The Role of Genetic Testing in the Evaluation of Thyroid Nodules. Thyroid Cancer and FNA. Thyroid Cancer. Pure Follicular Cancers.

(1)

The

Role

of

Genetic

Testing

in

the

Evaluation

of

Thyroid

Nodules

Jill E. Langer, MD Professor of Radiology The Perelman School of Medicine

University of Pennsylvania

SRU 2014

Cytology TSH

Risk factors Ultrasound

Where

does

Molecular

Analysis

of

FNA

Specimens

fit

into

the

evaluation

of

thyroid

nodules?

Thyroid Cancer and FNA



Each

thyroid

nodule

is

either

a

cancer

or

not

a

cancer



Thyroid

FNA

is

the

best

non

‐

surgical

diagnostic

tool

to

diagnose

malignancy



Thyroid

FNA

is

not

always

able

to

distinguish

a

cancer

from

a

non

‐

cancerous

lesion,

only

surgical

pathology

is

able

to

make

that

distinction

Thyroid Cancer



Thyroid

Epithelial

Tumors

are

derived

from

follicular

cells

(95%

of

all

cancers)



Well

‐

differentiated

Papillary

Cancer

(80%)



Pure

papillary

cancer



Follicular

Variant

of

Papillary

cancer



Well

‐

differentiated

Follicular

Cancer

(10%)



Poorly

‐

differentiated

Papillary

Cancer

(5%)



Anaplastic

Cancer

(

<

1%)



Medullary

cancers

are

derived

from

parafollicular

or

C

cells

(3

to

5%)

Pure Follicular Cancers



10%

of

all

thyroid

cancers

in

the

US;

higher

in

iodine

deficient

areas



female

>

male;

peak

in

6th

decade



87%

ten

‐

year

survival

(c/w

99%

for

PTC)



Histologically

similar

to

the

benign

follicular

adenoma

but

demonstrates

capsular

invasion

on

surgical

pathology



Of

all

follicular

tumors,

80%

benign

adenoma

and

20%

carcinoma

Follicular cancer

Capsular invasion

(2)

Thyroid FNA Cytology



Benign =

heterogeneous

population

of

follicular

cells,

no

atypical

nuclear

features,

often

colloid

and

macrophages



Malignant =

papillary

tissue

fragments,

abnormal

nuclear

features

such

as

irregular

contours

and

nuclear

grooves,

intranuclear

cytoplasmic

inclusions,

usually

indicates

a

papillary

cancer

Thyroid FNA Cytology



Non

‐

diagnostic

=

insufficient

number

of

cells

to

render

a

definite

diagnosis

(adequacy

often

defined

as

6

to

8

groups

of

cells,

each

group

of

at

least

10

cells)



Indeterminate

=

monotonous

follicular

cells

without

abnormal

nuclear

features

DDX:

benign

follicular

adenoma,

pure

follicular

cancer,

follicular

variant

of

papillary

thyroid

carcinoma,

benign

hyperplastic

nodule,

focal

area

of

chronic

lymphocytic

thyroiditis

Thyroid

FNA

Cytology

and

Risk

for

Cancer

Benign Malignant

Non‐diagnostic 2 ‐10% of FNAs Risk of cancer 1 to 10%

5 ‐15% of FNAs 99+% malignant

Papanicolaou Society of Cytopathology Task Force on Standards of Practice, 1997 60 ‐70% of FNAs

96+ % benign

Indeterminate

20‐30% of FNAs 15 ‐75% cancer risk

Standard

of

care

for

Indeterminate

Thyroid

Cytology



Lobectomy (majority of patients)

With completion thyroidectomy if malignant

histology, about 15 to 20%

Up to 85% unnecessary surgeries for benign

disease

Surgical risk of permanent hypoparathyroidism

and RLN injury (1 to 2 %)



Total thyroidectomy if high risk history,

bilateral nodules, patient preference,

cytologic

features

Bethesda

Classification

for

Thyroid

FNA

Cytology

Benign Malignant Non‐diagnostic Indeterminate

Suspicious

for

Malignancy

FLUS

or

AUS

Atypia of uncertain significance

Neoplasm

Baloch ZW et al, Diagnostic Cytopath 2008

Bethesda Classification for Thyroid

Cytology

Diagnostic Category Malignancy risk Nondiagnostic or unsatisfactory ~1-5%

Benign 2-4%

Atypia or follicular lesion of unknown significance (AUS/FLUS)

5-15%

Follicular neoplasm 15-30%

Suspicious for malignancy 60-75%

Malignant 97-100%

(3)

Bethesda Classification for Thyroid

Cytology

Diagnostic Category BETHESDA

Malignancy risk

Management Nondiagnostic or

unsatisfactory

~1-5% Repeat FNA

Benign 2-4% Observe with

US Follow up

Malignant 97-100% Total Thyd

Bethesda Classification for Thyroid

Cytology

Malignancy risk

Management Atypia or follicular lesion

of unknown significance (AUS/FLUS)

