Molecular Biologic Markers of Thyroid Cytology
Chan Kwon Jung, MD, PhD
Department of PathologyTHE CATHOLIC UNIVERSITY OF KOREA
Seoul St. Mary’s Hospital
October 22, 2012
Thyroid FNA results
Benign60%
Malignant10%
Indetermi-nate15-30%
Category Risk of Malignancy
I. Nondiagnostic or Unsatisfactory 1-4%
II. Benign 0-3%
III. Atypia of undetermined significance or Follicular lesion of undetermined significance
~5-15%
IV. Follicular Neoplasm or Suspicious for a Follicular Neoplasm
15-30%
V. Suspicious for Malignancy 60-75%
VI. Malignant 97-99%
The Bethesda System for Report-ing Thyroid Cytopathology
Use of molecular biomarkers
Improve the accuracy of fine-needle aspiration cytology
Provide prognostic information
Genetic alterations in thyroid cancer
Activating and inactivating somatic muta-tions,
Alteration in gene expression patterns,
MicroRNA dysregulation
Aberrant gene methylation
Thyroid can-cer
Follicle-derived
Well differentiated
carcinoma
Papillary carcinoma
Follicular carcinoma
Poorly differentiated
carcinoma
Undifferentiated (Anaplastic)
carcinoma
AC
PAX8-
PPA
Rγ
BRAFRAS
TP53β-cateninPI3KCAPTEN
Follicular cell
RAS
Follicular Carci-noma
Papillary Carci-noma
RET/PTC
PDC
Follicular Carci-noma
Papillary Carci-noma
Follicu-lar ade-noma
Follicu-lar ade-noma
TP53β-cateninPI3KCAPTEN
TP53β-cateninPI3KCAPTEN
TP53
TSHRGsα
BRAF mutations
BRAF is a serine-threonine kinase.
BRAF can be activated by point mutations, small in-frame deletions or insertions, or by chromosomal rearrangement
c.1799
BRAF Val600Glu (V600E)
98–99% of all BRAF mutations
papillary carcinomapoorly differentiated
carcinomaanaplastic carcinoma
GTG>GAG
Prevalence of BRAF mutations in different histologic variants of PTC
Classic papillary Tall cell variant Follicular variant
Review by Xing 60% 80% 10%
Seoul St. Mary’s Hospital
83% 100% 24%
Xing M. Endocr Relat Cancer 2005;12:245-62
Thyroid FNA studies
No. of samples
BRAF (+)
Final diagnosis in BRAF (+) samples
9 prospective studies
1814 159 (8.7%)
PTC = 159 (100%)
7 retrospective studies
685 291 (42.5%)
PTC = 291 (100%)
2 FNA on thy-roid specimens
267 131 (49.1%)
PTC = 130 (99.2%)Hyperplasia* = 1 (0.8%)
Total 2766 581 (21.0%
)
PTC = 580 (99.8%)
* Hyperplasia = atypical nodular hyperplasia
Review of all thyroid FNA studies using the BRAF mutation prior to 2009
Mehta V et al. Head Neck 2012 Sep 13. Epub
Review of all thyroid FNA studies using the BRAF mutation prior to 2009
Mehta V et al. Head Neck 2012 Sep 13. Epub
• 15% to 39% of BRAF-positive FNA samples fell into the nondiagnostic or “indeterminate” categories
• Several patients with preoperative benign FNA re-sults were found to be positive for BRAF mutation, and then confirmed as PTC after surgical removal of the thyroid gland
• The routine use of BRAF testing would further de-crease this false-negative rate.
ThinPrep
ThinPrep
Cell block using ThinPrep
Forward
Reverse
BRAF mutation test for diagnosis of malignancy in thyroid FNA
Author Methods Sensitivity Specificity Accuracy
FNA FNA &
BRAF
FNA FNA &BRAF
FNA FNA &
BRAFKim SW (2010)
DPO-based multiplex PCR
67.5%
89.6%
100%
99.3%*
90.9%
96.6%
Nam SY (2010)
Direct sequencing, allele specific PCR
79.1%
88.4%
100%
100% 92.6%
95.9%
Yeo MK (2011)
Pyrosequencing
71.2%
78.5%
100%
100% 93.9%
95.5%*Five false positive cases: 1 FA and 4 NH.
Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700Nam SY et al. Thyroid 2010;20:273-279Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560
False positive
50 DPO-PCR false positive cases: false positive rate 1.4%; specificity 98.6%
3 MEMO-sequencing false positive cases: false positive rate 0.08%; specificity 99.9%
Lee ST et al. J Clin Endocrinol Metab. 2012 ;97:2299-2306
False positive
• Ultra-sensitive molecular assays with analytical sensitivity <1% should not be used.
• Detection of very low-level mutations can be due to the error introduced during PCR, genetic heterogeneity, and presence of mutation in a very small proportion of cells.
