maria chiara zatelli sezione di endocrinologia dip. di scienze biomediche e terapie avanzate...

Maria Chiara ZatelliMaria Chiara ZatelliSezione di EndocrinologiaSezione di Endocrinologia

Dip. di Scienze Biomediche e Terapie AvanzateDip. di Scienze Biomediche e Terapie Avanzate Università di FerraraUniversità di Ferrara

Direttore: Prof. Ettore degli UbertiDirettore: Prof. Ettore degli Uberti

Analisi genetica del nodulo Analisi genetica del nodulo tiroideo:tiroideo:

implicazioni per l’esecuzione del implicazioni per l’esecuzione del linfonodo sentinellalinfonodo sentinella

Ferrara, 20 gennaio 2012Ferrara, 20 gennaio 2012

……molecular marker…?molecular marker…?

……a molecular marker (genetic marker) is a DNA a molecular marker (genetic marker) is a DNA sequence associated to a part of the genome, used in sequence associated to a part of the genome, used in molecular biology and biotechnology experiments to molecular biology and biotechnology experiments to identify a very highly specific DNA sequence…identify a very highly specific DNA sequence…

What relevance in thyroid nodules?What relevance in thyroid nodules?

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Fine-needle aspiration biopsy (FNAB) is Fine-needle aspiration biopsy (FNAB) is currently the most sensitive and specific currently the most sensitive and specific

tool tool for the presurgical differential diagnosis of for the presurgical differential diagnosis of

thyroid malignancythyroid malignancy

butbut

has also substantial limitationshas also substantial limitationsEszlinger et al. Mol Cell Endocrinol 2010;322:29

thyroid surgerythyroid surgery

75% benign 75% benign lesions lesions

5% malignant 5% malignant lesionslesions

20% follicular proliferation 20% follicular proliferation

FAFA FTCFTC fvPTCfvPTC

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

MOLECULAR BIOLOGYMOLECULAR BIOLOGY

study of biology at molecular levelstudy of biology at molecular level

pathologypathology

Does it improve Does it improve cytological fine needle cytological fine needle aspiration diagnosis of aspiration diagnosis of thyroid nodules?thyroid nodules?

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

RNARNA DNADNA

cytologycytology

somatic somatic mutation mutation analysisanalysis

rearrangemenrearrangement studiest studies

BRAFBRAFN-RASN-RASH-RAS H-RAS K-RASK-RAS

RET/PTC1 RET/PTC1 RET/PTC3 RET/PTC3

PPARPPAR/PAX8/PAX8

Highly feasible Highly feasible in clinical in clinical settingssettings

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

PAPILLARY CARCINOMAPAPILLARY CARCINOMA

DNADNA

BRAF V600E point mutationBRAF V600E point mutation

[K601E and V599Ins][K601E and V599Ins]

45-80% of PTC, mainly tall cell and 45-80% of PTC, mainly tall cell and classic hystology classic hystology

extrathyroidal invasion extrathyroidal invasion

higher stagehigher stage

recurrence (with reduced I up-take)recurrence (with reduced I up-take)

de-differentiationde-differentiation

resticted to PTCresticted to PTCNikiforova et al. Exp Rev Mol Diagn 2008, 8: 83

Lupi et al. J Clin Endocrinol Metab. 2007;92:4085

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

• do they improve diagnosis?do they improve diagnosis?genetic tests genetic tests

Kim et al. J Clin Endocrinol Metab, 2011, 96:658

BRAFV600E BRAFV600E molecular molecular

testtestCytologyCytology BRAFBRAF Cytology + BRAFCytology + BRAF

SensitivitySensitivity 100100 77.377.3 89.689.6 64.064.0 89.689.6 86.786.7

SpecificitySpecificity 36.436.4 98.898.8 95.595.5 100100 95.595.5 98.898.8

PPVPPV 92.992.9 92.192.1 99.499.4 100100 99.499.4 92.992.9

NPVNPV 100100 95.995.9 52.552.5 93.793.7 52.552.5 97.597.5

AccuracyAccuracy 93.393.3 95.495.4 90.290.2 95.495.4 90.290.2 96.996.9

K valueK value 0.510.51±±0.0.1111

0.810.81±±0.00.022

0.630.63±±0.00.077

0.760.76±±0.00.055

0.630.63±±0.00.077

0.880.88±±0.010.01

Zatelli et al . Eur J Endocrinol 2009, 161:467

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Kim et al. J Clin Endocrinol Metab, 2011, 96:658

