Molecular testing of thyroid nodules

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Molecular testing of thyroid nodules

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

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This talk

• Overview of molecular abnormalities in thyroid
  lesions
• Potential value
• Our own work

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Overview of molecular abnormalities(Nikiforov
YE, Modern Pathology 201124S34-43 Bhaijee F
Nikiforov YE. Endocr Pathol 201122126-133.Nikif
orova MN Nikiforov YE. Thyroid 2009913511361.

• Recent dramatic increase in understanding of
  molecular biology of thyroid cancer
• Main four
• BRAF and RAS point mutations
• RET/PTC and PAX8/PPAR? gene rearrangements
• Others
• PI3K/AKT signalling pathway - PDC
• TP53 and CTNNB1 mutations PDC, ATC
• TRK rearrangement PTC but rare

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Prevalence of mutations
Tumour type Mutation Prevalence
Papillary carcinoma (PTC) BRAF 40-45
Papillary carcinoma (PTC) RET/PTC 10-20
Papillary carcinoma (PTC) RAS 10-20 (usually FVPTC)
Follicular carcinoma (FC) RAS 40-50
Follicular carcinoma (FC) PAX8/PPAR? 30-35
Medullary carcinoma (MTC) Familial germline RET gt95
Medullary carcinoma (MTC) Sporadic somatic RET 40-50

• Nikiforov Arch Pathol Lab Med 2011135569-77
• Bhaijee Nikiforov Endocr Pathol 201122126-33
• Nikiforova Nikiforov Thyroid 200919(12)1351-61

Rivera et al, Modern Pathology 2010231191-2-1200 Follicular variant of PTC (FVPTC) Follicular variant of PTC (FVPTC)
Rivera et al, Modern Pathology 2010231191-2-1200 encapsulated infiltrative
BRAF 0 26
RAS 36 10
RET/PTC 0 10
PAX8/PPAR? 3.5 0
Like FA / FC Like classical PTC
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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

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BRAF - clinical and prognostic valueMelck et al
The Oncologist 2010151285-93 Yip et al.Surgery
20091461215-23 Xing et al J Clin Oncol
2009272977-2982.

• 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 gt65 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

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BRAF diagnostic value in cytologyAdeniran et
al Thyroid 201121(7)717-23. Bentz et al
Otolaryngol Head and Neck Surgery 2009140709-14

• 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
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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

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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

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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

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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

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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

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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

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PAX8/PPAR? gene rearrangement

• Fusion between PAX8 gene and perioxisome
  proliferator-activated receptor (PPAR?) gene
• Causes over-expression of PPAR? 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

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Gene expression profilesBorup et al
Endocr-Related Cancer 201017691-708. Maenhaut
et al Clin Oncol 201123282-288. Ferraz et al
Clin Endocrinol Metab 201196(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

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Review of 20 studies of genetic testing Ferraz
et al Clin Endocrinol Metab 201196(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
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Commercially available kits USA

• Sample in special preservative solution
• ? panel of 7 molecular markers

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Commercially available kits USA

• Sample ? cytopathology ?
• inadequate, benign or malignant report
• indeterminates ? gene expression

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Our own work in Newcastle

• Initial project
• Current BRAF pilot

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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/PPAR? rearrangements (RT-PCR-based assays)
  

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Example data NRAS codon 61
WT CONTROL
CODON 61 (Q61K) CONTROL
WT
WT
Q61K
Q61K
WT
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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

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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)

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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)

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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 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
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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

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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

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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?

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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

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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

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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

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• Thankyou for listening