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Yuri E. Nikiforov, MD, PhDDivision of Molecular & Genomic PathologyUniversity of Pittsburgh Medical Center
The role of Molecular Markers in Thyroid Nodule Management:
Then and Now
Disclosures
• Quest Diagnostics (consultant)• Service agreement between UPMC and
CBLPath to offer ThyroSeq for commercial use
Progress in Identifying Driver Mutations in Thyroid Cancer
Beginning of the molecular era…
Genetic Basis of Thyroid Cancer
Major pathways involved: MAPK, PI3K Main mutation mechanisms: Point
mutations and chr. rearrangements
Genomic Landscape of PTC
The Cancer Genome Atlas Research Network. Cell 159:676-690 (2014)
74%
15%
9%
Point mutations
Gene fusions
Copy number variations
Driver mutations define carcinogenic mechanisms BRAFV600E-like and RAS-like scores
The Cancer Genome Atlas Research Network. Cell 159:676-690 (2014)
Thyroid Differentiation Status in PTC
The Cancer Genome Atlas Research Network. Cell 159:676-690 (2014)
Lawrence MS et al. Nature. 2013 Jul 11; 499(7457): 214–218
Frequencies of Somatic Mutation in Different Tumors
Molecular Markers for Cancer Diagnosis
Diagnostic Category Risk Usualof cancer management
I. Nondiagnostic or Unsatisfactory 1‐4% Repat FNA with US
II. Benign 0‐3% Clinical follow‐up
III. Atypia of Undetermined Significance 5‐15% Repeat FNAor Follicular Lesion of UndeterminedSignificance (AUS/FLUS)
IV. Follicular Neoplasm or Suspicious 15‐30% Surgical lobectomyfor a Follicular Neoplasm (FN/SFN)
V. Suspicious for malignancy 60‐75% Total or lobectomy
VI.Malignant 97‐99% Total thyroidectomy
Baloch ZW et al. Diagn Cytopathol 2008;36:425Cibas ES & Ali SZ. Am J Clin Pathol 2009;132:658
The Bethesda System for Reporting Thyroid Cytopathology
• Gene mutations
• Gene expression (mRNA)
• Circulating TSHR mRNA
• miRNAs
• Proteomics
• Combination of marker types
Molecular Markers for Thyroid Cytology
Progress in Composition of Gene Mutation Panels
Single genes
Conventional sequencing
7-gene panel
Multi-gene panel
Next-Generation sequencing
Conventional sequencing
• Meta‐analysis of 47 studies• 9,924 FNA tested for BRAF V600E• Specificity – ~100%• Sensitivity – 52% (95%CI 39%‐64%)
Single gene tests - BRAF V600E
High specificity/PPV, very low sensitivity/NPV – good “rule in” test
J Clin Endocrinol Metab 94: 2092–2098, 2009
Small (7-gene) panels
Gene Fusions (RNA)
RET/PTC1
RET/PTC3
PAX8/PPARG
Gene Mutations (DNA)
BRAF
NRAS HRAS
KRAS
none
Small (7-gene) panels
High specificity/PPV, low sensitivity/NPV – good “rule in” test
Nishino M. Cancer Cytopathol 2016;124:14-27
Afirma Gene Expression Classifier (GEC)
N Engl J Med 2012; 367:705-715
Afirma Gene Expression Classifier
Alexander E et al. N Engl J Med (2012)
• Multi-institutional double-blind prospective study of indeterminate cytology FNA samples
• Sample size – 265 FNAs
Cytologicdiagnosis
n Sensitivity Specificity NPV PPV
AUS/FLUS 129 90% 53% 95% 38%
FN/SFN 81 90% 49% 94% 37%
SUSP 55 94% 52% 85% 76%
High sensitivity/NPV, low specificity/PPV – good “rule out” test
Afirma Gene Expression Classifier