RET/PTC rearrangement 10-20% of papillary thyroid
carcinomas
RET/PTC1 and RET/PTC3
Various prevalence and specificity:
1. Differences in specific age groups and in individuals exposed to ionizing radia-tion.
2. Heterogeneous distribution within the tumor
3. Various sensitivities of the detection methods used.
Review of all thyroid FNA studies using the RET/PTC mutation
All RET/PTC positive FNA samples were his-tologically proven PTCs
No false-positive results Highly specific biomarker for the diagnosis
of PTC
RAS mutations
Activating point mutation in codons 12, 13, and 61 of the NRAS, HRAS, and KRAS genes
Follicular thyroid neoplasms, both benign and ma-lignant
40-50% of conventional type follicular carci-noma
10-15% of oncocytic type follicular carcinoma 10-20% of papillary carcinoma
almost exclusively the follicular variant
30% of conventional type follicular adenoma <10% of oncocytic type follicular adenoma
Detection of RAS mutation indicates the presence of a tumor
RAS mutations
PAX8/PPARγ rearrangement
30-40% of conventional follicular carcinomas <5% of oncocytic carcinomas
2-13% of follicular adenomas: may be preinvasive (in situ) follicular carcinoma, or tumors where invasion was overlooked or not sampled during examination
1-5% of follicular variant of papillary carcinomas
Molecular testing of FNA sam-ples
Which patients should be tested?
Which biomarkers should be tested?
What is the cost of testing? How should testing be per-
formed?
Single marker test vs Multimarker pan-els
Korea Western
PTC Prevalence 95% 80-90%BRAF (+) rate >80% of PTC 30-50% of PTC
Molecular test BRAFBRAF, RAS,
RET/PTC, PAX8-PPARγ
Korean StudiesBRAF mutation test for diagnosis of malignancy in thyroid FNA
Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700Nam SY et al. Thyroid 2010;20:273-279Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560
Kim SW Nam SY Yeo MK
68%
79%71%
90% 88%79%
FNAFNA & BRAF
Kim SW Nam SY Yeo MK
91% 93% 94%97% 96% 96%
FNAFNA & BRAF
Sensitivity Accuracy
First two passes
3~4 FNA passes
400 μL nucleic acid preserva-tive solution
Cytologic evalua-tion
Indetermi-nate: AUS/FLUSFN/SFNSMC
Isolation of total nucleic acids
Molecular analy-sis:BRAF, HRAS, NRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPARγ
Resid-ual ma-terial
Nikiforov YE, et al. J Clin Endocrinol Metab 2011;96: 3390–7
A study with a panel of mutation analyses
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
Cancer risk based on cytology only
14%
Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPARγ )
PositiveNegativ
eCancer risk 88% 5.9%
AUS/FLUS (n=212)
Clinical management
Total thyroidecto
my
Lobectomy vs.
observation
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: 3390-7
Sensitivity 63%Specificity 99%PPV 88%NPV 94%Accuracy
94%
Cancer risk based on cytology only
27%
Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPARγ )
PositiveNegativ
eCancer risk 87% 14%
FN/SFN (n=214)
Clinical management
Total thyroidecto
myLobectomy
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: 3390-7
Sensitivity 57%Specificity 97%PPV 87%NPV 86%Accuracy
86%
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
Cancer risk based on cytology only
54%
Thyroid mutation panel (BRAF, RAS, RET/PTC, PAX8/PPARγ )
PositiveNegativ
eCancer risk 95% 28%
SMC (n=52)
Clinical management
Total thyroidecto
myLobectomy
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96: 3390-7
Sensitivity 68%Specificity 96%PPV 95%NPV 72%Accuracy
81%
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
Application of tumor specific mRNA/miRNA expression pat-terns in FNAC diagnosis
mRNA expression
Microarray studies revealed very distinct changes in the expression of certain genes
No single marker The aim of current approaches is to identify the
minimal number of discriminating genes
Afirma Gene Expression Classifier (Veracyte, South San Francisco, CA) evaluates mRNA ex-pression levels for 142 genes.
Gene Expression Classifier
A gene-expression classifier was used totest 265 indeterminate nodules
• Sensitivity 92%• Specificity 52%• Negative predictive values
AUS 95% Follicular neoplasm 94% Suspicious 85%
N Engl J Med 2012;367:705-15
A prospective, multicenter validation study in-volving 49 clinical centers in the USA: 4,812 FNAs from 3789 patients with thyroid nodules ≥1 cm in di-ameter over a 19-month period
Gene Expression Classifier
Patients with an indeterminate cytology, but benign gene expression classifier test results have a very low risk of can-cer.
The test requires two additional needle insertions during FNA biopsy and it is costly.
How much does the molecu-lar test cost?
In the USA Molecular panel testing (BRAF,
RET/PTC, and RAS): $650 Afirma Gene Expression Classifier:
$4,200 Thyroid surgery: $10,00 to
$15,000
MicroRNA small RNA sequences (19–25 nu-
cleotides) that function to regulate the expression of genes
regulate around 30% of the hu-man genome
development, apoptosis, cell pro-liferation, immune response, and hematopoiesis
tumor suppressor genes and oncogenes
miRs aberrantly expressed in human thyroid carcinomas of follicular cell origin
Endocrine-Related Cancer (2010) 17 F91–F104
Bethesda system
Nondiagnostic
Benign
AUS/FLUS
Follicular neoplasm
Suspicious for Malignancy
Malignant
Summary
Somatic muta-tion
Benign
-
+
BRAF or RET/PTC: PTC
PAX8/PPARγ: FTC
RAS: FTC, FA, fvPTC
Bethesda system
Nondiagnostic
Benign
AUS/FLUS
Follicular neoplasm
Suspicious for Malignancy
Malignant
Summary
gene ex-pression
Benign
Suspi-cious
Thank you for your atten-tion