Naoko Takeuchi

Methylation Analysis of Cell Cycle Control Genes in Adult T-Cell Leukemia/Lymphoma

Central to many cancers is the aberrant expression of genes that regulate the cell cycle including the cyclin-dependent kinase inhibitors known as p15INK4b and p16INK4a, p14ARF and the retinoblastoma (RB) protein. We performed a detailed analysis of the methylation status of these genes by methylation specific polymerase chain reaction (MSP) in tumor cells of 35 adult T-cell leukemia/lymphoma (ATL) patients. We found in nine of 35 cases (26%) at least one gene methylated. The frequency of p15INK4b methylation was 7 of 35 (20%). The incidence of methylation of p14ARF and p16INKa was two of 35 (6%) and one of 35 (3%), respectively. The RB gene was not found to be methylated in any of the ATL samples. The data indicate that inactivation of these cell cycle regulatory genes by hypermethylation is important in the development of ATL.

Loss of H19 imprinting in adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) is associated with the monoclonal integration of human T-lymphotropic virus type I (HTLV-I) proviral DNA into tumour cells, but the incidence of ATL is low (2–5%) among HTLV-I carriers; and those who develop the disease usually have a 40to 60-year latency period from the time of infection to progression to ATL. These phenomena suggest that additional mechanisms are involved in the progress of the disease, such as inactivation of tumour suppressor genes, or activation of oncogenes. Genomic imprinting is an epigenetic alteration in DNA. Loss of imprinting (LOI) results in loss of parental originspecific differential allele expression, and leads to activation of the normally silent copy of the gene. H19 is an endogenous gene showing maternal-specific monoallelic expression on 11p15. Previous studies have found that loss of H19 imprinting is associated with development of several kinds of cancers (Hashimoto et al, 1995; Hibi et al, 1996). Twenty ATL patients were diagnosed at the Department of Haematology, Nagasaki University School of Medicine. Bone marrow cells from nine patients with acute lymphoblastic leukaemia (ALL) were obtained from the German multicentre ALL Study Group. ATL cell lines KK1, SO4 and ST1 were kindly provided by Dr K. Tsukasaki (Nagasaki University School of Medicine, Japan). The presence of a polymorphic pattern and gene expression pattern of H19 were examined in each case using RsaI restriction fragment length polymorphic (RFLP) analysis as described previously (Hibi et al, 1996). Seven primary ALL samples, 11 ATL samples and two ATL cell lines were heterozygous for the RsaI polymorphism in H19 (Fig 1). These heterozygous samples were interpreted as informative cases that can be used in allele-specific expression analysis. The heterozygous samples in expression analysis were interpreted as having LOI. Seven of 11 (64%) informative ATL samples and two of two (100%) informative ATL cell lines showed LOI for H19 (Fig 1). LOI was found in three of four (75%) chronic ATL patients, and in four of seven (57%) acute ATL patients. In contrast, six of seven (86%) informative primary ALL samples did not have LOI. We have found that LOI of H19 is a frequent event in ATL. H19 encodes a polyadenylated RNA that lacks conserved open reading frames. No endogenous translation product has been identified; therefore, it was proposed that H19 RNA functions as a riboregulator. A recent study has found that H19 is activated by the E2F1 transcription factor, and overexpression of H19 confers a growth advantage in breast cancer cells (Berteaux et al, 2005). Moreover, the c-Myc oncogene induces the expression of H19 RNA (Barsyte-Lovejoy et al, 2006). Therefore, H19 may have an important role in transformation of many kinds of tumours. LOI of H19 was initially found in childhood tumours, including Wilms tumour (Rainier et al, 1995), hepatoblastoma, testicular germ cell tumour and choriocarcinoma (Hashimoto et al, 1995). It was later identified in adult tumours. These include oesophageal cancer (Hibi et al, 1996) and lung cancer. On the other hand, maintenance of imprinting has been identified in neuroblastoma, glioma and colorectal cancer (Hibi et al, 1996). These findings indicate that LOI of H19 is involved in several types of tumour. A previous study showed that H19 imprinting was maintained in both immature and mature haematopoietic cells (Morison et al, 2000); therefore, LOI identified in the current study, is not due to the immature phenotype of the affected cells. LOI was identified both in chronic and in acute ATL, indicating that it is associated with the initiation rather than the progression of ATL.

