroiditis vs lymphoma

� Molecular testing� Molecular testing

4

Immunocytochemistry� Primary vs secondary

� TTF-1 and thyroglobulin


� Calcitonin, chromogranin, synaptophysin, and mCEA� Calcitonin, chromogranin, synaptophysin, and mCEA

� PTC vs other follicular-derived lesions

� CK 19, HBME-1, Galectin-3

� None specific enough

� False positive staining in Non-neoplastic lesions such

as lymphocytic thyroiditis and post FNA reactive foci

An example of medullary carcinoma staining positive

for calcitonin (ThinPrep preparation)

Immunohistochemistry for cytokeratin 19 in a fine needle aspirate of papillary

carcinoma (Giemsa preparation with immunohistochemistry performed after

removal of the coverslip).

Anderson C E , McLaren K M J Clin Pathol 2003;56:401-405

Expression of various makers in

thyroid lesions

Diagnosis Galactin -3 HBME 1 CK 19

Carcinoma (n=85) 92% 87% 72%

PTC (n=67) 94% 85% 72%

Follicular (n=6) 66% 50% 50%

Hurthle cell (n=8) 88% 13% 50%

Anaplastic (n=4) 100% 0% 25%

Adenoma (n=21) 10% 10% 5%

Non neoplastic thyroid (n=102) 17% 7% 13%

Nodular goiter (n=29) 55% 24% 31%

Thyrotoxicosis (n=14) 7% 0% 0%

Normal (n=59) 0% 0% 7%

Prasad et al. Mod Pathol. 2005:14:169.

Molecular TestingMolecular Testing

9

1. None

2. BRAF only

Audience Response

What molecular testing does your laboratory offer for

thyroid FNA?

3. BRAF and Ras

4. A panel of markers (i.e. BRAF, Ras, RET/PTC, PAX8-PPRϒϒϒϒ)

Revised Management Guidelines for

Patients with Thyroid Nodules and

Differentiated Thyroid Cancer

American Thyroid Association, 2009

MAPK Signaling pathway� >70% of PTC found

to have mutations

involving one of

the genes in the

MAPK signaling

pathway

� Especially RET/PTC,

RAS, and BRAF

� Usually mutually

exclusive

Nikiforov Y. Mod Path. 2008:2: S37

Other mutations

� Follicular carcinoma

� RAS mutation

� PAX8-PPARγ rearrangement


� RET point mutation

Molecular alterations—frequency

Abnormality FA F CA PTC Comments

BRAF -- -- 40-60% •Classic and Tall cell

•Extranodal extension

and LN metastatsis

RET/PTC -- -- 20% •Classic

•Young onset and post

radiationradiation

•Type 1 (70%)

•Type 3 (30%)

RAS 20-40% 40-50% 10-20% •5% in goiter

•FV PTC

•Distant > LN metastasis

PAX8-PPARϒ

(t2,3)(q13;p25)

2-10% 20-40% 5-10% •FV PTC

•Younger age

•Aggressive behavior

Modified from Gillian CP. ANZ J Surg. 2010; 80: 33

BRAF � Found in ~45% (40-60%) of PTC

� Virtually all point mutations result in a valine-to-

glutamate at residue 600 (V600E)

� Highly prevalent in classical and tall cell variant� Highly prevalent in classical and tall cell variant

� Rare in follicular variant

Specificity of BRAF Detection in Thyroid FNA

Nature of studies No of

samples

BRAF

positive

Final diagnosis in

BRAF-positive samples

Prospectives 1814 159 100%

Retrospectives 685 291 100%

Research FNA of

surgically

removed thyroid

267 131 99.2%*

removed thyroid

Total 2766 581 99.8%

•The false positive is hyperplastic nodule with atypical nodular hyperplasia

Nikiforova & Nikiforov. Thyroid. 2009;19:1351

BRAF � Using SSCP, BRAF detected in 76% (28/37)

classical PTC (Yale data)

� Compared to direct sequencing, BRAF detected

in 65% (24/37)

� SSCP had an analytical sensitivity of 5% tumor cells

for a definite identification of BRAFfor a definite identification of BRAF

� Implemented reflex BRAF testing for equivocal

and positive thyroid FNA since Sept 09

� A total of 157 thyroid FNAs tested between Sept

2009 and Nov 2010

� 156 (99.4%) had sufficient DNA for BRAF testing

Demographic and characteristics of

patients and thyroid nodules

� ^10 patients had two nodules biopsied

� * the case with insufficient DNA was excluded

� # According to nodules

^10 patients had two nodules biopsied * the case with insufficient DNA was excluded# According to nodules

Adeniran et al. Thyroid 2011; 21:717

Histologic follow up results of thyroid

nodules with equivocal cytologic diagnosis


Over 50% of suspicious cases found to be follicular variant of PTC

Tissue follow up of thyroid nodules with

positive cytologic diagnosis according to

BRAF status


Histologic follow up with indeterminate

FNA and BRAF testing

Cytologic

diagnosis

(n=84)

