1. CpG island hypermethylation in lung neuroendocrine tumors PD-35 T. D’Adda, S. Pizzi, F. Inzani, C. Azzoni, L. Bottarelli, G. Pelosi *, M. Papotti §, C. Bordi, G. Rindi Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Anatomia ed Istologia Patologica, Università degli Studi di Parma; * Divisione di Patologia e Medicina di Laboratorio, Istituto Europeo di Oncologia ed Università degli Studi di Milano; § Dipartimento di Scienze Cliniche e Biologiche, Università di Torino

2. Background DNA methylation (the addition of a methyl group to the carbon-5 of cytosine residues) occurs almost exclusively at cytosines that are followed immediately by a guanine (CpG dinucleotides) stretches of these dinucleotides (“CpG islands”) are frequently located within the promoter regions of human genes and are usually free of methylation aberrant methylation within these “CpG islands” is associated with transcriptional inactivation of the corresponding gene alterations of DNA methylation seem to play an important role in the development of most cancers

3. Neuroendocrine (NE) tumors of the lung Lung NE tumors are an heterogeneous group of neoplasms with different biologic behavior and degrees of malignancy. They include:  typical (TC) and atypical carcinoids (AC), with low and intermediate malignant potential, respectively;  large cell neuroendocrine (LCNEC) and small cell lung carcinomas (SCLC), highly aggressive neoplasms The role of aberrant methylation has not been sistematically investigated in this family of tumors

4. TCAC LCNECSCLC

5. Materials The study was performed on 89 pulmonary NE tumors (formalin-fixed, paraffin-embedded):  21 typical carcinoids (TC)  20 atypical carcinoids (AC)  22 large cell neuroendocrine carcinomas (LCNEC)  26 small cell lung carcinomas (SCLC) Aim of the study To investigate the methylation status of a series of genes commonly involved in human cancerogenesis in the spectrum of NE tumors of the lung

6. DNA extraction from histologic sections amplification of the promoters of the following genes by methylation-specific PCR (MSP): APC, BRCA1, CST6, DAPK, FHIT, MGMT, p16, RAR ß, RASSF1A, Rb, RIZ1, RUNX3 Methylation-specific PCR (MSP) Methylation-specific PCR (MSP) includes two steps: 1. DNA modification by sodium bisulfite treatment (EpiTect bisulfite kit, QIAgen), which converts unmethylated cytosine residues in uracil, leaving methylated cytosines unchanged 2. double MSP reaction with two sets of primers, to unveil the methylation status of the promoter regions: set U, that anneals only to unmethylated sequences set M, that anneals only to the methylated ones Methods

7. MSP for RASSF1A promoter 1273456 cases 1-2 LCNEC, methylatedcasi 3-4 SCLC, methylated case 5 TC, methylatedcasi 6-7 AC, unmethylated  the presence of a 93 bp product after amplification with the primer set “M” means RASSF1A gene methylation;  methylated cases always show an amplificate (105 bp) with the primer set “U”, given the presence of normal tissue, unmethylated, intermingled with the tumor tissue from which DNA has been extracted

8.  MI was calculated, for each tumor, as the percentage of methylated genes over investigated genes;  median MI was calculated for each tumor type, being 24% for TC, 18% for AC, 35% for LCNEC and 45% for SCLC;  differences in median MI between tumor classes were all statistically significant (Mann-Whitney test), except for the comparison TC vs AC Results Methylation index (MI) is an indicator of the overall level of methylation

9. Overview of the frequencies of promoter methylation (percentage of methylated/investigated cases) per gene, in the different classes of lung NE tumors Frequency of promoter methylation (%)

10. Frequencies of promoter methylation above 40% were observed for BRCA1, FHIT, RASSF1A and RUNX3 in all NE tumor types Frequency of promoter methylation (%)

11.  higher methylation levels were observed in high grade tumors (LCNECs and SCLCs) as compared to carcinoids;  statistical significance (*) was reached for all comparisons, except for FHIT (Fisher’s exact test) Frequency of promoter methylation (%) * * * * * * *

12.  among high grade neoplasms, SCLCs showed higher methylation rate than LCNECs for all genes but APC;  the statistical significance was reached for p16 and RAR ß;  among lung NE tumors, RAR ß promoter was methylated only in SCLCs Frequency of promoter methylation (%) * p = 0.0094 * p = 0.0004

13.  among carcinoids, most frequently methylated genes were BRCA1, FHIT, RASSF1A and RUNX3;  TCs showed overall higher levels of methylation than ACs, although the differences were not statistically significant Frequency of promoter methylation (%)

14. Discussion  Hypermethylation is frequently observed in lung NE tumors  Hypermethylation of BRCA1, FHIT, RASSF1A and RUNX3 in both lower and higher grade NE tumors suggests the involvement of these genes in early stages of tumorigenesis  Higher degrees of methylation associate with higher grade NE tumors, suggesting accumulation of epigenetic defects with tumor progression