Genomic Revolution:Next Generation (Deep) Sequencing
Next Generation Sequencing
• Sequence up to 106-109 bases• Cost - $0.05-1 per 106 bases• Sensitivity - 3-5% of mutant allele
Conventional Sequencing
• Sequence up to 102-103 bases• Cost - $2400 per 106 bases• Sensitivity - 20-30% of mutant allele
Expansion of Molecular Panels Using Next-Gen Sequencing
PAX8/PPARγ
RASRET/PTC1RET/PTC3
BRAF
RET
PTEN
BRAF other
TSHRPIK3CA
AKT1
CTNNB1
TP53
13%
Novel mutations(eg. TERT, EIF1AX)
Novel gene fusions(eg. ALK, NTRK3)12%
90%65%
ThyroSeq® v.2 NGS Mutation Panel
Gene Fusions(mRNA) RETPPARG
NTRK1
NTRK3
BRAF
ALK
Other
Gene Mutations(DNA)BRAF RET
NRAS TSHR
HRAS AKT1
KRAS TP53
PIK3CA GNAS
PTEN CTNNB1
TERT EIF1AX
• 14 genes for mutations; 42 fusion types; 16 genes for expression
Gene expression(mRNA)PGK1KRT7TGTTF1NISCalcitonin
PTH
KRT20
Other
– pan-cell marker
Thyroid epithelial cells
– MTC– parathyroid
– metastatic
• 465 consecutive FNA samples with AUS/FLUS (Bethesda III) cytology at UPMC from May 2014‐March 2015
• Prospective study• Surgical outcome known for 96 patients
• Cancer prevalence after surgery – 22.5%
Nikiforov et al. Thyroid 2015;25:1217-23
ThyroSeq v2 Performance in AUS/FLUS (Bethesda III) Cytology Nodules
Sensitivity 91% (CI: 78-100%)Specificity 92% (CI: 86-98%)NPV 97% (CI: 93-100%)PPV 77% (CI: 61-93%)Accuracy 92% (CI: 86-97%)
Nikiforov et al. Thyroid 2015;25:1217-23
High specificity/PPV and high sensitivity/NPV – good “rule in” and “rule out” test
Cancer 2014, 120:3627-34
• Patients with FN/SFN (Bethesda IV) cytology and known surgical outcome seen at UPMC from Oct. 2013‐May 2014
• Retrospective and prospective groups
• Sample size: 143 consecutive FNA samples
• Cancer prevalence after surgery – 39/143 (27.3%)
143 consecutive FN/SFN nodules with surgery
Overall test performance
Retrospective groupn=91
Mutation NEGATIVE
n=64
Mutation POSITIVE
n=27
CANCERn=2
BENIGNn=62
BENIGNn=4
Sensitivity 92%Specificity 94%PPV 85%NPV 97%Accuracy 93%
CANCERn=23
Prospective groupn=52
Mutation NEGATIVE
n=37
Mutation POSITIVE
n=15
BENIGNn=35
BENIGNn=3
Sensitivity 86%Specificity 92%PPV 80%NPV 95%Accuracy 90%
CANCERn=2
CANCERn=12
Sensitivity 90% (CI: 80-99%)Specificity 93% (CI: 88-98%)NPV 96% (CI: 92-95%)PPV 83% (CI: 72-95%)
Accuracy 92% (CI: 88-97%)
Nikiforov et al. Cancer 2014,120:3627-34
ThyroSeq v2 NPV
Afirma NPV
Afirma PPV
ThyroSeq v2 PPV
Nikiforov et al. Thyroid 2015;25:1217-23
Test Performance in AUS/FLUS (Bethesda III) Cytology Nodules
ThyroSeq v2
Afirma
*Unless AF>30%
BRAFALKNTRK1,3PPARGRET/PTCTERTTP53
NRASHRASKRAS
PTENEIF1AX
TSHRGNAS
Cancer risk >95% 80% 20‐40% Very low*
Cancer risk associated with individual mutations
Cancer Risk in Nodules with RAS Mutations
RASmutation‐positive “benign” nodules
79%
Evidence for clonal neoplasm and early transformation to cancer
Nikiforov et al. Thyroid 2015;25:1217-23
Cancer Risk in Nodules with RAS Mutations
Medicine 2015;94:1-6
Total FNA samples 198
Total RAS+ 31
RAS+ Cancer 7 (23%)
RAS+ Benign 24 (77%)
BioMed Res Int 2015;2015:697068
Total FNA samples 132
Total RAS+ 27
RAS+ Cancer 26 (96%)
