1. Molecular Biologic Markers of Thyroid Cytology
Chan Kwon Jung, MD, PhD
Department of Pathology
THE CATHOLIC UNIVERSITY OF KOREA
Seoul St. Mary’s Hospital
October 22, 2012

2. Thyroid FNA results
Indeterminate
15-30%

3. The Bethesda System for Reporting Thyroid Cytopathology
Category
Risk of Malignancy
I. Nondiagnostic or Unsatisfactory
1-4%
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%

4. Use of molecular biomarkers
Improve the accuracy of fine-needle aspiration cytology
Provide prognostic information

5. Genetic alterations in thyroid cancer
Activating and inactivating somatic mutations,
Alteration in gene expression patterns,
MicroRNA dysregulation
Aberrant gene methylation

6. Thyroid cancer Follicle-derived Papillary carcinoma
Well differentiated carcinoma
Papillary carcinoma
Follicular carcinoma
Poorly differentiated carcinoma
Undifferentiated (Anaplastic) carcinoma

7. PDC AC Papillary Carcinoma Papillary Carcinoma TP53 β-catenin PI3KCA
PTEN
BRAF
RAS
RET/PTC
Follicular cell
TP53
β-catenin
PI3KCA
PTEN
PDC
TP53 AC
RAS
TP53
β-catenin
PI3KCA
PTEN
PAX8-PPARγ
Follicular adenoma
TSHR
Gsα
Follicular adenoma
Follicular Carcinoma
Follicular Carcinoma

8. 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

9. BRAF Val600Glu (V600E) 98–99% of all BRAF mutations
papillary carcinoma
poorly differentiated carcinoma
anaplastic carcinoma
GTG>GAG
c.1799

10. 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

11. Review of all thyroid FNA studies using the BRAF mutation prior to 2009
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 thyroid 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
Mehta V et al. Head Neck 2012 Sep 13. Epub

12. Review of all thyroid FNA studies using the BRAF mutation prior to 2009
15% to 39% of BRAF-positive FNA samples fell into the nondiagnostic or “indeterminate” categories
Several patients with preoperative benign FNA results 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 decrease this false-negative rate.
Mehta V et al. Head Neck 2012 Sep 13. Epub

13. ThinPrep

14. ThinPrep

15. Cell block using ThinPrep

16. Forward
Reverse

17. BRAF mutation test for diagnosis of malignancy in thyroid FNA
Author
Methods
Sensitivity
Specificity
Accuracy
FNA
FNA & BRAF
Kim 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%
92.6%
95.9%
Yeo MK (2011)
Pyrosequencing
71.2%
78.5%
93.9%
95.5%
*Five false positive cases: 1 FA and 4 NH.
Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700
Nam SY et al. Thyroid 2010;20:
Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560

18. 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 ;97:

19. 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.

20. RET/PTC rearrangement
10-20% of papillary thyroid carcinomas
RET/PTC1 and RET/PTC3
Various prevalence and specificity:
Differences in specific age groups and in individuals exposed to ionizing radiation.
Heterogeneous distribution within the tumor
Various sensitivities of the detection methods used.

21. Review of all thyroid FNA studies using the RET/PTC mutation
All RET/PTC positive FNA samples were histologically proven PTCs
No false-positive results
Highly specific biomarker for the diagnosis of PTC

22. RAS mutations
Activating point mutation in codons 12, 13, and 61 of the NRAS, HRAS, and KRAS genes

23. RAS mutations Follicular thyroid neoplasms, both benign and malignant
40-50% of conventional type follicular carcinoma
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

24. 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

25. Molecular testing of FNA samples
Which patients should be tested?
Which biomarkers should be tested?
What is the cost of testing?
How should testing be performed?

