Dr Sarah J Johnson
Consultant Cyto/histopathologist
Newcastle upon Tyne
MOLECULAR TESTING OF THYROID
NODULES
This talk
 Overview of molecular abnormalities in thyroid
lesions
 Potential value
 Our own work
Overview of molecular abnormalities
(NikiforovYE, Modern Pathology 2011;24:S34-43; BhaijeeF& NikiforovYE.EndocrPathol2011;22:126-133.
NikiforovaMN & NikiforovYE. Thyroid2009;9:13511361.
 Recent dramatic increase in understanding of
molecular biology of thyroid cancer
 Main four
 BRAF and RAS point mutations
 RET/PTC and PAX8/PPARγ gene rearrangements
 Others
 PI3K/AKT signalling pathway - PDC
 TP53 and CTNNB1 mutations – PDC, ATC
 TRK rearrangement – PTC but rare
Prevalence of mutations
Tumour type
Mutation
Prevalence %
Papillary carcinoma (PTC)
BRAF
40-45
RET/PTC
10-20
RAS
10-20 (usually FVPTC)
RAS
40-50
PAX8/PPARγ
30-35
Familial – germline RET
>95
Sporadic – somatic RET
40-50
Follicular carcinoma (FC)
Medullary carcinoma (MTC)
Nikiforov Arch Pathol Lab Med
2011;135:569-77
Bhaijee & Nikiforov Endocr
Pathol 2011;22:126-33
Nikiforova & Nikiforov Thyroid
2009;19(12)1351-61
Rivera et al, Modern
Pathology 2010;23:1191-21200
Follicular variant of PTC (FVPTC)
encapsulated
infiltrative
BRAF
0
26
RAS
36
10
RET/PTC
0
10
PAX8/PPARγ
3.5
0
Like FA / FC
Like classical PTC
BRAF point mutations
 Intracellular effector of MAPK signalling cascade
 Most V600E → activate BRAF kinase, stimulate MAPK pathway →
tumourigenic for thyroid cells
 1-2% - other mutations eg K601E
 BRAF V600E mutation
 quite specific for PTC and related tumour types
 60% classical PTC
 80% tall cell variant PTC
 10% FVPTC
 10-15% PDC
 20-30% ATC
 NOT in FC, MTC or benign nodules
 early in pathway
BRAF - clinical and prognostic value
Melcket al The Oncologist2010;15:1285-93;Yipet al.Surgery2009;146:1215-23;Xinget al J ClinOncol2009;27:2977-2982.
 Associated with aggressive tumour characteristics (V600E only)
 ETE, multicentricity, advanced stage, LN+, distant metastases, recurrence,
persistence, re-operations, tall cell morphology, lymphovascular invasion,
suspicious USS features
 especially >65 yrs
 Independent predictor of treatment failure, tumour recurrence,
tumour-related death
 Even in microPTC – associated with poorer clinicopathological
features (eg ETE, LN+) – exciting because management debated
 May relate to
 tendency to de-differentiate
 reduced ability to trap radio-iodine
 less responsive to TSH suppression
BRAF – diagnostic value in cytology
Adeniranet al Thyroid2011;21(7):717-23.Bentzet al OtolaryngolHead and Neck Surgery 2009;140:709-14
 BRAF mutation strongly correlates with PTC, independent of
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cytology
Improves accuracy, specificity and PPV for PTC
Specificity and PPV for PTC with BRAF-positivity = virtually
100%
Mixed results for sensitivity & NPV, can be low
Helpful in identifying PTC in “indeterminate” cytology samples
Could use to change management decision
Indeterminate
cytology
positive
Total thyroidectomy
+/ level VI LNs
negative
Diagnostic
hemithyroidectomy
BRAF test
BRAF –accuracy in cytology
 6 false positives for malignancy with BRAF analysis
 1 case in Korea – indeterminate cytology, BRAF-positive → histology of
“atypical nodular hyperplasia”
 5 when ultrasensitive testing used, not positive on repeat testing
 Recent meta-analysis – BRAF testing in 2766 samples
 581 BRAF-positive → 580 were PTC (some with benign cytology)
 rate of malignancy for BRAF-positivity = 99.8%
 frequency of indeterminate cytology in BRAF-positive samples = 15-39%
 Various techniques possible but need to avoid ultrasensitive
detection and methods that are not well validated → may risk
false positives
 BRAF detection in cytology also predicts aggressiveness
 BRAF-negativity with indeterminate cytology does not
eliminate need for diagnostic hemithyroidectomy
BRAF –therapeutic value
 Predicts aggressiveness →maybe consider more
aggressive treatment, more frequent follow-up,
but maybe not enough to act on yet
 Therapeutic target - BRAF inhibitors eg sorafenib
RAS - point mutations
 Family includes HRAS, NRAS, KRAS
 Propagate signals along MAPK and other signalling cascades
 Most frequent mutations in thyroid
 NRAS codon 61