5-15%

Follicular neoplasm 15-30%

Suspicious for malignancy 60-75%

Repeat

FNA

ATYPIA/FOLLICULAR LESION OF

UNDETERMINED SIGNFICIANCE (AUS/FLUS)

Baloch, Diagn Cytopathol 2003; Yassa Cancer 2007

0 20 40 60 80 100

Benign FLUS/AUS Foll Neo Susp PTC/PTC

Fr eque ncy (% )

Repeat FNA cytology in FLUS/AUS nodules

50% Benign

15% sPTC

Bethesda Classification for Thyroid

Cytology

Malignancy risk

Management AUS/FLUS on Second FNA ? Higher

than 5 -15% Surgery vs observation

Follicular neoplasm 15-30% ?? Hemi Thyd

Suspicious for malignancy 60-75% ? Total vs

Hemi- Thyd

Testing

Beyond

Cytology:

Additional

Testing

for

“Indeterminate”

FNA

Results



Genetic

analysis



Single Oncogene (BRAF)



Oncogene mutation panels

(Asuragen

TM

)



RNA

Expression



Micro RNA



Afirma

TM

gene expression classifier

Testing

Beyond

Cytology:

Additional

Testing

for

“Indeterminate”

FNA

Results



Goal is to better

predict malignancy

in

patients having thyroid surgery

and

perform total thyroidectomy for

indeterminate nodules with

very high

chance of cancer



Goal is to better

predict benignity to avoid

surgery

for the management of

indeterminate nodules with a

low chance

of malignancy

(4)

Nikiforov Nature Rev Endocrinol 2011 7:569

Oncogene

Analysis:

Point

Mutations

and

Chromosomal

Rearrangements

PAX8/PPAR Benign Nodules Follicular Carcinoma Papillary Carcinoma PDC and AC Number of cases reported 611 171 2140 125 BRAF V600E 1 (0.16%) 0 45% (30-83%) 21% (0-50%)

BRAF

Mutations

in

3047

Thyroid

Nodules:

Prevalence

and

Specificity

Meta‐analysis of literature 2003‐2007

Nikiforov Nature Rev Endocrinol 2011 7:569

PAX8/PPAR1-5%

PTC

with

Oncogenes

(70%)

0 10 20 30 40 50 60 70 80 90 100

Follicular neoplasm Susp for malignancy FLUS

Sp ec if icity (% )

Nikiforov (n=513) Cantara (n=95) Moses (n=137)

Mutation

panel

(BRAF,

RAS,

RET/PTC,

PPAR



)

HIGH specificity

Nikiforov J Clin Endocrinol Metab 2011 96:3390; Cantara J Clin Endocrinol Metab 2010 95:1365; Moses W J Surg 2010 34:2589 0 10 20 30 40 50 60 70 80 90 100

Follicular neoplasm Susp for malignancy FLUS

Se

n

sitivity

(%)

Nikiforov (n=513) Cantara (n=95) Moses (n=137)

Nikiforov J Clin Endocrinol Metab 2011 96:3390; Cantara J Clin Endocrinol Metab 2010 95:1365; Moses W J Surg 2010 34:2589

Mutation

panel

(BRAF,

RAS,

RET/PTC,

PPAR



)

Many cancers are not positive

NOT optimal sensitivity

FC

FA

~70% Follicular CA PAX8/PPAR RAS HRAS, NRAS codon 61 KRAS codons 12,13 20‐40%

FA

30% 40% 2‐13% Overlap of Follicular Adenoma and CA

(5)

BRAF

RET/PTC

100% CANCER

PPAR



RAS

False positives

15 8 40 0 5 10 15 20 25 30 35 40 45

RAS False positive rate

Frequenc

y

(%)

Nikiforov n=61 Cantara n=13 Moses n=10

Nikiforov J Clin Endocrinol Metab 2011 96:3390; Cantara J Clin Endocrinol Metab 2010 95:1365; Moses W J Surg 2010 34:2589

Proposed

Algorithm

by

Endocrine

Society

Nikiforov Y E et al. JCEM 2011;96:3390-3397



Should

not

use

to

AVOID

surgery

if

the

test

is

negative

not optimal sensitivity—you will miss cancers 

When

a

patient

is

going

to

surgery,

you

want

a

test

to

optimize

the

surgical

procedure

High specificityhigh positive predictive value Refer patients with cytology of suspicious for PTC who

test positive for the Oncogene panel for total

thyroidectomy instead of hemi‐thyroidectomy Malignancies with FN and FLUS less likely to be

mutation positive than sPTC

Mutation

panel

(BRAF,

RAS,

RET/PTC,

PPAR



)

1_Yip_J_Clin_Endocrinol_Metab₂₀₁₂_97:1905

When do we use Oncogene Panel

testing at Penn?



Cytology

suspicious

for

malignancy



Nodule

with

follicular

neoplasm

or

FLUSx2

cytology

when

observation

is

NOT

felt

to

be

an

option

Large nodules >3cm

Suspicious sonographic features 

?

