Assessment of p53 Functionality in Chronic Lymphocytic Leukemia by Different Assays; An Eric-Wide Approach.
Authors: te Raa Doreen , Malcikova Jitka , Mraz Marek , Pospisilova Sarka , Le Garff-Tavernier Magali , Merle-Beral Helene , Stankovic Tatjana , van Oers Marinus H. J. , Eldering Eric , Stilgenbauer Stephan , Zenz Thorsten , Kater Arnon P. 
Published: October 2019

Abstract
Abstract 2872

Introduction:
The DNA damage response axis plays a crucial role in chemoresistance in CLL, as indicated by the prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM). These deletions coincide with mutations in the remaining allele, although frequency, especially for ATM varies. Functional read-outs of the p53 axis might add clinical relevant information on the actual DNA damage response. Currently, different p53 function analyses are being developed. These assays are either based on measurement of (i) RNA expression levels of a single gene (RT-PCRp21) or gene sets (RT-MLPA), or protein expression levels (FACSp53-p21) after DNA damage by irradiation or etoposide/nutlin exposition or (ii) gene expression levels at base-line (RT-PCR miR34a). To what extent these different assays correlate is currently unknown.

Aim:
Detailed side-by-side analysis of available p53 functional assays in well characterized CLL samples

Methods:
Freshly frozen PBMC's of 15 different CLL samples (CD19/CD5>90%) were exchanged between 5 research groups that developed the following p53 functional assays: RT-PCRp21 (Ulm), RT-MLPA (bax, puma, p21 and CD95; Amsterdam), FACSp53-p21 (Paris) and RT-PCR miR34a (Salzburg/Brno). FISH-analysis (11q, 17p, 13q and 12) was performed on all samples. In addition mutations in TP53 were determined by FASAY, DHPLC and Sanger sequencing and ATM mutations were assessed by Sanger sequencing.

Results:
RNA expression levels showed significant correlation (p-value < 0,01) between the different P53 functional assays with high correlation coefficients (range: 0,7–0,94). Based on combination of FISH-analysis, FASAY and Sanger sequencing the investigated CLL samples could be distinguished into 6 different categories; 1. 17p- with TP53 mutation (n=5), 2. Sole TP53 mutation (n=1), 3. 11q- with ATM mutation (n=1), 4. 11q- in absence of TP53/ATM mutation (n=4), 5. 17p- with both TP53 mutation and ATM mutation (n=1) and 6. No 17p- and 11q- in absence of TP53/ATM mutation (n=3). All p53 functional assays showed absent to minimal induction of expression of respective target genes in samples with a cytogenetic abnormality combined with a TP53/ATM mutation (category 1, 3, 5). In contrast, samples without a cytogenetic abnormality in absence of TP53/ATM mutations (category 6) showed a marked increase in expression of respective target genes in all the assays. The patient with a sole TP53 mutation (category 2) showed a normal DNA damage response in all assays except for FACSp53-p21. Samples with an 11q- in absence of TP53/ATM mutation (category 4) showed high inter-assay variation. The two assays with predefined cut-off values (RT-MLPA and FACSp53-p21) assessed 13 samples equally (9 p53 dysfunctional and 4 p53 functional), except for 2 patients (category 2, 3 respectively). Reproducibility of these assays was tested by blinded sending of 3 samples that have been analyzed previously in this study. Except for p21, all assays showed 100% reproducibility.

Discussion:
For the first time a comparative side-by-side analysis of different available p53 functional assays was performed, and demonstrated strong correlations between the different assays. All assays could detect samples with expected disturbed and normal DNA-damage responses. To what extent these p53 functional assays could be of clinical relevance especially with respect to chemo-responsiveness, should be further studied in prospective studies.

Disclosures:
No relevant conflicts of interest to declare.