4. DISCUSSION
We enrolled a homogenous group of 374 children diagnosed with the four
main NHL subtypes and who were treated with modified BFM 95 regimens
over the past 6 years in south China. Compared to many studies, our
sample number was not small for survival analysis. The sex ratio was
approximately 3:1, with more males than females. BL and LBL were
observed more frequently than DLBCL and ALCL. At diagnosis, the dominant
majority of patients had advanced-stage disease and were stratified into
intermediate- or high-risk group. The incidence rate of BM involvement
was about twice that of CNS involvement. Generally, others have reported
similar clinical characteristics of pediatric NHL in both white and
Asian population.1, 27-31 Overall, the patients
responded well to the chemotherapy regimens at our center. The rates of
CR (86.1%), 5-year EFS (74.2%) and OS (85.7%) rate in the whole group
were comparable to that of different working groups.1,
32, 33 Although less than half of the patients in the present study
were genotyped for the C677T/A1298C polymorphism, the subgroup of 158
genotyped patients could be considered as a representative sample of the
whole group of pediatric NHL, as no significant difference was found in
any clinicopathological characteristics between the two subgroups with
or without MTHFR genotyping (TABLE 1). In this analysis, both
C677T and A1298C alleles were in H-W equilibrium. The mutation rate of
C677T was slightly greater than that of A1298C (47.5% vs. 37.7%).
C677T genotype frequencies were similar to that of other reports in
Asian pediatric patients with NHL.34, 35 Data on
A1298C are limited in Asia. However, some studies on North American and
European children with NHL reported a higher mutation rate of C677T and
A1298C.27, 36 Therefore, we confirm the high regional
and geo-graphic variability of MTHFR polymorphism prevalence in
previous publications. 8 The distribution of A1298C
genotypes was significantly different among the NHL subtypes (TABLE 2,P = 0.045); patients with DLBCL had the highest mutation rate
(58.3%), while patients with LBL had the lowest rate (28.9%).
The influence of MTHFR C677T/A1298C on the survival of patients
with cancer (non-hematologic and hematologic malignancy) has attracted
great attention from researchers. Regarding adult solid cancers,
available evidence on CRC is conflicting; different studies have
reported that both C677T and A1298C could be a positive or negative
prognostic factor or have no effects on survival of patients with
CRC.20-24 No significant differences in the OS were
found among the wild-type 677CC and variant CT/TT genotypes in patients
with glioma.37 Martha et al. reported that both the
C677T and A1298C polymorphisms were not significantly associated with
risk of death, but that the 677T variant allele may adversely affect
long-term survival in advanced breast cancer.38Lambrecht et al. found that the homozygous 677TT genotype was associated
with a higher relapse rate but was not predictive for OS in pediatric
osteosarcoma.39 Similarly, Xie et al. suggested that
genetic polymorphism of MTHFR C677T in the MTX metabolic pathway
had no obvious effect on progression-free survival and tumor necrosis
rate in sarcoma (age range, 5-52 years). 40 For
hematological malignancy, early studies on childhood ALL have reported
controversial results.16-18 However, a recent review
concluded that there was no association or opposite effects in the
majority of published data, and that these two polymorphisms are not
suitable predictors of outcome in pediatric ALL. 5However, the MTHFR 677TT homozygous genotype has been associated
with shorter survival in patients with myelodysplastic syndromes41.
Only a few studies have investigated the influence of the C677T and
A1298C polymorphisms on the survival of patients with NHL, with
controversial results, and there is a particular lack of evidence on
A1298C. In an Italian retrospective study with a total of 110 adult
patients with NHL treated according to CHOP (cyclophosphamide,
doxorubicin hydrochloride, vincristine sulfate, prednisone) or MACOP-B
(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,
bleomycin) protocol, survival analysis (log-ranks) implied that 677T
carriers had a significant reduced probability of 5-year EFS in the
whole group and in the MACOP-B–treated group (n = 68). The
difference was more obvious when 1298CC cases were excluded from the
reference group. However, no significant correlations were identified in
the CHOP- treated group(n = 42).7 D’Angelo et
al. also found that 677T carriers could have increased risk of relapse
and reduced disease-free survival compared with wild-type patients in
Caucasian and European children or adolescent NHL treated with LNH-97
and EURO LB-02 multiagent chemotherapy protocols developed by the AIEOP
(Associazione Italiana di Ematologia e Oncologia
Pediatrica).27 The multicenter NHL-BFM95 trial,
involving centers in Austria, Germany, and Switzerland (n = 484),
determined that the MTHFR 677 C > T polymorphism was
not associated with clinical characteristics (sex, age, tumor stage) and
did not appear to influence EFS in pediatric patients with
NHL.36
However, in our study, the C677T polymorphism was associated with
all-events incidence. Patients carrying the wild-type were more likely
to experience events. Moreover, K-M survival analysis revealed that 677T
allele could significantly increase the probability of 5-year EFS and
was associated with a non-significant increased probability of 5-year
OS. Therefore, the 677T variant allele might have played a protective
role in the survival of childhood NHL in the present analysis. The
influence of T variant allele on survival was more obvious when only
BL/DLBCL were included in K-M analysis. As no significant difference was
found when only LBL or ALCL were included, we assumed that the
significant differences in the whole group of NHL resulted from the
large proportion of BL/DLBCL cases (n = 81, 51.3%). Furthermore,
we speculated that cancer type, age, treatment regimens, and
particularly ethnicity might account in part for the discrepant results.
