1. 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

2. 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.

3. 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

4. PatientsN. Females89 Males12 Mean Age44.7 Single Nodule72 Multinodular29 AssaysN. FNCs101 HBME-1 assays48 BRAF assays48 HBME-1 and BRAF assays 5 MATERIAL AND METHODS

5. 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.

6. Table 1. CYTO-HISTOLOGICAL CORRELATION IN 101 THYROID FNCs Histology BenignMalignantTOT CytologyFollicular65*1075 Malignant12526 TOT6635101 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.

7. Table 2. HBME-1/LBC VERSUS HISTOLOGY: CORRELATION OF 53 CASES Histology BenignMalignantTOT HBME-1/ L B C Negative290 Positive7*1724 TOT361753 PPV = 70.8% O.A. : 87%; p < 0.001 *The 7 FNCs resulted in: 2 IPM Follicular Lesions, 2 Adenomas and 3 hyperplastic nodules)

8. 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 BenignMalignantTOT CytologyFollicular35439 Malignant1*1314 TOT361753

9. Table 4. BRAF mutation VERSUS HISTOLOGY: CORRELATION ON 53 CASES Histology BenignMalignant TOT BRAFWild-type33740 Mutated013 TOT332053 PPV = 100% O.A. = 87%; p<0.001

10. Table 5. CYTO-HISTOLOGICAL CORRELATION OF 53 CASES WITH BRAF DETERMINATION Histology BenignMalignantTOT CytologyFollicular337 *40 Malignant013**13 TOT332053 PPV = 100% O.A. = 87%; p<0.001 *4/7 cases showed BRAF-mutation; ** 4/13 cases resulted wild type

11. 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

12. 1c 1d 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

13. Case 2 PTC (Fig. 2a) and HBME1 Immunoreactivity (Fig. 2b) on LBC and on the corresponding surgical sample (Fig. 2c and 2d) 2a 2b2c 2d

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

15. 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