1. Molecular testing of thyroid nodules
Dr Sarah J Johnson
Consultant Cyto/histopathologist
Newcastle upon Tyne
Molecular testing of thyroid nodules

2. This talk Overview of molecular abnormalities in thyroid lesions
Potential value
Our own work

3. Overview of molecular abnormalities (Nikiforov YE, Modern Pathology 2011;24:S34-43; Bhaijee F & Nikiforov YE. Endocr Pathol 2011;22: Nikiforova MN & Nikiforov YE. Thyroid 2009;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

4. Prevalence of mutations
Tumour type
Mutation
Prevalence %
Papillary carcinoma (PTC)
BRAF
40-45
RET/PTC
10-20
RAS
10-20 (usually FVPTC)
Follicular carcinoma (FC)
40-50
PAX8/PPARγ
30-35
Medullary carcinoma (MTC)
Familial – germline RET
>95
Sporadic – somatic RET
Nikiforov Arch Pathol Lab Med 2011;135:569-77
Bhaijee & Nikiforov Endocr Pathol 2011;22:126-33
Nikiforova & Nikiforov Thyroid 2009;19(12)
Rivera et al, Modern Pathology 2010;23:
Follicular variant of PTC (FVPTC)
encapsulated
infiltrative
BRAF 26
RAS 36 10
RET/PTC
PAX8/PPARγ
3.5
Like FA / FC
Like classical PTC

5. 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

6. BRAF - clinical and prognostic value Melck et al The Oncologist 2010;15: ; Yip et al.Surgery 2009;146: ; Xing et al J Clin Oncol 2009;27:
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

7. BRAF – diagnostic value in cytology Adeniran et al Thyroid 2011;21(7): Bentz et al Otolaryngol Head and Neck Surgery 2009;140:709-14
BRAF mutation strongly correlates with PTC, independent of 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
positive
Total thyroidectomy +/ level VI LNs
Indeterminate cytology
BRAF test
negative
Diagnostic hemithyroidectomy

8. 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

9. 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

10. 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

11. 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

12. RET/PTC gene rearrangements
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

13. 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

14. 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

15. Gene expression profiles Borup et al Endocr-Related Cancer 2010;17: Maenhaut et al Clin Oncol 2011;23: Ferraz et al Clin Endocrinol Metab 2011;96(7):
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

16. Review of 20 studies of genetic testing Ferraz et al Clin Endocrinol Metab 2011;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
Indeterm-inate 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

17. Commercially available kits – USA
Sample in special preservative solution
→ panel of 7 molecular markers

18. Commercially available kits – USA
Sample → cytopathology →
inadequate, benign or malignant report
indeterminates → gene expression

19. Our own work in Newcastle
Initial project
Current BRAF pilot

20. Initial project – BSCC presentation 2011 S. Hardy, U. K. Mallick, P
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 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)

21. Example data – NRAS codon 61
WT CONTROL
CODON 61 (Q61K) CONTROL WT
Q61K WT WT
Q61K
The above is an example of traces generated for NRAS genotypes – WT (single peak at ~59oC), Q61K (WT peak at ~59oC with an additional peak at ~48oC). 21

22. Results – point mutations on thyroid histology cases
32 cases (patients), 36 blocks
6 non-neoplastic nodules /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% (BRAF v600E)
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

23. 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)
PDC 1 case, 2 slides 0/2 0%
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)

24. 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)

25. 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)
NRAS codon 61 5
3 FC (2 pts)
1 PTC
1 (NNN)
HRAS codon 61
2 FC (2 pts)
2 aPTC (1 tumour, 1 neg LN)
KRAS codon

26. Results as 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

27. Results as 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

28. 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
Mutation-specific targeted therapies?
To summarise our small initial validation study:
Molecular testing in thyroid samples is feasible and is highly likely to improve accuracy/sensitivity of cancer detection.
May reduce the need for diagnostic surgical procedures – reduced costs and less traumatic for patient.
Molecular analysis may have prognostic implications – mutation+ cancers often more aggressive and at higher risk of metastasis.
MAPK-based therapies currently being trialled in thyroid cancers – therefore molecular testing may have a role to play in a manner analogous to the current situation in other cancers e.g. lung.
NB. NPV = 61%.

29. 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

30. 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

31. 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

32. Thankyou for listening