• do they improve diagnosis?do they improve diagnosis?genetic tests genetic tests

BRAFV600E BRAFV600E molecular testmolecular test

BRAF mutation analysis BRAF mutation analysis may help especially in may help especially in

small nodulessmall nodulesEFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Section of EndocrinologySection of EndocrinologyUniversity of FerraraUniversity of Ferrara

(1436 (1436 ♀♀; ; 420420 ♂♂))age 52 age 52 ±± 0,32 (12-73) 0,32 (12-73)

2421 FNAs2421 FNAs

Biomolecular Biomolecular analysisanalysis

PathologyPathology

US & clinical evaluationUS & clinical evaluation

1856 pz1856 pz

suspected: 829suspected: 829

NON suspected: 1529NON suspected: 1529

BRAFV600E BRAFV600E molecular molecular

testtest

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

cytologycytology SS %% NSNS %%

tottot 829829 34,234,2 15921592 65,765,7

Malignant Malignant raterate 115115 13,913,9 103103 6,56,5

PTC/PTC/malignantmalignant 112112 97,497,4 9898 95,195,1

BRAF +BRAF + 6464 7,77,7 5050 3,13,1

BRAF +/BRAF +/MalignantMalignant

64/1164/1155

55,755,7 50/10350/103 48,548,5

FNAB per se detects FNAB per se detects a high percentage of a high percentage of malignant lesions in malignant lesions in unsuspected nodulesunsuspected nodules

Martina Rossi et al. submitted

All nodules should undergo FNABAll nodules should undergo FNAB

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

BRAFV600E BRAFV600E molecular testmolecular test CytologyCytology BRAFBRAF Cytology + Cytology +

BRAFBRAF

   SS NSNS SS NSNS SS NSNS

SensitivitySensitivity 76,76,88

69,469,4 56,656,6 5151 92,92,99

84,784,7

SpecificitySpecificity 99,99,77

99,999,9 100100 100100 99,99,77

99,999,9

PPVPPV 97,97,77

98,698,6 100100 100100 98,98,11

98,898,8

NPVNPV 96,96,55

9898 93,693,6 96,996,9 98,98,99

9999

AccuracyAccuracy 96,96,66

98,198,1 94,194,1 9797 98,98,88

9999

BRAF testing BRAF testing

significantly significantly

increases FNAB increases FNAB

sensitivity also in sensitivity also in

nodules clinically nodules clinically

non suspectednon suspected

BRAFV600E molecular test increases BRAFV600E molecular test increases diagnostic sensitivity for PTCdiagnostic sensitivity for PTC

15 PTC patients “rescued” 15 PTC patients “rescued” by BRAF analysisby BRAF analysis

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Martina Rossi et al . submitted

    BRAFV600E BRAFV600E molecular molecular testtest

SS %% NSNS %%

ACUSACUS 2266

3,13,1 7878 4,94,9

PTCPTC 66 75,075,0 1010 37,37,00

BRAF +BRAF + 55 19,219,2 00 0,00,0

FNFN 3355

4,24,2 6262 1,21,2

PTCPTC 55 25,025,0 9923,23,77

BRAF +BRAF + 22 5,75,7 66 9,79,7

BRAF testing identifies BRAF testing identifies

as malignant 10% of FNas malignant 10% of FN

Indication to total Indication to total thyroidectomythyroidectomy

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Martina Rossi et al . submitted

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Thyroid molecular markers increase Thyroid molecular markers increase diagnostic sensitivity of cytology for diagnostic sensitivity of cytology for

PTCPTCandand

influence surgical approachinfluence surgical approach

CONCLUSION -1CONCLUSION -1

• any prognostic any prognostic value?value?

genetic tests genetic tests

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Xing et al. J Clin Oncol. 2009;27:2977-82

BRAFV600E BRAFV600E molecular testmolecular test

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

BRAFV600E BRAFV600E molecular testmolecular test

PTCPTCpersistence/recurrence predictionpersistence/recurrence prediction

All PTCAll PTC Conventional Conventional PTCPTC

sensitivity sensitivity 68%68% 79%79%

specificity specificity 66%66% 60%60%

PPVPPV 36%36% 34%34%

NPVNPV 88%88% 92%92%Xing et al. J Clin Oncol 2009; 27:2977-82

BRAF mutation–positive patients BRAF mutation–positive patients are are significantly more likely to have PTC significantly more likely to have PTC

persistence/recurrencepersistence/recurrence

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

significantly reduced disease-free probability significantly reduced disease-free probability in BRAF+ patientsin BRAF+ patients