BRAF

results

Surgical Follow up Total P value

PTC Benign

Indeterminate

w/ MF pattern

Positive

(n=0)

0 0 0

w/ MF pattern

(INa)

(n=0)

< 0.001

Negative

(n=28)

2* 8 10

Indeterminate

w/ nuclear

atypia (INb)

Positive

(n=19)

16 0 16

Negative

(n=36)

9* 13 22

• These cases were follicular variant and diffuse sclerosing variant PTC

Adenirian et al. Acta Cytologica. In press

Performance of BRAF testing in

indeterminate FNA

Sensitivity Specificity PPV NPV

59% 100% 100% 66%

~ 20% increase in positive identification of PTC

Adenirian et al. Acta Cytologica. In press

Algorithm for managing patients with

thyroid FNA and BRAF testing

Ras Mutations

� Point mutations found in many human cancers and in most types of thyroid tumors

� K-RAS, H-RAS, N-RAS genes may be involved

� Hot spots - codons 12, 13 and 61� Hot spots - codons 12, 13 and 61

� N-RAS codon 61 mutations most common in thyroid tumors

Mechanism of RAS Activation by Point Mutation

RASGDP Mutations

codons

• 12/13 in exon 1

Inactive

RASGTP

DownstreamEffectors

• 12/13 in exon 1 affecting GTP binding domains

• 61 in exon 2 affecting GTPase domain

Active

Molecular Pathways Activated by RAS

RASGRB2

SOS

Y1062

SHC

FRS2

RET

P

P

P

P

PLC Ral /Cdc42

DAG

PKC

AKT

Rho

Rac

B-RAF

MEK

ERK

PI3K

JNK

P70S6K

MEKK1

BCL

BAD

Apoptosisc-Jun, Fos ,

c-Myc , Elk-1

Incidences of Ras Mutations in Thyroid

Lesions

Thyroid Lesions Incidence of Ras Mutations

Follicular Carcinoma 40-50%

Papillary Carcinoma 10-20%*Papillary Carcinoma 10-20%*

Follicular Adenoma 20-40%

Goiter (Adenomatous Hyperplasia) 0-3%

•Almost exclusively in follicular variantLiu et al. Thyroid 2004: 14: 616

Nikiforvoa and Nikiforvoa. Thyroid 2009: 19: 1351

RAS mutations and

indeterminate FNA

� 64 Indeterminate thyroid FNAs (including FLUS, FN,

and suspicious) with positive RAS mutation and

surgical follow up

� Results of surgical follow up� Results of surgical follow up

Malignant 84%

PTC 52

Follicular carcinoma 4

Neoplastic Follicular Adenoma 11 16%

Non-neoplastic 0 0%

Nikiforov et el. JCEM. 2009;94:2092

Our Approach

Positive BRAF (regardless of RAS mutation)

Total thyroidectomy

� Perform RAS mutation in addition to BRAF mutation analysis for all indeterminate cases

IndeterminatePositive

RAS/Negative BRAF

Lobectomy

Negative BRAF + Negative RAS

Repeat FNA

Our Approach� Not recommended for cases with the diagnosis of

“Follicular Neoplasm”

� Up to 20% of RAS positive cases are Follicular

Adenoma and occasionally Adenomatous

HyperplasiaHyperplasia

� Current approach is lobectomy +/- intraoperative

consultation—No significant impact on patient

management

Alternate approach to molecular testing

in thyroid FNA

� Performing a panel of mutations on ALL thyroid FNAs

� The panel includes

� BRAF (V600E)

� RAS (k-,n-, & h-)

� RET/PTC rearrangement (RET/PTC 1 & RET/PTC 3)

ϒ

� RET/PTC rearrangement (RET/PTC 1 & RET/PTC 3)

� PAX8-PPRϒ

Performance of Molecular

testing using a panelReference Number of FNA

Samples

Specimens

types

Number of mutations

identified

1 470 All types 32 (7%)

2 400 All types 50 (13%)

3 285 Only those with

surgical FU

67 (29%)

4 432 All types 61 (14%)

1. Nikiforov et el. JCEM. 2009;94:2092.

2. Moses et al. WJ Surg. 2010;34:2589.

3. Cantara et al. JCEM. 2010;95:1365.

4. Mathur et al. Surgery. 2010; 148:1170.

Diagnostic

category

Numbers Mutation status Follow up

Malignant Follicular

Adenoma

Negative

Positive 79 Positive 26 26 0 0

Negative 53 53 0 0

Suspicious 81 Positive 52 50 0 2

Negative 29 10 4 15

Indeterminate 115 Positive 22 21 1 0

Negative 93 29 29 35

Negative 99 Positive 13 9 4 0Negative 99 Positive 13 9 4 0

Negative 86 5 10 71

Non-diangostic 53 Positive 14 12 2 0

Negative 39 4 11 24

Total 427 Positive 127

(30%)