RAS+ Benign 1 (4%)
JCEM 2013;98:E829
all RAS 40%
J Clin Endocrinol Metab 100: 2743–2750, 2015
• Combination of 7-gene mutation panel (miRInform) and miRNA expression classifier based on 10 miRNA genes (ThyraMIR)
• Validated in 109 FNA samples with AUS/FLUS and FN/SFN cytology• Cancer prevalence 32%• Multi-center study (12 endocrinology centers)
J Clin Endocrinol Metab 100: 2743–2750, 2015
Performance of combined 7-gene mutation panel (miRInform) and expression of 10 miRNA genes test
Cancer Cytopathol 2016;124:14-27
Nishino M. Cancer Cytopathol 2016;124:14-27
*Depending on the type of mutation Nikiforov et al. Cancer 2014, 120:3627-34Nikiforov et al. Thyroid 2015, 25:1217-23Data on file; UPMC MGP lab
FNA
FLUS (BC III)
FN/SFN(BC IV)
SUSP(BC V)
Cancer Risk 10‐15% 50‐70%
ThyroSeq v2 ‐ +
Therapeutic Surgery
Modified Cancer Risk 3% 75‐99%*
Cytology
20‐30%
Observation
‐ +
Therapeutic Surgery
4% 75‐99%*
Observation
‐ +
Therapeutic Surgery
20% 90‐99%*
LobectomyRecommended Management
Cancer risk and proposed patient management based on combination of cytology and ThyroSeq test
Molecular Markers for Cancer Prognostication
Yip et al. Ann Surg 262:519‐25 (2015)
Mean follow‐up 33 ± 21.2 months
BRAF V600E status and tumor recurrence and mortality
Gene Panels for Tumor Prognostication
Xing M et al. JAMA (2013)Xing M et al. JCEM (2005) Kim TY et al. Clin Endocrinol(2006)
N=219 N=203
All Variants of PTC included Conventional PTC Only All Patients with PTC
N=1849
BRAF V600E mutation
N Sensitivity Specificity NPV PPVMortalityTufano et al. Medicine (2012)
2167 83% 51% 99% 5%
RecurrenceXing M al. JAMA (2013)
1849 66% 54% 87% 25%
Thyroid Cancer Prognostication Aggressive Tumors Have Multiple Mutations
Ricarte-Filho JC et al. Cancer Res (2009)
Papillary Carcinomas
Nikiforova M et al. JCEM (2013)
n=57BRAFNRASHRASKRAS
PIK3CATSHRTP53
0 20 40
• 469 patients with FCDTC• Mean follow‐up 7.8±5.8 years
Melo M et al. JCEM (2014)
Aggressive Tumors Have TERT Mutations
Follow‐up (years)
FCDTC Papillary Carcinoma Follicular Carcinoma
Cum Survival
TERT wt TERT wtTERT wt
TERT mutTERT mut TERT mut
Univariateanalysis
Multivariate analysis
Univariateanalysis
Multivariate analysis
Presence (%) OR (95%CI) P value OR (95%CI) P value Presence (%) OR (95% CI) P value OR (95%CI) P value
TERT wt 34 (12.5) 1 <0.001 1 0.002 48 (24.6) 1 0.001 1 0.007
TERT mut 10 (43.5) 5.36 (2.18‐13.18)
4.60 (1.73‐12.21)
10 (62.5) 5.10 (1.76‐14.78)
4.68 (1.54‐14.27)
Thyroid Cancer Prognostication
Xing M. et al. JCO (2014)
PTC (all types) PTC (conventional type)
BRAF+TERTBRAF+TERT
III. Aggressive Tumors Have TERT MutationsThyroid Cancer Prognostication
Molecular Signature
• BRAF + TERT• Multiple driver mutations (eg. BRAF and PIK3CA)
• TP53• TERT
• ALK fusions• NTRK1 fusions• NTRK3 fusions• BRAF V600E
• RET/PTC • RAS• PTEN• BRAF K601E• PAX8/PPARG
Risk of Recurrence
Preoperative cancer risk stratification based on molecular profiling
High Risk
Low Risk
Intermediate Risk
Case #170 yo F with 1.2 cm nodule
FNA Cytology: • Atypia of Undetermined significance
Case #474 yo F with 3.7 cm nodule
