HER-2/neu status in human pancreatic adenocarcinoma: comparative study of expression, amplification and aneuploidy.

Truini M 1, Ferro P 2,3, Boccardo S 1, Bacigalupo B 2, Salvi S 1, Franceschini MC 2,4, Gorji N 2, Dessanti P 2, Pistillo MP 5, Roncella S 2, Fedeli F 2. 1 S.C. Anatomia e Citoistologia Patologica, Istituto Nazionale per la Ricerca sul Cancro, (IST); Genova. 2 S.C. Anatomia ed Istologia Patologica e Citodiagnostica, ASL5 Spezzino, La Spezia. 3 AIL “Sez. Francesca Lanzone”, La Spezia. 4 Comitato Assistenza Malati e Lotta contro i Tumori, Sarzana (SP). 5 S.C. Genetica dei Tumori (Lab. Tumori Mammari), IST, Genova.

Pancreatic adenocarcinoma (PAC) is one of the most lethal cancer in Italy and in western countries. Therefore, the development of novel therapeutic strategies for PAC is a main focus of current investigations. Trastuzumab recombinant monoclonal antibody is widely used for the treatment of patients with metastatic breast cancer that over-expresses HER-2/neu oncogene. The same therapy could be proposed also for PAC because gene amplification and over- expression of Her-2/neu have been described in a variety of cases, although with controversial results. Background

The purpose of this study was to evaluate, at protein and genomic level, the status of HER-2/neu in PAC and to correlate it with biological markers of tumours. Aim

To investigate the HER-2/Neu status in PAC we analyzed 45 paraffin-embedded tissue sections from PAC patients, 16 of which were primary tumours (10 with nodal metastasis), 19 from hepatic metastasis and 10 from metastatic lymph nodes at initial diagnosis. Correlation between HER-2/neu status with stage, grade and expression of Ki-67 (Mib-1) proliferation index was also investigated. Materials and Methods 1

HER-2/neu protein expression was analysed by immunohistochemical staining (IHC) of p185 membrane protein using the 4B5 monoclonal antibody. Antigens were localized by means of a Ventana Medical System/ultra view TM DAB detection kit and the immunostaining was performed using an automated Immunostainer BenchmarkXT (Ventana Medical Systems, S. A. Strasbourg, France). Gene status (amplification of HER-2/neu and poliploidy of chromosome 17, Chr.17) was analyzed by Fluorescente In Situ Hybridization (FISH) (kit Pathvysion, Vysis - Abbott). SpectrumOrange fluorescent-labeled probe specifically hybridizes to the HER-2/neu gene locus and SpectrumGreen fluorescent-labeled probe specifically hybridizes to the centromeric region of Chr. 17. Materials and Methods 2

Our results showed over-expression of p185 membrane protein in 11/45 (24.4%), gene amplification in 2/45 (4.4%) and aneuploidy of Chr.17 in 6/45 (13.3%) of cases studied respectively (Tab.1). Over-expression of p185 was found in 3/16 (18.7%) primary tumours (2/6 PAC without lymph node infiltration and 1/10 PAC with lymph node infiltration) (Tab.2, Fig.1) and in 8/19 (42.1%) hepatic metastatic lesions (Tab.3, Fig.2). Amplification of the Her-2/neu oncogene was restricted to the hepatic metastatic lesion (2/19, 10.5%) (Tab.3, Fig.2). At last, Chr.17 aneuploidy was found in 4/19 (44.4%) hepatic metastasis (Tab.3, Fig.2) and in 2/10 (20.0%) metastatic lymph nodes (Tab.4, Fig.3). No association was found between Her-2/neu status and tumour stage, grade or Ki-67 cycling index. Results

Positive cases (%) n° of cases p185 Over-expression HER-2/neu Amplification Chr. 17 Aneuploidy 4511 (24.4)2 (4.4)6 (13.3) Table 1. Summary of HER-2/neu status in PAC.

Positive cases (%) n° of cases p185 Over-expression HER-2/neu Amplification Chr. 17 Aneuploidy 163 (18.7)0 (00.0) Figure 1. Expression of p185 in a representative case of PAC without genetic alteration. Table 2. Summary of HER-2/neu status in primary PAC. HEIHC FISH

Positive cases (%) n° of cases p185 Over-expression HER-2/neu Amplification Chr. 17 Aneuploidy 198 (42.1)2 (10.5)4 (44.4) Figure 2. Expression of HER-2/neu in a representative case of hepatic metastasis of PAC that shows over-expression of p185 and amplification of HER-2/neu gene. Table 3. Summary of HER-2/neu status in hepatic metastasis of PAC. HEIHC FISH

Positive cases (%) n° of cases p185 Over-expression HER-2/neu Amplification Chr. 17 Aneuploidy 100 (00.0) 2 (20.0) Table 4. Summary of HER-2/neu status in PAC cells of metastatic lymph node. Figure 3. Aneuploidy of Chr. 17 in a representative case of metastatic limph node. HEIHC FISH

Our study confirms that a subset of PAC was characterized by amplification of HER-2/neu gene or aneuploidy of Chr.17, both associated/or not to the over-expression of p185. HER-2/neu gene amplification was mainly related to recurrent metastasis rather than to primary tumours and over-expression of p185 was not always associated to genetic alterations. The use of Trastuzumab may find clinical application in the targeted therapy of PAC with over-expression of p185 or amplification of HER- 2/neu gene. Conclusions