Effect of Polymorphisms in p53 Pathway Proteins and B-Chronic Lymphocytic Leukemia Disease Progression,

Abstract 3888

In B-CLL, mutations in the IGHV genes encoding the BCR are correlated with disease aggressiveness: patients with unmutated BCR (U-CLL) typically have a worse prognosis than those with mutated BCR (M-CLL). Evidence that many U-CLL express BCR with specificity toward apoptotic cell antigens suggests that dying cells provide pro-survival signals for U-CLL clones. Thus progression of U-CLL may depend both on mechanisms that ensure the clone's survival and those that promote cell death.

A single nucleotide polymorphism (SNP) within the p53 gene at codon 72 significantly affects p53 function: p53-72R (CG C=Arg) is substantially better at inducing apoptosis than p53-72P (CC C=Pro). Although p53-72P is linked with worse outcomes in a number of malignancies, p53-72R has been linked to heightened B-CLL incidence (Leuk Res. 2006;30:1113–8) and to diagnosis as U-CLL (Br J Haematol. 2011;153:533–5). Pro-apoptotic p53-72R may be fostering the apoptotic self antigen needed for BCR signaling in U-CLL. MDM2 is a ubiquitinase with a major role in keeping p53 levels low. Following DNA damage, p53 is phosphorylated by ATM and protected from MDM2. A SNP within the promoter affects MDM2 RNA/protein levels: SNP309G > SNP309T. In some B-CLL cohorts, the SNP309G allele was associated with disease progression (Eur J Haematol. 2010;85:251–6; Blood 2010;115:4191–7).

We investigated a cohort of 93 Caucasian B-CLL patients from the New York City metropolitan area for expression of the above p53 and MDM2 SNPs. We examined whether the B-CLL-expressed SNPs differ from Caucasian controls in the same geographic area (Lupus 2009;18:61–6). Additionally, we examined whether U-CLL and M-CLL patients in this cohort vary in SNP expression and assessed whether the above SNPs influence disease progression, as determined by time to first treatment (TTFT) and overall survival (OS).

Isolated genomic DNA underwent PCR and pyrosequencing for the p53 SNP at codon 72 (rs1042422) and the MDM2 SNP309 (rs2279744) using primers selected by PSQ Assay Design. Primers used for p53: forward = Biotin-5'-GAAGACCCAGGTCCAGATGA and reverse = 5'-CCGGTGTAGGAGCTGCTG, resulting in an 82 bp PCR product; sequencing = 5'-GGTGCAGGGGCCACG. Primers used for MDM2: forward = 5'-ATTTCGGACGGCTCTCGC and reverse = Biotin-5'-CTAGTGACCCGACAGGCACCT, resulting in a 147 bp PCR product; sequencing = 5'-GGGCTGCGGGGCCGCT. Pyrosequencing was done on a PSQ HS96A instrument as per manufacturer's guidelines using PyroMark Gold Q96 Reagents.

Our cohort of B-CLL was not significantly different from controls in distribution of the p53 SNP. However, there was a higher proportion of MDM2 SNP309G/G in B-CLL compared to controls (CLL=31%; normal=16%; p=0.0238). A p53-72R/R genotype was evident in a lesser proportion of U-CLL than M-CLL, albeit the difference was not statistically significant (n=80, p=0.1860). On the basis that U-CLL with p53-72R/R genotype might be favored if the clone underwent a deletion of p53-augmenting ATM, we compared the p53-72 SNP genotype of U-CLL ± FISH evidence of ATM deletion (11q22.3) (patients with 17p13 p53 deletion were excluded). Interestingly, the p53-72R/R genotype was significantly greater in U-CLL patients with ATM deletion than those without (n=27; p=0.0002).

Comparison of p53-p72 SNP to TTFT showed trends in agreement with Majid, et al. (Br J Haematol. 2011;153:533–5). B-CLL patients with p53-72P/P had a shorter TTFT (P/P=41 months; R/R=97 months; n=88). However, due to limited power, this did not reach significance (p=0.1531). Also consistent with the above-cited study, the single M-CLL with p53-72P/P had the shortest TTFT, while M-CLL with p53-72R/R had the longest (n=48; p<0.0001).

Comparison of MDM2 SNP309 to TTFT in all studied B-CLL patients suggested that those with SNP309G have a shorter TTFT than those with SNP309T (G/G=44 months; T/T+G/T=111 months), but the differences did not reach significance (p=0.1048). However, when considering mutation status, U-CLL with SNP309G/G had the shortest TTFT (U-CLL G/G=33 months; n=42; p=0.0079). With our cohort, there was no statistically significant correlation between the p53 or MDM2 SNPs and CD38 level or OS. Taken together, our study suggests that the genotype of these functionally relevant SNPs may influence B-CLL subtype and disease progression.