Dr Sarah J Johnson Consultant Cyto/histopathologist Newcastle upon Tyne.

Dr Sarah J JohnsonConsultant Cyto/histopathologistNewcastle upon Tyne

This talk

Overview of molecular abnormalities in thyroid lesions

Potential value Our own work

Overview of molecular abnormalities(Nikiforov YE, Modern Pathology 2011;24:S34-43; Bhaijee F & Nikiforov YE. Endocr Pathol 2011;22:126-133.

Nikiforova MN & Nikiforov YE. Thyroid 2009;9:1351 1361.

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 mutationsTumour type Mutation Prevalence %

Papillary carcinoma (PTC) BRAF 40-45

RET/PTC 10-20

RAS 10-20 (usually FVPTC)

Follicular carcinoma (FC) RAS 40-50

PAX8/PPARγ 30-35

Medullary carcinoma (MTC) Familial – germline RET >95

Sporadic – somatic RET 40-50

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;23:1191-2-1200

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 valueMelck et al The Oncologist 2010;15:1285-93; Yip et al.Surgery 2009;146:1215-23; Xing et al J Clin Oncol 2009;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 cytologyAdeniran et al Thyroid 2011;21(7):717-23. 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

Indeterminate cytology BRAF test

positive

negative

Total thyroidectomy +/ level VI LNs

Diagnostic hemithyroidectomy

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

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 profilesBorup et al Endocr-Related Cancer 2010;17:691-708. Maenhaut et al Clin Oncol 2011;23:282-288. Ferraz et al Clin Endocrinol Metab 2011;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 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

miRNA

Malignancy risk down from 20%

to 8-10%

Cohort with 3% malignancy

risk

?follow up with USS + repeat FNA

Commercially available kits – USASample in special preservative solution

→ panel of 7 molecular markers

Commercially available kits – USASample → cytopathology →

inadequate, benign or malignant report indeterminates → gene expression

Our own work in Newcastle

Initial project

Current BRAF pilot

Initial project – BSCC presentation 2011S. 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

Q61KWT

WT CONTROL CODON 61 (Q61K) CONTROL

WT WTQ61K

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% (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

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)

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 mutations13 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 outcomesmutation 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)2 aPTC (1 tumour, 1 neg LN)

0

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

•

• Mutation-specific targeted therapies?

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