118

(93%)

7

(6%)

2

(1%)

Negative 300

(70%)

101

(34%)

54

(18%)

145

(48%)




Performance of Molecular

testing using a panelReference Sensitivity Specificity PPV NPV

1 62% 100% 97% 95%

2 38% 65% 42% 65%

3 80% 100% 100% 90%




Prognostic Molecular

Markers

BRAF and Prognosis� Correlate with aggressive tumor characteristic

� Extrathyroidal extension

� Nodal metastases

� Resistant to radioiodine

� Tumor recurrence� Tumor recurrence

BRAF: Invasion and LNs

BRAF: Recurrence

Kaplan-Meier estimate of recurrence-free probability of PTC in patients with or without BRAF

mutation.

A, Analysis of a multicenter series consisting of 219 cases, mainly Caucasian patients. Log-rank

test: �2 O 4.0, P O 0.04. [Xing et al., 2005 (83).]

B, Analysis of a Korean series consisting of 203 patients. Log-rank test: �2 O 4.60, P O 0.037.

[Kim et al., 2006 (71), with permission from Wiley–Blackwell.]

BRAF: Higher Stage Disease

� BRAF is an

Independent

Prognostic Factor

associated with

Worse Disease and

Poorer Outcomes

Xing, M: BRAF Mutation

in Thyroid Cancer

BRAF mutation analysis and FNA

BRAF +ve BRAF –ve

Histologic subtype

Follicular variant 7% 51% p<0.05

Classic 22% 11% p<0.05

Tall cell variant 30% 6% p<0.05 Tall cell variant 30% 6% p<0.05

Extrathyroidal extension 56% 15% p<0.05

Mean size (cm) 1.9 2.3 p<0.05

LN involvement (Level VI) 47% 19% p<0.05

Yip et al. Surgery 2009; 146: 1215

Pathologic features of 60 papillary thyroid

carcinomas


Histologic Features of Papillary Thyroid

Carcinoma with and without BRAF Mutation

Morphologic features BRAFmutation

Positive

BRAFmutation

Negative

P Value

Tumor Capsule (intact or

infiltrated)

12 (35%)1 15 (68%)2 0.02

Infiltrative Growth 32 (94%) 13 (59%) 0.002

Stromal fibrosis, sclerosis or

desmoplasia

33 (97%) 15 (68.1%) <0.001

Plump tumor cells with 22 (65%) 6 (27%) 0.01Plump tumor cells with

moderate amount of

eosinophilic cytoplasm

22 (65%) 6 (27%) 0.01

Classic fully developed nuclear

features

33 (97%) 5 (22.7%) 0.0001

Lymphovascular invasion 4 (11.7%) 4 (18%) NS

Psammoma bodies 17 (50%) 5 (22.7) 0.05

Stromal calcifications 24 (70%) 10 (45%) 0.09

Cystic change 8 (23.5%) 3 (13.6%) NS

Extrathyroidal Extension 4 (11.7%) 4 (18%) NS

Finklestein et al. Presented at the 2011 USCAP meeting

Potential impact of BRAF status

on patient management

� Initial surgical management

� Total thyroidectomy

� Possible central lymph node dissection

� Initial radioiodine treatment� Initial radioiodine treatment

� Higher does of radioactive iodine

1. Archival slides with or without microdissection

2. Lavage fluid from needles washing after direct preparing direct smears

Audience Response

What kind of specimen does your laboratory used

for molecular testing for thyroid FNA?

preparing direct smears

3. Cell block

4. Obtaining additional pass to be collected in preservative solution

Specimen types for molecular testing

Specimen types Comments

Scraping all cellular

materials from archival

slides

• Ability to select slides containing optimal numbers

of tumor cells

• Destruction of diagnostic materials on the original

slides

• Labor intensive with microdissectionMicrodissecting from

archival slides archival slides

Lavage fluid from

washing the needles

after preparing direct

smears

• Quantity and composition of cellular materials

unknown—Compromise sensitivity

• Discordance of BRAF mutation status between

matched FNA and FFPE samples

• Storage up to 3 weeks after collection

Obtaining additional

pass to be collected in

preservative sol’n

• Quantity and composition of cellular materials

unknown—Compromise sensitivity

• Discordance of BRAF mutation status between

matched FNA and FFPE samples

• Required different collection protocol

Conclusions & Future Directions

� Molecular testing improved diagnostic accuracy for

thyroid FNA

� High PPV for cancer except RAS

� Need to define algorithms of patient management based

on cytology and molecular testing

� Prognostic markers

� BRAF mutation signifies more aggressive tumor

behavior—different therapeutic regime

� Therapeutic targets

� Novel inhibitors of MAP kinase pathway

classifying your thyroid fna specimens using...

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