Study of gene promoter methylation in acute promyelocytic leukaemia

by Chim, Chor-sang

ABSTRACT

Study of Gene Promoter Methylation in Acute Promyelocytic Leukaemia

17

Abstract

18

DNA methylation, catalyzed by DNA methyltransferase, involves the addition of a methyl group to the carbon 5 position of the cytosine ring in the CpG dinucleotide and results in the generation of methylcytosine.

In cancer, aberrant methylation of specific gene

promoter

CpG

islands

results

in

transcriptional

silencing, and serves as an alternative mechanism of gene inactivation.

Apart from cellular oncogenes,

dysregulation of

tumor

suppressor

genes

is

an important event and CDKN2B (P15INK4B)

in

carcinogenesis. CDKN2A (p16INK4A)

are

two closely linked tumor suppressor genes located at

chromosome 9p21,

encoding for p16 and p15 cyclin-

dependent kinase inhibitors.

While most tumor suppressor genes such as Rb and p53 are inactivated by point mutation in one allele and deletion of the other, the major mechanism of p15 gene inactivation in AML is through aberrant methylation of the 5' promoter region.

Acute

promyelocytic

leukaemia

(APL)

is

characterized by t(15;17) with generation of a chimeric

Abstract 19

PML/ RARa gene responsible for leukemogenesis. Recently, all-trans retinoic acid (ATRA) and arsenic trioxide have been shown to induce differentiation and apoptosis of the neoplastic promyelocytes.

Studies were performed to test the hypotheses:

following

1. p15 and p16 are frequently methylated in APL

Diagnostic bone marrow DNA from 26 APL patients was

studied

with

methylation-specific

polymerase

chain

reaction (MSP) with primers for methylated (M-MSP) and

unmethylated

(U-MSP)

DNA.

Validity

of

M-MSP

was

confirmed

by

sequencing

of

selected

diagnosis,

nineteen

patients

(73.1%)

samples. showed

At p15

methylation, whereas only three patients (11.5%) showed

p16 methylation, methylation.

all

of who had concomitant p15

2. Adverse prognostic impact of p15 methylation on APL

Disease-free (DFS) and overall (OS) survivals were correlated with p15 methylation status at diagnosis. The five-year DFS of patients with p15 methylation was significantly inferior to those without (15% versus

Abstract

20

62.5%, p multivariate

0.02); and this remained significant in

analysis

that

included

age,

sex,

morphologic subtypes, and presenting leukocyte counts in addition to p15 methylation status.

3. p15 methylation serves as a disease marker of MRD

MSP for sensitivity of

p15 gene 10-5?Six

methylation has a

maximum

patients with serial

marrow

samples during the course of disease were evaluated with MSP for p15 methylation at diagnosis and follow-up.

Persistent

p15

methylation

preceded

subsequent

hematological negative MSP

relapses

in

two,

and

conversion

to

for p15 methylation

correlated with

prolonged survival in another four patients.

4. Multiple genes are methylated in addition to p15 and p16

The methylation status of a panel of nine genes including p15, p16, p73, VHL, E-CAD, Caspase 8, MGMT,

RAR~, and estrogen receptor (ER) was analyzed by MSP in the diagnostic bone marrow samples of 23 APL patients. Methylation of p15, RAR~, ER and p16 was detected in 17

(73.9%) , 10 (43.4%) , 5 (21.7%) and 3 (13%) patients at

Abstract

21

diagnosis but not in p73, VHL, E-CAD, caspase 8 and MGMT.

In conclusion, preferential methylation of selected genes (such as P15, ER and RARf3) occurs in APL. p15 methylation might be of prognostic significance, and is a potential marker of MRD in APL.