Xing et al. J Clin Oncol. 2009;27:2977-82

BRAF V600E genetic testBRAF V600E genetic test

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

significantly increased mortalitysignificantly increased mortalityin BRAF+ patientsin BRAF+ patients

Elisei et al. J Clin Endocrinol Metab. 2008;93:3943

BRAF V600E genetic testBRAF V600E genetic test

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Thyroid molecular markers may predict Thyroid molecular markers may predict patients outcomepatients outcome

CONCLUSION -2CONCLUSION -2

• any surgical any surgical relevance ?relevance ?

genetic tests genetic tests

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

BRAF mutationBRAF mutation

0

10

20

30

40

50

60

BRAF (+)34

BRAF (-)69

n.

of

PTC

LN met (+) LN met (-)

20/26 BRAF+ LN

is a significant risk is a significant risk factor for locoregional factor for locoregional lymph node metastasislymph node metastasis

Kim et al. Ann Surg 2006;244: 799

is not necessary for is not necessary for locoregional lymph node locoregional lymph node

metastasismetastasis

lymph node metastases lymph node metastases may show may show de novode novo

mutationsmutations

Vasko et al. J Clin Endocrinol Metab 2005; 90: 5265

103 PTC

!! also in microPTC !!!! also in microPTC !!Lin et al. Ann Surg Oncol 2010;17:3294

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

extrathyroidal invasionextrathyroidal invasionlymph node lymph node metastasismetastasis

local neck recurrence local neck recurrence PTC recurrencePTC recurrencecomplicationscomplications

surgical strategysurgical strategylobectomylobectomy

total thyroidectomytotal thyroidectomy

lymph node dissectionlymph node dissection no dissectionno dissection

BRAF mutationBRAF mutation

High prognostic impactHigh prognostic impact

Xing Endocrine Reviews 2007; 28: 742

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

BRAF mutationBRAF mutation

Indication for sentinel lymphnodeIndication for sentinel lymphnode

Yip et al Surgery 2009; 146: 1215

PREDICTOR OF LYMPHNODE METASTASESPREDICTOR OF LYMPHNODE METASTASES

decision decision makingmaking

BRAFV600E BRAFV600E molecular testmolecular test more aggressive more aggressive

surgerysurgery

neck lymph node neck lymph node dissectiondissection

less less aggressive aggressive

surgerysurgery

+-

BRAF mutation-basedBRAF mutation-based

risk stratificationrisk stratification

PTC managementPTC management

Xing et al. J Clin Oncol. 2009;27:2977-82

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

EFE 2012EFE 2012

Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

Positivity of thyroid molecular markers Positivity of thyroid molecular markers as indication for sentinel lymphnode as indication for sentinel lymphnode

procedureprocedure

CONCLUSION -3CONCLUSION -3

May address patients with May address patients with

persistent/recurrent disease persistent/recurrent disease

to therapy withto therapy with

BRAF-specific inhibitorsBRAF-specific inhibitors

Cantwell-Dorris et al. Mol Cancer Ther 2011, 10: 385

BRAF V600E genetic testBRAF V600E genetic test

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

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Thyroid molecular markers & sentinel lymphnodeThyroid molecular markers & sentinel lymphnode

CONCLUSIONSCONCLUSIONS

increase cytology diagnostic sensitivity for PTC increase cytology diagnostic sensitivity for PTC

may predict patients outcomemay predict patients outcome

influence surgical approachinfluence surgical approach

their positivity is an indication for sentinel lymphnode their positivity is an indication for sentinel lymphnode

procedureprocedure

Thyroid molecular markers Thyroid molecular markers

THANKSTHANKS

Ettore degli UbertiEttore degli Uberti

Section of EndocrinologySection of EndocrinologyDept. of Biomedical Sciences and Advanced TherapiesDept. of Biomedical Sciences and Advanced Therapies

University of FerraraUniversity of Ferrara

Laboratorio di Laboratorio di Fisiopatologia Fisiopatologia

EndocrinaEndocrina

Ambulatorio Ambulatorio Ecografia/Ecografia/

AgoaspiratiAgoaspirati

Personale Personale MedicoMedicoTecnicoTecnico

InfermieristicoInfermieristico

Assegnisti di Assegnisti di ricercaricerca

DottorandiDottorandiStudentiStudenti

DegenzaDegenzaDay HospitalDay HospitalAmbulatoriAmbulatori