Genetic polymorphisms in the tumour necrosis factor locus in childhood acute lymphoblastic leukaemia
Seisho Takeuchi, MD, Department of Medicine, Kochi Medical School, Okohcho, Nankoku, Kochi 783–8505, Japan. E-mail: firstname.lastname@example.org
Summary. Genetic polymorphisms in the tumour necrosis factor (TNF) locus influence the outcome of non-Hodgkin's 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.
Tumour necrosis factor (TNF) and lymphotoxin-α (LTα) play an important role in normal lymphoid tissue function (Le Hir et al, 1996). TNF is also important in the inflammatory process. Previous studies in patients with malignancy have shown that excessive TNF production may cause poor nutrition, inhibit the immune response, and produce anaemia, therefore hampering the patient's ability to tolerate the tumour and the treatment (Tracey et al, 1988). Other data indicate that TNF may promote the growth of lymphoid cells (Cordingley et al, 1988). Indeed, patients with malignant lymphoma often have high TNF levels, which are associated with poor prognosis (Warzocha et al, 1997).
The TNF and LTα genes reside within the class III region of the major histocompatibility complex. Several studies have shown that individual differences in the gene products were linked to polymorphic markers in this region. A polymorphism that directly affects the expression of TNF is a point mutation resulting in a G to A transition at position −308 of the TNF promoter (Wilson et al, 1997). The presence of guanine (G) defines the common variant TNF1, and the presence of adenine (A) defines the less common variant TNF2. The presence of the TNF2 allele results in higher levels of TNF expression. A polymorphism that affects LTα expression was found in the first intron of the gene at nucleotide position +252 (Messer et al, 1991). The less common variant guanine (G) is cleaved by NcoI and is defined as LTα (5·5 kb); and the common variant adenine (A) is non-cleaved and defined as LTα (10·5 kb). The presence of LTα (5·5 kb) allele was shown to result in higher LTα production. A recent study found that the presence of either the TNF or LTα high-producing alleles contributed to a higher rate of relapse and shorter survival in non-Hodgkin's lymphoma (NHL) (Warzocha et al, 1998). However, data are not available as to whether these polymorphisms influence the outcome of childhood acute lymphoblastic leukaemia (ALL).
Patients. In total, 214 primary ALL DNAs were obtained from patients participating in the ongoing German Berlin-Frankfurt-Münster (BFM) group Multicentre Trial of Childhood ALL (ALL-BFM 90/95) (patients were diagnosed between April 1990 and March 1995). The series was selected only on the basis of availability of DNA after routine diagnostic tests. However, those tested were similar to the total group of patients enrolled on the ALL-BFM 90/95 trial (n = 2813) with respect to the distribution of sex, age at diagnosis, central nervous system (CNS) manifestation, immunophenotype, as well as response to chemotherapy, whereas the median white blood cell count (WBC) was higher in the test panel (20·2 × 109/l vs. 10·5 × 109/l). Informed consent was obtained from the patients, their parents, or both, as appropriate. Clinical information for up to 8 years from initial diagnosis was available for all of the 214 children in this study.
Laboratory methods. The genotype was performed as described previously (Warzocha et al, 1998). The TNF gene, which includes the polymorphic site at the nucleotide position −308, was amplified by the allele-specific polymerase chain reaction (PCR). A region containing the polymorphism at the nucleotide position +252 of LTα was amplified by PCR, followed by NcoI restriction fragment length polymorphism.
Statistical analysis. Distribution and allele frequency and their associations with clinical characteristics were compared using the chi-square test or the U-test. An effect was considered statistically significant if the P-value was 0·05 or less.
Results and discussion
We analysed the allele frequencies of TNF (−308) and LTα (+252) polymorphisms in 214 childhood ALL samples. As both polymorphisms are associated with increased production of TNF (Warzocha et al, 1998; Fitzgibbon et al, 1999), we decided to analyse these polymorphisms as haplotypes rather than as individual markers. The low-risk haplotype was defined by the presence of less than two alleles associated with either high TNF or LTα production. The high-risk haplotype was defined by the presence of either two or more alleles associated with increased TNF or LTα production.
No statistically significant association was found between the presence of a given TNF/LTα haplotype and clinical characteristics such as sex, WBC counts at diagnosis, CNS involvement, immunophenotype or response to chemotherapy (Table I). Interestingly, the high-risk haplotype was associated with older age at diagnosis (P = 0·02), although the exact reason for this is unclear. It may imply that older ALL patients have a different biological background from those in the younger age group, which could contribute to an unfavourable prognosis in these older patients. This is in contrast to ALL with a TEL/AML-1 fusion, which is associated with a younger age group (Mclean et al, 1996). The five-year event-free survival did not differ significantly between low-risk haplotype (77·2%) and high-risk haplotype (76·9%). These data suggest that genetic polymorphism in the TNF locus has a limited effect on any clinical features and outcome of childhood ALL. This type of investigation could be better performed in molecularly defined subtypes of leukaemia. However, the number of patients analysed in the current study was too small to divide patients into these subtypes.
Table I. Clinical characteristics of 214 childhood acute lymphoblastic leukaemia (ALL) patients according to risk groups defined by status of tumour necrosis factor (TNF)/lymphotoxin (LT) polymorphic haplotype.
| < 10||173 (81)||128 (85)||45 (71)|| |
| ≥ 10||41 (19)|| 23 (15)||18 (29)||0·02|
| < 20||106 (50)|| 75 (50)||31 (49)|| |
| ≥ 20||108 (50)|| 76 (50)||32 (51)||0·95|
| No||195 (91)||138 (97)||57 (97)|| |
| Yes||6 (9)|| 4 (3)|| 2 (3)||0·83|
| T-ALL||34 (16)|| 24 (16)||10 (16)|| |
| Precursor B-ALL*||180 (84)||127 (84)||53 (84)||1·00|
|Response to chemotherapy†|
| Good||184 (89)||130 (89)||54 (88)|| |
| Poor||23 (11)|| 16 (11)|| 7 (12)||0·91|
|Event-free survival at 5 years|| ||(77·2 ± 3·8)||(76·9 ± 5·5)||0·63|
A recent study showed that the presence of either TNF or LTα high-producing alleles contributed to a higher rate of relapse and shorter survival of NHL (Warzocha et al, 1998). We found previously that a genetic polymorphism leading to increased TNF synthesis may enhance susceptibility to adult T-cell leukaemia/lymphoma among human T-lymphotropic virus type I carriers (Tsukasaki et al, 2001). In contrast, no association was noted between TNF haplotype distribution and prognostic factors such as age at diagnosis, presence of extranodal disease, or bone marrow infiltration, as well as disease outcome in individuals with follicular lymphoma (Fitzgibbon et al, 1999). Also, TNF polymorphism was not associated with disease outcome of either Hodgkin's disease or chronic lymphocytic leukaemia (Wihlborg et al, 1999). TNF may have functional importance in several types of lymphomas, probably by mediating apoptosis, or by stimulating proliferation of these cells, but this is not the case in childhood ALL.
Supported in part by National Institutes of Health grants, C. and H. Koeffler Fund, Joseph Troy Trust, Parker Hughes Trust, Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, and Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan. H.P.K. is a member of the Jonsson Comprehensive Cancer Center and holds the endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center/UCLA School of Medicine.