Non

‐

diagnostic

cytology

x

2

Cantara et al: 14/53 nodules with nondx cytology had

mutations ₁₂_cancers

Cantara J Clin Endocrinol Metab 2010 95:1365

Testing

Beyond

Cytology:

Additional

testing

for

“Indeterminate”

FNA

Results



Oncogene

testing

BRAF

Oncogene mutation panels (AsuragenTM₎



RNA

Expression



Micro

RNA



Afirma

TM

gene

expression

classifier

Hypothesis

of

GEC



A

novel,

molecular

diagnostic

test

could

be

developed

with

high

Negative

Predictive

Value

(NPV)

for

malignancy.



When

applied

to

cytologically

indeterminate

thyroid

nodules,

a

benign

gene

expression

classifier

(GEC)

result

would

accurately

(6)

Development of a gene expression classifier (GEC) to accurately identify benign thyroid nodules in patients with indeterminate FNA cytology

A multidimensional algorithm required to separate complex data sets Whole Transcriptome approach using

microarray technology

Suspicious

Molecular Classifier Trained and Validated to Distinguish Benign vs.

Suspicious Nodules

Benign

Feasibility  Development  Validation

On Whole Genome Exon Array, went from 22K to

3K genes relevant to thyroid neoplasia

From 3K set, selected final 142 genes and locked final

algorithm

Tested final 167 gene expression classifier on

independent samples

AFIRMA DEVELOPMENT

Chudova D, et al. J Clin Endocrinol Metab 2010. ₃₂ 167 genes N u m ber of Genes 22K genes 3K genes N Engl J Med 2012 367:206

Results by Cytopathology Diagnosis

N (sample size) = 47 129 81 55 55

N/A

Alexander EK, et al. NEJM 2012.

Diagnostic Category Malignancy

risk

Management

Non-diagnostic or

unsatisfactory ~1-5% Repeat FNA

Benign 2-4% Observe with US

Follow up

FLUS x 2 GEC Negative FN in low risk patient

5-6% Observe with US

Follow up

Afirma

in

action—



51 endocrinologists surveyed 9/11-3/12



368 pts with indeterminate cytology and benign GEC



Pattern of surgical referral evaluated

8 74 0 20 40 60 80 100

With benign GEC Traditional paradigm

% re co mme n d ed fo r su rg er y p<0.001

(7)



2011-2012—US FNA of 1112 nodules

Nondx 9.7%

Benign 73%

Malignant/suspicious for malignancy 9.3%

_{FLUS/AUS or follicular neoplasm 8%--89 pts and offered} GEC testing

30 not submitted (pt declined, MD choice) 6 NO result (insuff RNA)

53

with

GEC

results

89

AUS/FLUS/FN

McIver 2012 ATA abstract

Afirma

TM

in action—Mayo clinic

Afirma

TM

in action—Mayo clinic

53

nodules

with

GEC

results

13

(24%)

BENIGN

40

(76%)

SUSPICIOUS

27 surgery ₁₃ no surgery

23 (85%) BENIGN

4 (15%) CANCER

McIver ATA abstract 2012



Different cancer prevalence in FLUS/FN

 NEJM 25% vs. Mayo 15%



Different proportion of GEC suspicious results

in FLUS/FN

NEJM 57-60% vs Mayo 78%



Real world application may vary . . .

Afirma

TM

in

action—Mayo

clinic

Positive predictive value

Mayo 14% vs. NEJM 38%

NPV

and

PPV

for

Afirma

TM

GEC

based upon N Engl J Med 2012 367:206

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 NPV Prevalence of malignancy 90% Sensitivity and 52% Specificity

0 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 PPV 38% 94%

Courtesy of Bryan McIver

Negative

Predictive

Value

falls

at

higher

disease

prevalence

0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Prevalence of malignancy NP V 95% Sensitivity 90% Sensitivity 80% Sensitivity

Courtesy of Bryan McIver

When

do

we

use

GEC

testing

at

Penn?

Gene expression classifier

HIGH NEGATIVE

PREDICTIVE VALUE

FLUS/AUS

Follicular/Hürthle cell neoplasm

Cancer risk <25%

BENIGN

OBSERVE

SUSPICIOUS

(8)

Molecular Analysis



Oncogene

testing

identifies

indeterminate

nodules

with

a

high

risk

of

malignancy

Used for patients undergoing surgery

Suspicious for PTC, suspicious nodules with FN

or non‐dx cytology and suspicious

sonographic appearance



m

‐

RNA

Gene

expression

classifier

has

a

high

NPV

Used when observation rather than surgery is

the goal

FLUS x 2, small FN, no suspicious US features

Evaluation

of

thyroid

nodules

2012

History, physical examination, TSH Thyroid US with risk stratification for FNA

FNA cytology sample

Cytology

•Benign

•Malignant

•Nondx*

Cytology

•Follicular lesion of undetermined significance

•Follicular/Hürthle cell neoplasm •Suspicious for malignancy

Result reported Molecular analysis

Result reported

To

reflect

back

50

years

.

New England Journal of Medicine 1964

2014 Ultrasound FNA Cytology Molecular analysis

Thank

you