IMMUNOCYTOCHEMICAL AND MOLECULAR HELP IN THYROID LIQUID BASED CYTOLOGY.

G. Simone, M. Liuzzi, G. Achille, S. Russo, F. Palma, G. Giannone, V. Rubini, C. Quero, G. Grammatica.

NCI “Giovanni Paolo II” - Bari ( Italy)

22nd EUROPEAN CONGRESS OF PATHOLOGY NATIONAL CONGRESS SIAPEC-IAP Florence, September 4-9 2009

INTRODUCTION

Fine Needle Cytology (FNC) is the most important tool in the diagnosis of thyroid nodules.It has been demonstrated that Liquid Based Cytology (LBC) improves the quality of the smears and the diagnostic accuracy, also because of well preserved cellularity useful to immunocytochemical (ICA) or molecular assays (MA). The aim of the study is to verify the potential help of ICA or MA on LBC, when applied on indeterminate or malignant Thyroid FNCs.

MATERIAL AND METHODS

101 Patients who underwent thyroid FNCs entered the study 89 females and 12 males (mean age: 44.7; Fem 46.4;

Males 43); Echography: 72 nodules were single, whereas 29 nodules were the major in multinodular thyroids

The material was processed in Thin Prep 2000 TM.

48 Samples were available for HBME-1 using ICA 48 Different samples were available for BRAF gene mutation (V600E) detection, using MASA technique 5 Samples of the same Patients were available for HBME-1

and BRAF determination 101 FNCs were classified according to SIAPEC classification.

Surgical samples of all FNCs were available for cyto-histological correlation

Patients N.

Females 89

Males 12

Mean Age 44.7

Single Nodule 72

Multinodular 29

Assays N.

FNCs 101

HBME-1 assays 48

BRAF assays 48

HBME-1 and BRAF assays

5

MATERIAL AND METHODS

RESULTS

Out of 101 FNCs, 75 were classified as “Follicular Lesions” (THY 3) and 26 as Papillary Thyroid Carcinoma (PTC). The cyto-histological correlation showed an Overall Agreement (O.A.) of 89.1% (p<0.001) (Tab.1). In 53 LBCs, the prevalence of HBME-1 expression was 45.2%(Tab. 2) and the O.A. with histological diagnosis resulted 87%, versus 91% according to Cytology (Tab 3).In the other 53 cases, the prevalence of BRAF mutation was 24.5% (Tab. 4) and the O.A. with histological diagnosis resulted 87% (p <0.001), the same than according to Cytology (Tab 5).The Positive Predictive Value for PTC was 96.2%, 70.8% and 100% for Cytology, HBME-1/ICA and BRAF/MA, respectively. In 5 cases, where both HBME-1 and BRAF mutation were investigated, 2 PTCs were HBME-1 positive and BRAF mutated whereas, the remaining 3 cases (2 Adenomas and 1 Hyperplastic Nodule) were negative for both the markers.

Table 1. CYTO-HISTOLOGICAL CORRELATION IN 101 THYROID FNCs

Histology

Benign Malignant TOT

Cytology Follicular 65* 10 75

Malignant 1 25 26

TOT 66 35 101

Cytology PPV = 96.2%

O.A.: 89.1%; p < 0.001

*36 Thy 3 resulted in Adenomas and 29 in Hyperplastic Nodules in adenomatous goitre.

Table 2. HBME-1/LBC VERSUS HISTOLOGY: CORRELATION OF 53 CASES

Histology

Benign Malignant TOT

HBME-1/

L B C

Negative 29 0 29

Positive 7* 17 24

TOT 36 17 53

PPV = 70.8%

O.A. : 87%; p < 0.001

*The 7 FNCs resulted in: 2 IPM Follicular Lesions, 2 Adenomas and 3 hyperplastic nodules)

Table 3. CYTO-HISTOLOGICAL CORRELATION OF 53 CASES WITH HBME-1 IMMUNOCYTOCHEMICAL ASSAY

PPV = 92.9%

O.A. = 91%; p<0.001

*1 False Positive case was HBME-1 Negative

Histology

Benign Malignant TOT

Cytology Follicular 35 4 39

Malignant 1* 13 14

TOT 36 17 53

Table 4. BRAF mutation VERSUS HISTOLOGY: CORRELATION ON 53 CASES

Histology

Benign Malignant

TOT

BRAF Wild-type 33 7 40

Mutated 0 13 13

TOT 33 20 53

PPV = 100%

O.A. = 87%; p<0.001

Table 5. CYTO-HISTOLOGICAL CORRELATION OF 53 CASES WITH BRAF DETERMINATION

Histology

Benign Malignant TOT

Cytology Follicular 33 7 * 40

Malignant 0 13** 13

TOT 33 20 53

PPV = 100%

O.A. = 87%; p<0.001

*4/7 cases showed BRAF-mutation;** 4/13 cases resulted wild type

1a 1b

Fig. 1a: Inderterminate Thyroid FNA (See also * in Table 5 ) resulted in papillary cancer at histology, as observed in thin layer (LBC). Fig. 1b: The same case of 1a, as observed in conventional smear (Thin Prep, Cytic Co., Papanicolau Stain, 400x)

Case 1

1c1d

Fig. 1c: Histological control: Papillary Thyroid Cancer (HE 400x)Fig. 1d: BRAF mutation as detected by PCR, Mutant Allele Specific Amplification (MASA) technique: the electrophoretic run

Case 2

PTC (Fig. 2a) and HBME1 Immunoreactivity (Fig. 2b) on LBC and on the corresponding surgical sample (Fig. 2c and 2d)

2a 2b

2c 2d

Case 2: V600E exon 15 BRAF mutation as detected by direct sequencing

Our study showed that Immunocytochemical and molecular assays can be successfully applied on monolayered smears, on well preserved cells obtained by LBC; they could increase diagnostic accuracy in thyroid FNC.However, these markers evidenced some limits in relation to the lower specificity and PPV of HBME1 (Positive also in follicular adenomas) as well as for the low sensitivity of the BRAF mutation.In our experience, the HBME 1 appears to be still usefull in reducing the diagnosis of “ Indeterminated Nodule”.Moreover, in particular follicular lesions, when a diagnosis has to be confirmed, MASA technique showed to be an available tool, according to the absolute PPV of the BRAF mutation in PTC

CONCLUSIONS

22nd EUROPEAN CONGRESS OF PATHOLOGY NATIONAL CONGRESS SIAPEC-IAP Florence, September 4-9 2009