26. Single marker test vs Multimarker panels
Korea
Western
PTC Prevalence % %
BRAF (+) rate >80% of PTC % of PTC
Molecular test
BRAF
BRAF, RAS, RET/PTC, PAX8-PPARγ

27. Korean Studies BRAF mutation test for diagnosis of malignancy in thyroid FNA
Sensitivity
Accuracy
Kim SW et al. J Clin Endocrinol Metab 2010;95:3693–3700
Nam SY et al. Thyroid 2010;20:
Yeo MK et al. Clinical Endocrinology 2011; 75, 555–560

28. A study with a panel of mutation analyses
First two passes
Cytologic evaluation
3~4 FNA passes
Indeterminate:
AUS/FLUS
FN/SFN
SMC
Residual material
Molecular analysis:
BRAF, HRAS, NRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPARγ
400 μL nucleic acid preservative solution
Isolation of total nucleic acids
Nikiforov YE, et al. J Clin Endocrinol Metab 2011;96: 3390–7

29. AUS/FLUS (n=212) 14% Thyroid mutation panel
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
AUS/FLUS (n=212)
Cancer risk based on cytology only
14%
Thyroid mutation panel
(BRAF, RAS, RET/PTC, PAX8/PPARγ )
Sensitivity 63%
Specificity 99%
PPV 88%
NPV 94%
Accuracy 94%
Positive
Negative
Cancer risk
88% %
Clinical management
Total thyroidectomy
Lobectomy vs. observation
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:

30. FN/SFN (n=214) 27% Thyroid mutation panel
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
FN/SFN (n=214)
Cancer risk based on cytology only
27%
Thyroid mutation panel
(BRAF, RAS, RET/PTC, PAX8/PPARγ )
Sensitivity 57%
Specificity 97%
PPV 87%
NPV 86%
Accuracy 86%
Positive
Negative
Cancer risk
87% %
Clinical management
Total thyroidectomy
Lobectomy
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:

31. SMC (n=52) 54% Thyroid mutation panel
Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA
SMC (n=52)
Cancer risk based on cytology only
54%
Thyroid mutation panel
(BRAF, RAS, RET/PTC, PAX8/PPARγ )
Sensitivity 68%
Specificity 96%
PPV 95%
NPV 72%
Accuracy 81%
Positive
Negative
Cancer risk
95% %
Clinical management
Total thyroidectomy
Lobectomy
Nikiforov YE, et al. J Clin Endocrinol Metab 2011, 96:

32. Application of tumor specific mRNA/miRNA expression patterns in FNAC diagnosis

33. 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 expression levels for 142 genes.

34. Gene Expression Classifier
A prospective, multicenter validation study involving 49 clinical centers in the USA: 4,812 FNAs from 3789 patients with thyroid nodules ≥1 cm in diameter over a 19-month period
A gene-expression classifier was used to
test 265 indeterminate nodules
Sensitivity %
Specificity 52%
Negative predictive values AUS % Follicular neoplasm 94% Suspicious 85%
N Engl J Med 2012;367:705-15

35. Gene Expression Classifier
Patients with an indeterminate cytology, but benign gene expression classifier test results have a very low risk of cancer.
The test requires two additional needle insertions during FNA biopsy and it is costly.

36. How much does the molecular 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

37. MicroRNA
small RNA sequences (19–25 nucleotides) that function to regulate the expression of genes
regulate around 30% of the human genome
development, apoptosis, cell proliferation, immune response, and hematopoiesis
tumor suppressor genes and oncogenes

38. miRs aberrantly expressed in human thyroid carcinomas of follicular cell origin
Endocrine-Related Cancer (2010) 17 F91–F104

39. - + Summary Bethesda system BRAF or RET/PTC: PTC PAX8/PPARγ: FTC
Nondiagnostic
Benign
AUS/FLUS
Follicular neoplasm
Suspicious for Malignancy
Malignant
Benign -
Somatic mutation +
BRAF or RET/PTC: PTC
PAX8/PPARγ: FTC
RAS: FTC, FA, fvPTC

40. Summary Bethesda system Nondiagnostic Benign AUS/FLUS Benign
Follicular neoplasm
Suspicious for Malignancy
Malignant
Benign
gene expression
Suspicious

41. Thank you for your attention