Molecular Biologic Markers of Thyroid Cytology

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



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%


27%


PositiveNegativ

eCancer risk 87% 14%

FN/SFN (n=214)

Clinical management

Total thyroidecto

myLobectomy



86%



54%


PositiveNegativ

eCancer risk 95% 28%

SMC (n=52)

Clinical management

Total thyroidecto

myLobectomy



81%


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

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Molecular Biologic Markers of Thyroid Cytology