Loss or Mutation of TP53 Is Highly Associated with Complex Aberrant Karyotype and Chromosomal Translocations in Chronic Lymphocytic Leukemia.

Abstract In chronic lymphocytic leukemia (CLL) cytogenetic aberrations such as del(17p) and del(11q) predict inferior outcome. In addition, complex aberrant karyotypes as well as chromosomal translocations as defined by metaphase cytogenetics were suggested as poor prognostic markers for overall survival. We screened 194 consecutive CLL patients for del(17p)/TP53-deletion by fluorescence in situ hybridization (FISH) and for TP53-mutations by denaturing high performance liquid chromatography (DHPLC) and subsequent direct sequencing of aberrant fragments. In addition 160 of these CLL patients were analyzed by classical metaphase cytogenetics to determine the incidence of TP53-aberration in different cytogenetic subgroups. Interphase FISH on 194 samples detected TP53-deletions in 9.3% (n=18) of cases. In parallel, exons 3–9 of the TP53 gene were screened by DHPLC and an aberrant pattern was detected in 9.8% (n=19) of cases. TP53-mutations were confirmed and further characterized by direct sequencing in 16 of the 19 cases. The residual 3 samples had an aberrant pattern in DHPLC for the amplicon of exons 8–9 which pointed to a small population of TP53-aberrant cells which was beyond the detection limit of sequencing. 16 of 18 (89%) cases with TP53-deletion were accompanied by a TP53-mutation affecting the residual allele. 3 samples with TP53-mutations had no deletion of one TP53 allele. Therefore, the overall incidence of TP53-aberrations was 10.8 % (21/194) with a significant association of TP53-deletion and TP53-mutation (p<0.0001). Metaphase cytogenetics was performed on 160 CLL samples. A complex aberrant karyotype defined by ≥ 3 aberrations was identified in 14% of samples (22/160). The incidence of TP53-aberrations in this cytogenetic subgroup was 50% (11/22) and therefore significantly higher than in other cytogenetic subgroups (p<0.0001). Among 160 samples with cytogenetic analysis 49 (31%) exhibited translocations. We divided these translocations into subgroups with karyotypes carrying balanced translocations only (n=18), carrying unbalanced translocations only (n=20) as well as karyotypes with both balanced and unbalanced translocations (n=11). Within the entire group of translocations the incidence of TP53-aberration was 27% (13/49). The incidence of TP53-aberrations was 5.5% (1/18) in the group with only balanced translocations, 40% (8/20) in the group with only unbalanced translocations and 36% (4/11) where balanced and unbalanced translocation occurred in combination. When the latter two groups with unbalanced translocations were combined TP53-aberration occurred in 39% (12/31) of cases. Altogether the association of TP53-aberration with translocations was strong (p<0.0001) especially with unbalanced translocations (p<0.0001) whereas no coherency with balanced translocations could be demonstrated (p>0.05). Furthermore, translocations were detected in 91% (20/22) and unbalanced translocations in 82% (18/22) of complex karyotypes. The association of translocations, in particular unbalanced translocation with complex aberrant karyotype was significant (for both p<0.0001). In conclusion: Loss of TP53 and TP53 mutations occur with a frequency of 9.3% and 9.8%, respectively and are significantly associated. A highly significant association of TP53-aberrations with complex aberrant karyotypes and unbalanced translocations was observed. We hypothesize that TP53-aberrations might contribute to genetic instability leading to accumulation of cytogenetic aberrations especially unbalanced translocations.

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Prognostic Impact of NOTCH1 Hotspot Mutation in TP53-Mutated Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.3283.3283 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3283-3283

Author(s):

Barbara Kantorova ◽

Jitka Malcikova ◽

Veronika Navrkalova ◽

Jana Smardova ◽

Kamila Brazdilova ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Tp53 Mutation ◽

Direct Sequencing ◽

Lymphocytic Leukemia ◽

The Other ◽

Wild Type ◽

Tp53 Mutations ◽

Time To First Treatment ◽

Hotspot Mutation

Abstract Introduction A presence of activating mutations in NOTCH1 gene has been recently associated with reduced survival and chemo-immunotherapy resistance in chronic lymphocytic leukemia (CLL). However, a prognostic significance of the NOTCH1 mutations with respect to TP53mutation status has not been fully explained yet. Methods An examined cohort included 409 patients with CLL enriched for high risk cases; in 121 patients consecutive samples were investigated. To determine the TP53 mutation status, a functional analysis of separated alleles in yeast (FASAY, exons 4-10) combined with direct sequencing was performed; the ambiguous cases were retested using an ultra-deep next generation sequencing (MiSeq platform; Illumina). The presence of NOTCH1 hotspot mutation (c.7544_7545delCT) was analyzed using direct sequencing complemented by allele-specific PCR in the selected samples. In several patients harboring concurrent NOTCH1 and TP53 mutations, single separated cancer cells were examined using multiplex PCR followed by direct sequencing. A correlation between mutation presence and patient overall survival, time to first treatment and other molecular and cytogenetic prognostic markers was assessed using Log-rank (Mantel-cox) test and Fisher's exact test, respectively. Results The NOTCH1 and TP53 mutations were detected in 16% (65/409) and 27% (110/409) of the examined patients, respectively; a coexistence of these mutations in the same blood samples was observed in 11% (19/175) of the mutated patients. The detected increased mutation frequency attributes to more unfavorable profile of the analyzed cohort; in the TP53-mutated patients missense substitutions predominated (75% of TP53 mutations). As expected, a significantly reduced overall survival in comparison to the wild-type cases (147 months) was observed in the NOTCH1-mutated (115 months; P = 0.0018), TP53-mutated (79 months; P < 0.0001) and NOTCH1-TP53-mutated patients (101 months; P = 0.0282). Since both NOTCH1 and TP53 mutations were strongly associated with an unmutated IGHV gene status (P < 0.0001 and P = 0.0007), we reanalyzed the IGHV-unmutated patients only and interestingly, the impact of simultaneous NOTCH1 and TP53 mutation presence on patient survival was missed in this case (P = 0.1478). On the other hand, in the NOTCH1 and/or TP53-mutated patients significantly reduced time to first treatment was identified as compared to the wild-type cases (41 months vs. 25 months in NOTCH1-mutated, P = 0.0075; 17 months in TP53-mutated, P < 0.0001; and 18 months in NOTCH1-TP53-mutated patients, P = 0.0003). The similar results were observed also in the subgroup of the IGHV-unmutated patients, with the exception of patients carrying sole NOTCH1 mutation (P = 0.2969). Moreover, in the NOTCH1-TP53-mutated patients an increased frequency of del(17p)(13.1) was found in comparison to the TP53-mutated patients only (72% vs. 56%); this cytogenetic defect was not detected in the patients with sole NOTCH1 mutation. Our results might indicate, that NOTCH1 mutation could preferentially co-selected with particular, less prognostic negative type of TP53 defects. Notably, in our cohort the NOTCH1 mutation predominated in the patients harboring truncating TP53 mutations localized in a C-terminal part of the TP53 gene behind the DNA-binding domain (P = 0.0128). Moreover, in one of the NOTCH1-TP53-mutated patients the analysis of separated cancer cells revealed a simultaneous presence of NOTCH1 mutation and TP53 in-frame deletion in the same CLL cell. In contrast, in the other examined NOTCH1-TP53-mutated patient the concurrent NOTCH1 mutation and TP53 missense substitution (with presumed negative impact on patient prognosis) were found in different CLL cells. Conclusions The parallel presence of NOTCH1 hotspot mutation might be detected in a significant proportion of TP53-mutated patients and it seems to be associated with less prognostic unfavorable TP53 mutations. Nevertheless, these preliminary data should be further confirmed in a large cohort of patients. This study was supported by projects VaVPI MSMT CR CZ.1.05/1.1.00/02.0068 of CEITEC, IGA MZ CR NT13493-4/2012, NT13519-4/2012 and CZ.1.07/2.3.00/30.0009. Disclosures Brychtova: Roche: Travel grants Other. Doubek:Roche: Travel grants Other.

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Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up

Blood ◽

10.1182/blood-2008-04-154070 ◽

2008 ◽

Vol 112 (8) ◽

pp. 3322-3329 ◽

Cited By ~ 272

Author(s):

Thorsten Zenz ◽

Alexander Kröber ◽

Katrin Scherer ◽

Sonja Häbe ◽

Andreas Bühler ◽

...

Keyword(s):

Tp53 Mutation ◽

Direct Sequencing ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Clone Size ◽

Tp53 Mutations ◽

Long Term Follow Up ◽

Serial Samples

AbstractThe exact prognostic role of TP53 mutations (without 17p deletion) and any impact of the deletion without TP53 mutation in CLL are unclear. We studied 126 well-characterized CLL patients by direct sequencing and DHPLC to detect TP53 mutations (exons 2-11). Most patients with 17p deletions also had TP53 mutations (81%). Mutations in the absence of 17p deletions were found in 4.5%. We found a shorter survival for patients with TP53 mutation (n = 18; P = .002), which was more pronounced when analyzed from the time point of mutation detection (6.8 vs 69 months, P < .001). The survival was equally poor for patients with deletion 17p plus TP53 mutation (7.6 months, n = 13), TP53 mutation only (5.5 months, n = 5), and 17p deletion only (5.4 months, n = 3). The prognostic impact of TP53 mutation (HR 3.71) was shown to be independent of stage, VH status, and 11q and 17p deletion in multivariate analysis. Serial samples showed evidence of clonal evolution and increasing clone size during chemotherapy, suggesting that there may be patients where this treatment is potentially harmful. TP53 mutations are associated with poor sur-vival once they occur in CLL. The de-monstration of clonal evolution under selective pressure supports the biologic significance of TP53 mutations in CLL.

