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Molecular testing of thyroid nodules

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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

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