Screening for TP53 Mutations Identifies Chronic Lymphocytic Leukemia Patients with Rapid Disease Progression.

Screening for cytogenetic aberrations with a selected panel of FISH probes has identified important prognostic subgroups in chronic lymphocytic leukemia (CLL). Good prognosis CLL patients with deletions of the long arm of chromosome 13 (del13q) as sole aberration are opposed by patients with deletions of the short arm of chromosome 17 (del17p). The tumor suppressor gene TP53 is located at 17p13 and loss of TP53 is hypothesized to be at least partially responsible for the poor prognosis of del17p CLL patients. However, it is not clear, if the loss of other genes in the deleted region contributes to the poor prognosis. In addition, the degree of overlap between the patient populations defined by del17p and TP53 mutation is only poorly defined. Therefore, we characterized peripheral blood or bone marrow samples of 193 CLL patients by FISH analysis and screened for TP53 mutations by two methods, i.e. by denaturing high performance liquid chromatography (dHPLC) and by a microarray-based resequencing assay, the AmpliChip p53 Test. PCR products of exons 3–9 of TP53 were screened by dHPLC and aberrant fragments were analyzed by direct sequencing, whereas the entire coding region including the splice sites of exons 2–11 were analyzed with the AmpliChip p53 Test. The overall incidence of TP53 mutations by both methods was 13.5% (26/193), whereas the incidence of del17p by FISH was 9.3% (18/193). Interestingly, 17 out the 18 del17p samples carried a TP53 mutation suggesting that loss of TP53 does indeed play a pivotal role in the poor prognosis of del17p. At least 9 samples carried a TP53 mutation only. The AmpliChip p53 Test detected 32 mutations in 25 patients compared to 24 mutations detected in 20 patients by dHPLC/direct sequencing. The AmpliChip p53 Test, which is designed to detect single nucleotide substitutions and single nucleotide deletions, did not detect 3 mutations (1 1-bp deletion, 1 4-bp deletions, 1 single nucleotide insertion). The method of dHPLC followed by direct sequencing did not call 10 single nucleotide mutations. Of these, 1 mutation was located in exon 10 not included in the dHPLC screening. The remaining 9 mutations were detected by dHPLC analysis, but failed to be called by direct sequencing. The clinical course of patients with TP53 aberrations (n=20) (del17p and/or TP53 mutation) was compared to 113 patients lacking these abnormalities. Patients with TP53 aberration had a highly significantly decreased time to progression compared to patients without TP53 aberration (p<0.001, median 13.2 vs. 64.4 months). This difference remained significant when analysis was restricted to patient samples without prior therapy (p<0.001, median 9.2 (n=13) vs. 70.6 months (n=101)). As FISH analysis for del17p is the standard approach to detect TP53 aberrations, we compared the clinical course of patients with del17p (n=8) to patients with TP53 mutation (without del17p) (n=7) vs patients without TP53 aberration (n=113). This analysis resulted in a median time to progression of 9.2 vs 22.4 vs 63.4 month, respectively (p<0.001). The data of the present analysis suggest that TP53 mutation might be one of the factors conferring poor prognosis to CLL patients. Likewise, we identified 9 samples (4.7%) with TP53 mutation alone with poor clinical course that would have escaped detection by FISH analysis.