MDM2 gene SNP309 T/G and p53gene SNP72 G/C do not influence diffuse large B-cell non-Hodgkin lymphoma onset or survival in central European Caucasians

The cohort consisted of 311 patients from whom genomic DNA-samples were available that had biopsy-confirmed, aggressive NHL according to the Revised European-American Lymphoma Classification (translated into the World Health Organisation classification) and were treated in the NHL-B1 and B2 study [15, 16] of the German High Grade Non-Hodgkin's lymphoma study group (DSHNHL). A subgroup of these patients was diagnosed with diffuse-large B-cell lymphoma (DLBCL; n = 205). Patients were excluded from the study if the diagnosis of aggressive or very aggressive lymphoma was not confirmed or if the diagnosis was changed into indolent lymphoma or no lymphoma at all by a panel of five expert hematopathologists in a blinded central pathology review. Other criteria for exclusion are summarized elsewhere [15, 16]. Table 1 outlines the clinico-pathological characteristics and table 2 the histopathological diagnoses of the patients. Blood donors (n = 512) from the Institute for Transfusion Medicine, University of Saarland Medical School, served as controls. DNA from patients diagnosed with B-NHL was collected at the University of Göttingen during the study period.

Genomic DNA was isolated from whole blood with the QIAamp Blood Kit (Qiagen, Hilden). DNA was diluted in water to a final concentration of 15 ng/μl to use 5 μl (45 ng) per reaction. The mutation tests were performed in the LightCycler 1.2 (p53) or LightCycler 480 (MDM2) instrument, using the FastStart DNA Master Hybridization Probes kit with 3 mM MgCl₂ (Roche Diagnostics, Mannheim) in a total volume of 20 μl, and analyzing the melting curve of the hybridization probes releasing from the PCR product. The analysis for the p53 codon 72 mutation was performed as described [17]. For the detection of the Mdm2 polymorphism rs2279744, we used 0.5 μM of the the primers mdmF^mt 5'ggCTgCggggCTgCT-3'(position 2565–2579 in Genbank AF527840), changing base 2575C to T (underlined), and primer mdmR 5'-CCAATCCCgCCCAgACTAC-3'(2611–2637), plus 0,25 μM of the detection probes, consisting of the 3'-terminal fluorescein-labeled Sensor(T) CTgCTTCggCgCg_gATgATCgCAg–FL (position 2575(---)2607), specific for the T allele (underlined) also containing the base 2575T and a gap for the target sequence positions 2588–2596, and the 5'-LightCycler Red 640 labeled and 3'-phosphorylated anchor probe 640-CCTgTCgggTCACTAgTgTgAACgCTg–PH (2611–2637)(TIB MOLBIOL, Berlin). After an initial denaturation at 95°C for 12 min 30 s, amplification was performed using 45 cycles of denaturation (95°C, 5 sec, ramp rate 4.4°C), annealing (60°C, 10 sec, ramp rate 2.2), and extension (72°C, 20 sec, ramp rate 4.4°C). Fluorescence was measured at the end of the annealing period of each cycle. After the amplification a melting curve was generated: the PCR mixture was heated to 95°C for 20 sec, ramp rate 4.4°C/s, cooled to 40°C, 20 sec, ramp rate 1.5°C/sec and then slowly heated to 85°C with one acquisition per °C. The fluorescence signal was monitored continuously during the temperature ramp and then plotted against the temperature. These curves were transformed to derived melting curves (-d(F2)/dT vs. T).

The MDM2 target sequence is extremely rich in strong GC bases (70%). This is known to cause difficulties in PCR amplification. In particular, we found stem loop CCGC–GCGG, spanning the polymorphism, to have a Tm of 82°C (OLIGO 6.0, MBI). This stem loop was excluded from the PCR fragment by introduction of one base substitution C2575T which had to be changed also in the sensor probe. The segment around the mutated base contains only G and C bases (85%), making a probe based analysis difficult. A very short probe will fail to bind the mismatched allele whereas a longer probe will reach extremely high binding temperatures, causing poor differentiation of the variants in the melting curve analysis. To overcome these difficulties we followed the procedures of [18], introducing a 9 nucleotides gap ggaggtccg in the sensor probe, resulting in a 24 mer sequence with a GC content of 67%.

Out of the NHL B1/2 trial patients, 311 DNA samples were available for analysis. The allelic frequencies were compared with Fisher's exact test. Mann-Whitney U test was used to test for differences among the onset of NHL by age. Event-free survival (EFS) was defined as the time from the beginning of therapy to either disease progression; initiation of salvage therapy; or additional (off-protocol) treatment, relapse, or death. Overall survival (OS) was defined as the time from first day of treatment to death from any cause. In EFS and OS analyses, a Cox multivariate analysis was done to adjust for known adverse risk factors, defined by the International Prognostic Index IPI, and additionally, for bulky disease (tumors > 7.5 cm anywhere). EFS and OS were estimated with the Kaplan-Meier method and were compared with the log-rank test. Differences between groups were regarded as significant for p values less than 0.05 (two-sided). Statistical analyses were performed with R 2.5.1. [19].

Among the 311 patients diagnosed with NHL, no difference in the onset by age between genders was detectable (p = 0.33). Specifically, male patients were diagnosed, on average, at the age of 58 years (range: 23–75 years) and female patients at the age of 59 years (range: 24–75 years)(Fig. 1). Similarly, in the subgroup of 205 patients diagnosed with DLBCL, no difference in the onset by age was detectable (p = 0.18). Men were diagnosed at the age of 57 years (range: 23–75 years) and women at the age of 59 years (range: 24–75 years)(figure 1).

Among 512 healthy central Europeans, 14% were homozygous for SNP309 G, and in the cohort of 311 central European patients diagnosed with NHL, 19% were homozygous; the genotype frequencies did not deviate from those expected under the Hardy-Weinberg equilibrium. Stratification according to SNP309 T/G genotypes failed to reveal differences in the onset of NHL by age in men (p = 0.25) and women (p = 0.29), and also in the onset of DLBCL by age in men (p = 0.19) and women (p = 0.82) (figure 2). Similar genotype frequencies were observed in the controls and the male NHL and DLBCL patient groups (figure 3). Previous work by Bond and colleagues had indicated that estrogen signalling can co-operate with the G-allele of SNP309 in lymphomagenesis in women, documented by stratification of the cohort in pre-menopausal women up to 51 years and older women [11]. In our study, no significant difference was detected between female NHL or DLBCL patients diagnosed by 51 years of age and those diagnosed later (figure 3A). Specifically, among all NHL patients, four of 31 women diagnosed by 51 years, and 26 of 105 women diagnosed later, exhibited the G/G genotype (p = 0.35). For women diagnosed with DLBCL, these numbers were 3/20 and 19/70, respectively (p = 0.59).

Next, we examined whether SNP309 T/G can influence prognosis. For this purpose, Kaplan-Meier plots for EFS and OS, stratified according to genotypes, were calculated (figure 4A). Cox proportional hazard analysis to adjust for IPI-factors (age >60; ex. involvement >1; ECOG 2–4; Stage III-IV; LDH > ONV) and bulky disease showed no difference between the genotype groups G/G and T/T. (An independent influence of SNP309 T/G on EFS/OS could not be detected in this multivariate analysis). The results of the Cox regression analyses are summarized in Table 3.

The allelic frequencies of the p53 gene SNP72 G/C polymorphism have been reported to vary widely between the ethnicities [20]. For European/North American Caucasians the frequency of the minor (C) allele coding for proline at position 72 was between 22 and 30% in the various studies, comparable with the frequency observed in our controls (26%) [21], and references therein). Among the patients diagnosed with NHL or DLBCL, the C allele was found with a frequency of 25%, respectively. Again, all genotype frequencies were as expected under the Hardy-Weinberg equilibrium.

Since it was conceivable that our female patients ≤ 51 years of age at the time of diagnosis, instead of having a reduced p53 response due to the SNP309 G/G genotype as was observed in the Ashkenazi Jewish cohort [11], have an apoptosis-impaired p53 associated with the SNP72 C/C genotype [22], we analyzed the SNP72 genotype frequencies by the same methods. No significant difference was detected between female NHL or DLBCL patients diagnosed by 51 years of age and those diagnosed later (figure 3B). Specifically, in the NHL group, two of 31 women diagnosed by 51 years and three of 105 women diagnosed later, exhibited the C/C genotype. For women diagnosed with DLBCL, these numbers were 2/20 and 2/70, respectively.

Finally, we examined whether SNP72 G/C has an influence on prognosis. Kaplan-Meier plots were calculated for EFS and OS, stratified according to genotypes (figure 4B). Cox proportional hazard analysis to adjust for IPI-factors (age >60; ex. involvement >1; ECOG 2–4; Stage III-IV; LDH > ONV) and bulky disease showed no difference between the genotype groups C/C and G/G. (Again, an independent influence of SNP72 G/C on EFS/OS could not be detected in this multivariate analysis). The results of the Cox regression analyses are summarized in Table 3.