Utilitàdelle tecniche di biologia molecolare nella diagnosi delle neoplasie delle ghiandole salivari

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1 Utilitàdelle tecniche di biologia molecolare nella diagnosi delle neoplasie delle ghiandole salivari Scuola Nazionale di Surgical Pathology Corso di Patologia del Distretto Testa-Collo Bologna, Febbraio 2015 Ospedale Bellaria, Aula Magna Padiglione Tinozzi

2 Ibridazione in situ fluorescente (FISH) La FISH (Fluorescence in situ hybridization) èuna tecnica che utilizza sonde marcate con fluorocromi per evidenziare geni, cromosomi o parte di essi.

3 FISH Localizzazione di geni ed il rilevamento morfologico diretto di difetti o anomalie genetiche in patologie ereditarie Analisi sia su campioni ematologici che tumori solidi. Verifica di aneuploidie Trisomia 21, neoplasie) diagnosticamente importanti (es. Studio delle metafasi mediante colorazione dei cromosomi - Cariotipizzazione

4 Materiale adatto per la FISH Materiale fresco 1. Colture cellulari 2. Materiale congelato 3. Sezione da paraffina (max. 5µm) 4. Nuclei isolati da paraffina

5 La sonda o probe : Èun frammento di DNA o RNA che viene marcato. La sonda èdata da sequenze di DNA clonato, DNA genomico, prodotti di PCR e oligonucleotidi sintetici. La lunghezza ideale da 50pb in su. Cosa può marcare una sonda: Marcatura Telomero gene Marcatura centromero Marcatura del gene e eventuale amplificazione del segnale Marcatura Telomero Whole painting

6 Applicazione FISH nelle ghiandole salivari J Oral Pathol Med. 1995Oct;24(9): A fluorescence in situ hybridization (FISH) analysis with centromere-specific DNA probes of chromosomes 3 and 17 in pleomorphic adenomas and adenoid cystic carcinomas. Li X 1, Tsuji T, Wen S, Mimura Y, Wang Z, Sasaki K, Shinozaki F. Aberrations of chromosomes 3 and 17 were studied by FISH using centromere-specific DNA probes in 11 salivary adenoid cystic carcinomas (ACC) and 8 salivary pleomorphic adenomas (PA), with 3 lymph nodes as controls.. These findings suggest that polysomy of both chromosomes occurs during the development of salivary gland tumors, and its frequency is increased in adenoidcystic carcinoma as compared to pleomorphic adenoma. In addition, monosomy of chromosome 17 could possibly be significant in salivary gland tumors.

7 Applicazione FISH nelle ghiandole salivari Oncol Rep. 2005Nov;14(5): Dual FISH analysis of benign and malignant tumors of the salivary glands and paranasal sinuses. Götte K 1, Ganssmann S, Affolter A, Schäfer C, Riedel F, Arens N, Finger S, Hörmann K. We examined 58 of these tumors by dual fluorescence in situ hybridization (FISH) with centromere-specific probes on six chromosomes (3, 7, 9, 11, 17, and 18) fornumerical changes... In summary, the occurrence of polysomy might reflect a step towards malignancy in tumors of the salivary glands and paranasal mucosa. Polysomy of chromosome 11 could be defined as typical for all investigated histological types of malignant tumor in this region of the head and neck.

8 Applicazione FISH nelle ghiandole salivari A growing number of both benign and malignant SGTs are characterized by recurrent genetic alterations, particularly chromosome translocations. Pleomorphic adenoma (PA) Rearrangements involving 8q12 (39%), with the t(3;8)(p21;q12) translocation representing about half the cases Sporadic clonal changes involving chromosomal segments other than 8q12 or 12q13-15 (23%) Apparently normal karyotype (30%) Rearrangements of 12q13-15 (8%)

9 Applicazione FISH nelle ghiandole salivari Warthin tumor (WT) Three distinct groups of WT have been defined based on cytogenetic aberrations: One with a normalkaryotype, A second with numerical changes only (loss of Y chromosome or trisomy or monosomy 5), A third group involving structural changes with one or two reciprocal translocations Mucoepidermoid carcinoma (MEC) More than 55%of the MEC cases expressed the MECT1-MAML2 The second most common chromosomal abnormality was single or multiple trisomies+7, +8, and +X Other recurrent abnormalities found were deletions of the terminal part of 6q. Apart from these abnormalities, the t(11;19)-negative MECs showed a heterogeneous pattern of rearrangements with no obvious recurrent aberrations

10 Applicazione FISH nelle ghiandole salivari Adenoid cystic carcinoma Adenoid cystic carcinoma is characterized by a t(6;9)(q22-23;p23-24)translocation. The translocation fuses exon 14 of MYBgene, on chromosome 6q22-23, to the last coding exons of NFIBgene, on chromosome 9p Most breakpoints occur in intron 14 of MYB and intron 8 of NFIB. Mammary analogue secretory carcinoma (MASC) Characterized by ETV6-NTRK3 fusion gene

11 Criterio Diagnostico: Mammary analogue secretory carcinoma (MASC) was recently identified as a distinct salivary gland neoplasm, morphologically resembling intercalated duct cell predominant acinic cell carcinoma (AciCC). FISH for ETV6translocation was performed with commercial break-apart ETV6 probes (Abbot Molecular,Des Plains, IL, USA) FISH for MAML2translocation was performed using commercial break-apart MAML2 probes (MEC 1 Dual Color Break-apart Probe, ZytoVision, Bremerhaven, Germany).

12 From acinic cell carcinoma (AciCC) to Mammary analogue secretory carcinoma (MASC) his histological impression was confirmed by ETV6 FISH, which showed 63 of 66 (95.5%) analyzed cells harbor the ETV6 translocation (Fig. 2d).

13 From acinic cell carcinoma (AciCC) to mucoepidermoid carcinoma MEC. MAML2 FISH showed 34 of 39 (87.2%) cells with a classic translocation pattern (Fig. 3c).

14 Criterio Prognostico:

15 (D) Disomy of genomic PTEN in acinic cell carcinoma (ACCC). (E) Hemizygous deletion of PTEN in acinic cell carcinoma (ACCC) (F) Homozygous deletion of PTEN in SDC. (I) Cluster amplification of HER2 in salivary duct carcinoma (SDC). (J) Cluster amplification of EGFR in mucoepidermoid carcinoma (MEC).

16 PTEN function is reduced in different types of salivary gland cancer indicating unfavourable prognosis. Its association with EGFR and HER2 signalling might affect targeted therapy.

17 Criterio Predittivo:

18 Pogressive malignant salivary glands tumors (MSGT) treated with the dual EGFR/ Her2 tyrosine kinase inhibitor lapatinib Eligible patients with ACC and non-acc were treated in two separate cohorts in a multicenter phase II trial with lapatinib given as 1,500 mg orally once daily and continuously until disease progression, unacceptable toxicity, patient refusal, or physician s decision to withdraw the patient (18). Conclusions HER2 to CEP17 FISH ratio may predict which patients with MSGT have an increased likelihood to benefit from lapatinib. The finding of HER2:CEP17 ratio as a predictive marker of efficacy to lapatinib warrants further investigation.

19 L ANALISI FISH PUO ESSERE DI AIUTO AL CLINICO DANDO INFORMAZIONI: DIAGNOSTICHE PROGNOSTICHE PREDITTIVE

20 Grazie Per l attenzione