Case #262 yo F with 2.1 cm nodule
Case #341 yo M with 1.6 cm nodule
Case #1: 70 year-old woman with incidentally noted 1.2 cm thyroid nodule
Cytology: Satisfactory/FLUS• Cancer risk 5‐15%
Left lobe normal, no risk factors
Molecular Testing:
Observation:Neck exam and ultrasound stable at 5 years follow‐up
ThyroSeq v2 (2014)No mutations identified• Cancer risk ‐ 3%
7‐gene panel (2010)No mutations identified• Cancer risk ‐ 6%
Case #2: 62 year-old woman with 2.1 cm thyroid nodule
Total thyroidectomy:PTC with extrathyroidal extension
Cytology:Follicular lesion of undetermined significance (FLUS) (Bethesda III) due to low cellularity • Cancer risk 5‐15%
ThyroSeq:BRAF V600E mutation is identified(9% AF/ 18% cells with mutation)• Cancer risk ~99%
Case #3: 41 yo male with left inferior pole thyroid nodule
Cytology:Groups of microfollicles with atypia (Bethesda III‐IV)• Cancer risk for FLUS 5‐15%, FN – 20‐30%
Parathyroid exploration, left thyroidectomy:Parathyroid adenoma, left inferior, intrathyroidal
US: 1.6 x 1.2 x 1.2 cm predominantly solid, isoechoic nodule in left inferior pole, not convincingly border forming
ThyroSeq:‐Mutations NOT identified‐ High expression (92%) of the parathyroid hormone (PTH) gene DETECTED
• Strong evidence for parathyroid tissue
US: Solitary 3.7 x 2.6 x 2.8 cm, solid, isoechoic, hypervascular, circumscribed nodule with calcifications
Cytology: Hurthle cell nodule (Bethesda III/IV)Cancer risk for FLUS 5‐15%, FN – 20‐30%
Case #4: 74 yo female with solitary right lobe nodule, recently increased in size
ThyroSeq:Positive for NRAS (Q61R); TP53 (R175H); PIK3CA (E545K) mutations
Total thyroidectomy:Oncocytic follicular carcinoma with capsular and multifocal vascular invasion (3 foci)
Case #170 yo F with 1.2 cm nodule
FNA Cytology: • Atypia of Undetermined significance
Case #474 yo F with 3.7 cm nodule
Case #262 yo F with 2.1 cm nodule
Case #341 yo M with 1.6 cm nodule
Observation Therapeutic surgery
Dx of parathyroid disease
Therapeutic surgery Risk prediction
Precision MedicinePrecision Medicine refers to the tailoring of medical treatment to the individualcharacteristics of each patient. It does not literally mean the creation of drugs ormedical devices that are unique to a patient, but rather the ability to classifyindividuals into subpopulations that differ in their susceptibility to a particulardisease, in the biology and/or prognosis of those diseases, or in their response to aspecific treatment. (National Research Council)
AcknowledgementsNikiforov’s Research LaboratoryLindsey KellyRebecca Leeman-Neill Chan Kwon JungViktoria EvdokimovaManoj GandhiRaffaele Ciampi
University of Pittsburgh PathologyMarina NikiforovaPaul OhoriRaja Seethala
Endocrine SurgerySally CartyLinwah YipMichael Stang (now at Duke)Kelly McCoy
Head and Neck SurgeryRobert FerrisUmamaheswar Duvvuri
EndocrinologyShane LeBeauStephen Hodak (now at NYU)Christopher Coyne
RadiologyMitchell Tublin
PharmacologyDanny Altschuler
Grants/Research support:• NIH, ACS, University of Pittsburgh Cancer Institute• Philanthropic support from grateful patients