Allelotype analysis in relapsed childhood acute lymphoblastic leukemia

We performed for the first time the allelotype of relapsed childhood acute lymphoblastic leukemia (ALL). A total of 38 cases were screened for loss of heterozygosity (LOH) using 71 markers. In all, 26 (68%) patients showed LOH on at least one chromosomal arm, indicating that LOH is a frequent event at relapse. The most frequent loss was found on chromosomal arm 9p at the p16/INK4a locus (39%). LOH at the TEL gene locus on chromosomal arm 12p also occurred often (25%). Frequent loss was observed on chromosome arms 4q (20%), 6q (21%), and 17q (20%). Sequential analysis (i.e. samples obtained from both initial diagnosis and relapse) shows that some patients (63%) have the identical LOH status at both phases, suggesting the presence of the same clone. Other samples (37%) showed distinct LOH alterations, indicating clonal evolution at relapse. Despite the heterogeneous and complex changes, some shared LOH loci occurred in these matched samples, suggesting that many of the same tumor-suppressor genes are aberrant at both phases. In summary, novel tumor-suppressor genes on chromosome arms 4q, 6q, and 17q, as well as the p16 and TEL genes, have an important role in the relapse of childhood ALL.

Genetic polymorphisms in the tumour necrosis factor locus in childhood acute lymphoblastic leukaemia.

Summary. Genetic polymorphisms in the tumour necrosis factor (TNF) locus influence the outcome of non‐Hodgkins lymphoma (NHL). We investigated whether these polymorphisms might contribute to the clinical course of childhood acute lymphoblastic leukaemia (ALL). Genomic DNA from 214 childhood ALL patients was analysed. Patients with a high‐risk haplotype were older than patients with low‐risk haplotype (P = 0·024). No statistically significant associations were found between TNF haplotype and sex, WBC counts, central nervous system involvement, immunophenotype, response to chemotherapy, and event‐free survival. These data suggest that genetic polymorphisms in the TNF locus have a limited effect on the outcome of childhood ALL.

P53 codon 72 polymorphism is associated with disease progression in adult T‐cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATL) is associated with the monoclonal integration of human T-lymphotropic virus type I (HTLV-I) proviral DNA into tumour cells, but the incidence of ATL is low (2–5%) among HTLV-I carriers and those who develop the disease usually have a 40to 60-year latency period from the time of infection to progression to ATL. These phenomena suggest that additional mechanisms are involved in the progression of the disease, such as inactivation of tumour suppressor genes. P53 is one of the most important tumour suppressor genes that is involved in multiple pathways including apoptosis, cellular transcriptional control and cell cycle regulation. A common polymorphism of P53 at codon 72 results in either a variant protein with a proline (Pro) residue (CCC) or an arginine (Arg) amino acid (CGC) (Zhang et al, 2003). Recent studies have found that this polymorphism is associated with susceptibility or progression of several kinds of cancers (Zhang et al, 2003). However, data are not available as to whether this polymorphism plays a role in the pathogenesis of ATL among HTLV-I carriers. Human T-lymphotropic virus type I carriers (n 1⁄4 81) were identified in the general population in an endemic region for HTLV-I, the Nagasaki district of Japan, after a mass medical and laboratory screening for individuals with anti-HTLV-I serum antibodies. ATL patients (n 1⁄4 87) were diagnosed at the Department of Haematology, Nagasaki University School of Medicine. HTLV-I negative controls (n 1⁄4 89) were included in the analysis. All subjects were Japanese. The genotype was performed as previously described (Zhang et al, 2003). Allele frequency of the P53 codon 72 polymorphism was not statistically different between 89 HTLV-I negative controls and 81 healthy HTLV-I carriers (P 1⁄4 0Æ677) (Table I). These data suggest that genetic polymorphism in the P53 locus does not play a role in the infection of the HTLV-I. The allele frequency was not statistically different between 87 individuals with ATL and 81 healthy HTLV-I carriers (P 1⁄4 0Æ627). These data suggest that this polymorphism does not play a role in the development of ATL among HTLV-I carriers. We previously found that a genetic polymorphism of the tumour necrosis factor gene enhanced susceptibility to ATL among HTLV-I carriers (Tsukasaki et al, 2001). In contrast, polymorphisms at the methylenetetrahydrofolate reductase locus did not alter susceptibility to ATL (Takeuchi et al, 2004). The frequency of the P53 codon 72 genotype in clinically defined subtypes of ATL is shown in Table I. Of note, the frequency of the arginine homozygous allele was higher in acute ATL than in chronic ATL (P 1⁄4 0Æ0396). These results suggest that the P53 polymorphism plays an important role in the progression of ATL. Previous studies showed that mutation of the P53 gene in acute ATL was higher than that in chronic ATL (Hatta & Koeffler, 2002). We have previously reported that the frequency of adenomatous polyposis coli (APC) promoter methylation in acute type ATL (57%) was much higher than in chronic type ATL (13%) (P 1⁄4 0Æ03) (Yang et al, 2005). Several differences occur among the P53 variants in their ability to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis and to repress the transformation of primary cells (Thomas et al, 1999). Polymorphism of the P53 gene is associated with increased susceptibility to the development of gastric, urinary tract and breast cancers (Zhang et al, 2003). Taken together, P53 may have functional importance in several types of malignancies including ATL, probably by modulating its efficiency as a transcription factor.