 HRAS codon 61
 Found in
 10-20% PTC – mostly FVPTC
 40-50% FC
 20-40% FA – but ?precursors for FC
 some hyperplastic nodules but clonal so ?neoplasm
 less in oncocytic tumours
RAS - point mutations
 Prognosis
 some association with dedifferentiation and worse outlook
 but also associated with eFVPTC – indolent behaviour
 Finding RAS mutation in thyroid nodule
 strong evidence for neoplasia
 but does not establish diagnosis of malignancy
 RAS mutation in cytology
 PPV for malignancy 74-88%
 helpful when cytology difficult such as FVPTC
RET/PTC gene rearrangements
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RET highly expressed in C cells, not follicular cells
But activated by RET/PTC rearrangement
11 types, RET fusion to different genes
Commonest in thyroid cancer - RET/PTC1 & RET/PTC3
All fusions activate MAPK signalling pathway
Variation in expression – needs to be “clonal”, ie majority
Clonal RET/PTC - reasonably specific for PTC
 10-20% PTC in adults
 50-80% PTC after radiation exposure (RET/PTC1 – classical PTC,
RET/PTC3 – solid type PTC)
 40-70% PTC in children and young adults
 Non-clonal RET/PTC – no diagnostic implications
RET/PTC- prognosis and diagnosis
 PTC with RET/PTC - younger age, classical PTC histology,
high rate LN metastases
 But varied views on overall prognostic value
 Detection of clonal RET/PTC = strong indication PTC
 Histology – not useful because classical so diagnosis clear
 In FNA – can improve pre-operative diagnosis PTC but
can have false positives
PAX8/PPARγ gene rearrangement
 Fusion between PAX8 gene and perioxisome proliferator




activated receptor (PPARγ) gene
Causes over-expression of PPARγ protein
Found in
 30-40% conventional FC
 less often in oncocytic carcinomas
 5-38% FVPTC
 2-13% FA – often thick capsule, ?pre-FC or misdiagnosed
Often - younger age, smaller tumour, more frequent vascular
invasion
Detection in histology not diagnostic of malignancy but should
prompt exhaustive search for capsular or vascular invasion
Detection in FNA – typically malignant but numbers low
Gene expression profiles
Borupet al Endocr-RelatedCancer2010;17:691-708.Maenhautet al ClinOncol2011;23:282-288.Ferrazet al ClinEndocrinolMetab2011;96(7):2016-2026
 mRNA
 no ideal marker of PTC
 lack of markers to distinguish FC from FA
 slight difference between radiation-induced PTC and not
 ?can measure different background susceptibilities to
radiation
 microRNAs
 easier to extract from FNA than mRNA
 possible future diagnostic potential
 PTC & FC have different profile to normal thyroid
Review of 20 studies of genetic testing
Ferraz et al ClinEndocrinolMetab2011;96(7):2016-2026
 Highest sensitivity with panel of markers
 BUT more FP with panel than with single marker
 Best if done on same material as used for cytology, not extra
 Suggest
Indeterminate
cytology
Panel of
markers
Negative
group
Malignancy risk
down from
20% to 8-10%
miRNA
Cohort with
3% malignancy
risk
?follow up
with USS +
repeat FNA
Commercially available kits – USA
Sample in special preservative solution
→ panel of 7 molecular markers
Commercially available kits – USA
Sample → cytopathology →
 inadequate, benign or malignant report
 indeterminates → gene expression
Our own work in Newcastle
 Initial project
 Current BRAF pilot
Initial project – BSCC presentation 2011
S. Hardy, U.K. Mallick, P. Perros, S.J. Johnson, A. Curtis and D Bourn
Aim: to set up and validate assays for detection of molecular markers in
thyroid samples
Retrospective – archival histology then cytology
Panel of markers:
• BRAF codon 600
• HRAS codon 61
on extracted DNA
• KRAS codons 12/13
(melt curve analysis)
• NRAS codon 61
• RET/PTC rearrangements
on extracted RNA
• PAX8/PPARγ rearrangements
(RT-PCR-based assays)
Example data – NRAS codon 61
WT CONTROL
CODON 61 (Q61K) CONTROL
WT
Q61K
WT
WT
Q61K
Results – point mutations on thyroid histology cases
32 cases (patients), 36 blocks
6 non-neoplastic nodules
0/6
0%
5 follicular thyroid adenoma (FA)
0/6
0%
5 follicular thyroid carcinoma (FC)
1/5
20% (NRAS codon 61)
7 papillary thyroid carcinoma (PTC)
1/6
17%
4 “aggressive” PTC (aPTC)
4/4
100% (BRAF v600E)
3 poorly differentiated carcinoma (PDC)
1/3
33% (NRAS codon 61)
1 SCC
1/1
