PD-35
CpG island hypermethylation in
lung neuroendocrine tumors
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
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
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
TC
AC
LCNEC
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
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)
DNA extraction from histologic sections
Methods
amplification of the promoters of the following genes by
methylation-specific PCR (MSP):
APC, BRCA1, CST6, DAPK, FHIT, MGMT, p16, RARß,
RASSF1A, Rb, RIZ1, RUNX3
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
MSP for RASSF1A promoter
1
2
3
4
cases 1-2 LCNEC, methylated
case 5 TC, methylated
5
6
7
casi 3-4 SCLC, methylated
casi 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
Median Methylation Index (MI)
50
40
Results
Methylation index
(MI) is an indicator
of the overall level
of methylation
30
20
10
0
TC
AC
LCNEC
SCLC
 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
Frequency of promoter methylation (%)
TC
AC
LCNEC
SCLC
100
80
60
40
20
0
APC
BRCA1
FHIT
MGMT
p16
RARb
RASSF
RUNX3
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 (%)
100
80
60
40
20
0
BRCA1
FHIT
TC
AC
RASSF
LCNEC
RUNX3
SCLC
Frequencies of promoter methylation above 40% were
observed for BRCA1, FHIT, RASSF1A and RUNX3 in all
NE tumor types
Frequency of promoter methylation (%)
Low Grade NETs
High Grade NETs
100
*
*
80
*
60
40
*
*
20
*
*
0
APC
BRCA1
FHIT
MGMT
p16
RARb RASSF RUNX3
 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 (%)
80
60
* p = 0.0004
* p = 0.0094
100
40
20
0
APC
BRCA1
FHIT
LCNEC
MGMT
p16
RARb
RASSF
RUNX3
SCLC
 among high grade neoplasms, SCLCs showed higher
methylation rate than LCNECs for all genes but APC;
 the statistical significance was reached for p16 and RARß;
 among lung NE tumors, RARß promoter was methylated
only in SCLCs
Frequency of promoter methylation (%)
80
60
40
20
0
APC
BRCA1
FHIT
TC
MGMT
p16
RASSF
RUNX3
AC
 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
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