Long-term study of the clinical significance of loss of heterozygosity in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in childhood, with a widely variable outcome. Differences in the behavior and prognosis of the leukemia suggest that ALL can be divided into several biologic subgroups. We analyzed the loss of heterozygosity (LOH) of 6q, 9p, 11q and 12p using 31 microsatellite sites to determine their overall frequency and clinical value. We have studied 244 primary ALL samples obtained from the Multicenter Trial ALL-BFM 90 of Childhood ALL group. These patients have now been followed clinically for over 8 years. LOH occurred in 169 (69%) individuals in the following frequencies: 6q, 49 patients (20%); 9p, 97 patients (40%); 11q, 29 patients (12%); 12p, 60 patients (25%). Clinical data showed that those with 6q LOH were younger (P = 0.01) and had lower WBC counts (P = 0.02); patients with 9p LOH more frequently had CNS involvement (P = 0.01) and T cell phenotype (P = 0.0001); individuals with 11q LOH had a good response to induction chemotherapy (P = 0.02); those with 12p LOH were younger (P = 0.005), frequently had precursor B ALL (P = 0.001), and had a longer event-free survival (P = 0.05). Taken together, these data confirm that LOH is a very frequent alteration in childhood ALL.

Frameshift mutations in caspase-5 and other target genes in leukemia and lymphoma cell lines having microsatellite instability.

Microsatellite instability (MSI) was examined in 49 lymphoid leukemia/lymphoma cell lines. Frameshift mutations within eight MSI target genes were analyzed in the 12 cell lines and nine childhood acute lymphoblastic leukemia (ALL) samples with MSI. Six cell lines (50%) showed mutations within the coding repeats of the BLM, caspase-5, TCF-4, and PTEN genes. No mutations were identified in the nine childhood ALL samples with MSI. This is the first report that describes mutations of these genes in hematological malignancies. Mutations, presumably caused by an abnormality of the DNA repair genes, may be selected for during the establishment of cell lines.

Mutations in the retinoblastoma-related gene RB2/p130 in adult T-cell leukaemia/lymphoma

The retinoblastoma (Rb) family consists of the tumor suppressor Rb/p105 and related proteins p107 and Rb2/p130. Although the involvement of the RB / p105 gene in Adult T-cell leukaemia/lymphoma (ATL) has been studied, no mutational data is reported regarding the RB2 / p130 gene in ATL. We screened for mutations of the RB2 / p130 gene. Mutation was detected in 1 of 41 primary ATL sample. This is the first report describing mutation of the RB2 / p130 gene in ATL, suggesting that RB2/p130 may be involved in the development of ATL, and may behave as a tumor suppressor gene in T lymphocytes.