100% (NRAS codon 61)
1 metastatic struma ovarii
1/1
100% (NRAS codon 61)
• ie. pattern as expected
• Concordance between different blocks from same tumour
(BRAF v600E)
Results – point mutations on cytology slides
Cases with molecular result available on histology:
NNN
2 cases, 4 slides
1/3
50% cases (NRAS codon 61)
FA
1 case, 1 slide
0/1
0%
FC
4 cases, 7 slides
2/6
50% cases (1 NRAS, 1 HRAS)
PTC
2 cases, 6 slides
1/3
17% (NRAS codon 61)
aPTC
3 cases, 9 slides
4 tumour
3/3
100% (2 BRAF V600E, 1 HRAS codon 61)
5 LN/bed
1/3
50% cases (HRAS but in neg LN)
0/2
0%
PDC
1 case, 2 slides
Cases with no molecular result available on histology:
Thy4 (histol = FA)
0/1
0%
Thy3 (histol = FC)
0/1
0%
Thy3f (histol = FC), 4 slides
2/2
100% (NRAS,HRAS)
Results as cancer patients
23 cancer cases
 21 molecular results on histology
 9/21 mutations
 5 of 9 had molecular tests on cytology: 2 fails, 3 positive matches
 2 no molecular result on histology
 1/2 mutation on cytology
ie. cytology found mutations in 57% (4/7)
Results as mutations
13 cases with mutations (on cytology and/or histology)
 12 malignant outcome
 1 benign outcome
9 histology cases with mutations – all malignant outcomes
11 cytology slides with 12 mutations - 7 patients - 6 malignant outcomes
mutation
No of mutations
outcome
malignant
benign
BRAF V600E
2
2 aPTC (2 pts)
0
NRAS codon 61
5
3 FC (2 pts)
1 PTC
1 (NNN)
HRAS codon 61
5
2 FC (2 pts)
0
2 aPTC (1 tumour, 1 neg LN)
KRAS codon
0
0
0
Results as cytology slides
 37 cytology slides
 29 thyroid, 4 LN, 4 recurrences
 Most were DQ slides
 Failure rate 9 of 37 = 24%
 1 LBC slide (SurePath) - paired DQ worked
 2 cyst fluid only (LN met) – failed (same case histology worked)
 2 unsatisfactory slides (1 thyroid, 1 bed) – a paired US worked
 1 with lots blood & colloid – paired slide worked
 2 Thy3f
 1 Thy5
Results as cytology slides
 37 cytology slides
 24 slides with histology mutation result available
 9 in agreement for no mutation
 4 in agreement for presence of mutation
 5 discordances – mutations in cytol not histol, 4 malignant outcomes
 11 cytology pairs (2 slides from same specimen)
 4 matches – 1 fail, 1 NRAS, 2 no mutation
 7 mismatches – 3 with one fail, 2 NRAS v fail, 1 NRAS v no mutation, 1
BRAF V600E v HRAS codon 61
 1 of 4 slides from same specimen
 2 fail, 1 NRAS & HRAS, 1 HRAS only
Conclusions from initial study
• Molecular testing for DNA point mutations is feasible in stained thyroid
cytology samples
• PPV 92% for malignant outcome
• BUT
• not always successful result
• not always match of cytology with cytology, or cytology with histology
• can have multiple mutations in one sample and/or tumour
• can have mutations in negative LN cytology sample from cancer case
• can have mutations in non-neoplastic nodules
• Next step – prospective BRAF testing for 12 months
• Molecular testing also feasible in histology of thyroid cancers – possible
future role for individualised treatment and prognostication
Current BRAF Pilot
 Prospective
 12 months BRAF testing on cytology reported
as Thy3a, Thy3f, Thy4 and Thy5 PTC
 No result to clinician, no action on result
 Will then
 correlate with surgical and histological outcome
 assess whether BRAF result would have influenced
management decision
BRAF Pilot – results so far
 Tested 14 cytology slides from 13 patients
 Slide types
 12 DQ – all worked, even with heavy bloodstaining
 1 ICC for Tg on destained DQ – worked
 1 SurePath LBC – failed
 Outcomes
 2 BRAF V600E mutations
 LN5 met PTC (histol = classical & follicular variant, pT3 pN1b)
 Thyroid Thy5 PTC (histol = classical multifocal, pT1b pN1b)
 11 wild type
 7 Thy3a - 1 with histol = FA
 3 Thyf - 1 with histol = dominant nodule with contralat PTC
 1 Thy5 ATC vs MM – histol = ATC
Summary points for whole talk
 Molecular testing of thyroid cytology and
histology specimens is feasible in routine labs
 Diagnostic aims
 single stage theraeutic surgery for cancers
 avoiding diagnostic hemithyroidectomies for benigns
 BRAF mutation shows most promise
 diagnostically, prognostically & therapeutically
 Other mutations and rearrangements
 diagnostically & prognostically – less predictive
 Also likely future role for microRNA studies
Thankyou for listening