Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

• MeSH
• Alleles MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell blood   genetics   pathology   MeSH
• Humans MeSH
• Mutation MeSH
• DNA Mutational Analysis instrumentation   methods   MeSH
• Neoplastic Cells, Circulating pathology   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Genes, Reporter genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Transfection instrumentation   methods   MeSH
• High-Throughput Nucleotide Sequencing instrumentation   methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Adult MeSH
• Genes, p53 * MeSH
• In Situ Hybridization, Fluorescence MeSH
• Polymorphism, Single Nucleotide MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Aged MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

• MeSH
• Drug Resistance, Neoplasm genetics   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   genetics   mortality   MeSH
• Genes, p53 genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Kaplan-Meier Estimate MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Mutational Analysis MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• DNA Damage genetics   MeSH
• Prognosis MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• Gene Silencing MeSH
• Immunoglobulin Variable Region genetics   MeSH
• Vidarabine analogs & derivatives   therapeutic use   MeSH
• Blotting, Western MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

PURPOSE: There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. PATIENTS AND METHODS: We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. RESULTS: A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). CONCLUSION: The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

• MeSH
• Time Factors MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   mortality   MeSH
• Gene Deletion MeSH
• DNA-Binding Proteins chemistry   genetics   MeSH
• Adult MeSH
• Genes, p53 MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Adolescent MeSH
• Mutation MeSH
• Prognosis MeSH
• Protein Binding MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

• MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   genetics   physiology   MeSH
• Chromosome Deletion MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   genetics   pathology   MeSH
• Adult MeSH
• Doxorubicin pharmacology   MeSH
• Cohort Studies MeSH
• Leukocytes, Mononuclear drug effects   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosomes, Human, Pair 11 genetics   MeSH
• Mutation genetics   MeSH
• Retrospective Studies MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Cell Survival drug effects   physiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH