TP53 Mutation in Waldenstrom Macroglobulinemia

Background. Waldenstrom macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow (BM) infiltration of clonal lymphoplasmacytic cells, which produce a monoclonal immunoglobulin M. MYD88L265P mutation may be considered as a founder event because of it high frequency in WM. WM cells may acquire additional genetic hits that may potentially promote disease progression: CXCR4 or CD79B mutations, copy number variation,…TP53 is a tumor suppressor gene that functions as regulator influencing cellular responses to DNA damage. Little is known regarding TP53 alteration in WM. Our aim was to screen TP53 mutation in a large cohort of WM at diagnosis to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa) and to identify clinical and biological characteristics.

Method. BM samples of 125 WM (mean age: 67 years) were analyzed at diagnosis. Tumoral DNA was extracted following CD19 B cell selection. TP53 mutations were analyzed by targeted NGS to scan the coding exons of TP53. MYD88L265P, CD79A, CD79B, and CXCR4mutations were analyzed by sanger sequencing and/or NGS. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 62 cases. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess P53 and p21 expression after nutlin3a exposition to characterize functional mutant of TP53. Viability and cell growth of treated cells were determined using the MTS assay.

Results. We have identified TP53 mutations using NGS in 7.3 % of WM (6 non-sense, 3 frameshift mutations located in the DNA binding domain) (TP53^mut WM). The mutation load of TP53 varied from 13% to 98.9% (mean: 62.0%) using the variant allele frequency in NGS. We next examined the effects of nutlin-3a which is an mdm2 inhibitor on WM patients CD19+ cells genotyped for TP53 mutation. Nutlin-3a increased the expression of p53 and p21 in TP53^Wild WM patients using flow cytometry (n=6). In contrast, in TP53^MutWM cells, no significant variation of p53, p 21 and viability using MTS assay was observed suggesting the presence of functional mutation of TP53. The minimal deleted region of 17p in 17p deleted (TP53^Del) samples was mapped using SNP array and contained 79 genes, among which was systematically comprised the loss of TP53. A high correlation between TP53 mutation and deletion 17p (p<10⁶) was observed. One case of CN-LOH was observed at TP53 locus (1,6% of cases). Overall, we have identified alteration of TP53 locus including mutation, deletion and copy neutral loss of heterozigosity in 11, 2% of WM. Using SNP array, we found a relationship between deletion 17p, alteration of TP53 locus including mutation, UPD or del17p (TP53^Alt) and TP53^Mutand a greater frequency of genomic aberrations in WM compared toTP53^wild (p=0.01, p=0.024 and p=0.06 respectively). A higher frequency of WM patients with more than 3 CNA identified by SNPa was observed in TP35^Mut group (p=0.03) and del17p group (p<0.00001). No association was observed between TP53^Mut and CXCR4 and MYD88mutations.

We thoughtto identify clinical and biological characteristics of WM according to TP53^Mutand/or Del17pfeatures. With a median follow-up of 5 years, 33 (26%) patients had died. 69% of cases were treated. Front line therapy included rituximab-based regimens in 76%, alkylating agent in 78%, fludarabine in 6%. The WM with TP53alteration, irrespective of TP53^Mut or del17p, displayed features of adverse prognosis in regards to higher serum levels of b2m (89% versus 40%, p=0.012), and also greater IPSSWM score 2 and 3 (50% versus 30%, and 43% versus 30%, p=0.041, respectively). Importantly, the presence of TP53alteration, irrespective of TP5^Mut or del17p, was associated to poor outcome in overall survival in our series, TP53alteration (p=0.003), del17p (p=0.002), and TP53^Mut(p=0.015). TP53 alteration prognostic value was independent of CXCR4 or MYD88L265Pmutations.

Conclusion: A low frequency of TP53 mutation was observed in WM at diagnosis. We identified a genomic signature associated to their presence. In addition, a pejorative prognostic value of TP53 mutation was observed in WM highlighted the need of new therapeutic in this sub group of WM.