A large meta-analysis concluded that the C677T polymorphism showed a
statistically significantly increased risk for whites, but a decreased
risk for Asians6. Therefore, it is also possible that
the C677T polymorphism has an opposite influence on the survival of
European and Chinese pediatric NHL even when patients are treated using
the same chemotherapy protocol. The reduced MTHFR activity due to the
677T variant allele might have positive effects on survival of pediatric
NHL in the Chinese population.
Interestingly, the A1298C polymorphism has been associated with
increased NHL risk for in Asians.6 In the present
study, except for NHL subtype, no significant associations of A1298C in
clinical characteristics (age, sex, stage, risk, BM/CNS involvement) and
outcome (EFS and OS) could be detected. However, a fact that should not
be neglected is that MTHFR enzymatic activity is affected by the A1298C
polymorphism to a lesser extent than by C677T, and the remaining
activity in the 1298CC homozygous genotype still represents 60% of the
normal status despite of the substitution of glutamate for alanine in
the amino acid sequence.5, 9, 10 Furthermore, due to
partial linkage disequilibrium, it is possible, but indeed very rare,
for 677T and 1298C variant alleles in cis to coexist, supporting the
hypothesis that triple mutations (677TT/1298AC or 677CT/1298CC) or
double homozygous conditions (677TT/1298CC) are probably de novo
recombinant events.42 In the present study, virtually
all patients with 677TT (n = 14) had the wild-type 1298AA
genotype. For these reasons, it was difficult to observe a clear
allele–dosage effect for the variant 1298C being better accounted for
in homozygous conditions. This could partly explain the lack of
significant associations between the A1298C polymorphism and survival in
our study, base on the difficulty in determining the effects of 1298
variant itself on survival.
Our results indicated that patients with ALCL had a significantly higher
probability of experiencing events than patients with the other three
subtypes, while patients with DLBCL had a relatively superior survival.
Overall, patients with ALCL have inferior EFS compared to the other
three types.1, 30, 43 Besides, we found that age, sex,
and BM involvement did not affect survival, but CNS involvement,
advanced stage, and intermediate/high risk predicted inferior survival
in pediatric NHL. Similarly, a report from BFM Group study NHL-BFM95
(n = 505; age range, 1.4–19.7 years) revealed that CNS-positive
patients had remarkably lower 3-year EFS compared to the whole group
(69% vs. 89%), and that stage and risk were adversely associated with
3-year EFS.31 Besides, a large multicenter
retrospective study of 749 pediatric patients with NHL in east Asia
suggested that BM involvement does not affect treatment outcome, while
CNS involvement and advanced stage (≥ stage III) were associated with
poor outcomes (P < 0.05).30 More
recently, Choeyprasert et al. identified advanced disease stage as a
significant poor prognostic factor for 5-year EFS of pediatric NHL in
Thailand.29
We are aware of the limitations of our study, given the retrospective
nature and relatively small sample size enrolled for analyzing the
influence of MTHFR on the survival of pediatric NHL. Moreover,
not all newly diagnosed pediatric NHL at our center during the study
period were included for analysis, as a small group of them did not
receive modified BFM 95 regimens, but were alternatively treated with an
adult protocol such as the R(rituximab)-CHOP regimen.
In conclusion, we found that the MTHFR C677T polymorphism and
some clinicopathological characteristics including, NHL subtype, CNS
involvement, stage, and risk at diagnosis, are significantly related to
the survival of pediatric patients with NHL treated with modified BFM 95
regimens. The 677T allele could increase the probability of 5-year EFS
and 5-year OS. No evidence showed that the MTHFR A1298C
polymorphism influenced the EFS or OS rate in pediatric patients with
NHL. Further larger studies are encouraged to verify these results
properly.