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The TP53 Codon72 Polymorphism Is Associated with TP53 Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1783.1783 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1783-1783 ◽

Cited By ~ 1

Author(s):

Vera Grossmann ◽

Valentina Artusi ◽

Susanne Schnittger ◽

Frank Dicker ◽

Sabine Jeromin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mutation Rate ◽

Tp53 Mutation ◽

Genome Project ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Employment Equity ◽

Tp53 Mutations ◽

Equity Ownership ◽

Exon 4

Abstract Abstract 1783 TP53 is one of the most important cell-cycle regulator genes and its tumor suppressor activity is fundamental in cellular responses. Mutations in TP53 are known to influence clinical outcome in diverse diseases. In particular, a relationship between TP53 mutations and a poor prognosis has been established in chronic lymphocytic leukemia (CLL), which is one of the most commonly diagnosed lymphoid malignancies in Western countries. Thus far, it has been demonstrated that TP53 mutations are associated with codon72 polymorphism in different diseases e.g. breast cancer, lung cancer, head and neck squamous cell carcinoma, and that this variant could determine cancer susceptibility. In this study, we investigated the overall TP53 mutation rate in 511 CLL and focused on the codon72 polymorphism (rs1042522) in exon 4 (transcript-ID: ENST00000269305). We initially examined the published available 1000 Genome Project results of the European cohort: from a total of 283 genomes analyzed, 137 showed an ARG/ARG genotype (48%), 124 an ARG/PRO genotype (43%) and 22 a PRO/PRO genotype (7.7%). Secondly, in order to determine a potential association between this polymorphic variant and mutations in the TP53 gene, we investigated 511 thoroughly characterized patients with CLL, all diagnosed by immunophenotyping in our laboratory. For molecular analyses, all cases were analyzed for TP53 mutations (exon 4 to exon 11) either by DHPLC and subsequent Sanger sequencing (n=210/511), or using a sensitive next-generation amplicon deep-sequencing assay (n=301/511) (454 Life Sciences, Branford, CT). We observed the occurrence of the three distinct genotypes (ARG/ARG, ARG/PRO, PRO/PRO) of codon72 in the CLL cohort and detected ARG/ARG as the most common genotype (63%), followed by ARG/PRO (31.7%), and PRO/PRO (5.3%); very similar to the distribution of the codon72 polymorphism in the 1000 Genome Project data. Moreover, mutations in TP53 were detected in 63/511 patients resulting in an overall mutation rate of 12%, which reflects the expected mutation rate in this disease. Importantly, as already demonstrated in other malignancies, we here present that also in CLL patients harboring a PRO/PRO genotype a significantly higher frequency of TP53 mutations (9/27, 33%) was observed compared to ARG/ARG (41/321, 13%, P=.037) and ARG/PRO (13/163, 8%, P=.012). With respect to the clinical outcome we confirmed a generally poor survival for the TP53 mutated cases as compared to TP53 wild-type patients (n=23 vs. 189 with clinical data available, alive at 7 years: 29.6% vs. 88.1%; P<.001). Moreover, the impact of the three distinct genotypes on outcome was analyzed. However, no correlation was detectable, neither in the cohort of TP53 mutated cases (P=.225) nor in the TP53 wild-type patients (P=.190). In summary, we demonstrated a significant association between the codon72 allelic variant and TP53 mutation rate in our CLL cohort. Patients with a PRO/PRO genotype showed a significantly higher frequency of TP53 mutations than all other genotypes. However, no prognostic impact of codon72 allelic variant was observed, neither in the TP53 wild-type nor in the TP53 mutated cohort. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

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Single Cell Analysis Proves the Coexistence of NOTCH1 and TP53 Mutations within the Same Cancer Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.2913.2913 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2913-2913

Author(s):

Barbara Kantorova ◽

Jitka Malcikova ◽

Kamila Brazdilova ◽

Marek Borsky ◽

Karla Plevova ◽

...

Keyword(s):

Single Cell ◽

Tp53 Mutation ◽

Single Cell Analysis ◽

Direct Sequencing ◽

Gene Mutations ◽

Lymphocytic Leukemia ◽

Cell Analysis ◽

Tp53 Mutations ◽

First Time ◽

Clonal Composition

Abstract Introduction Mutations in NOTCH1 and especially TP53 genes represent potent drivers of chronic lymphocytic leukemia (CLL) progression and chemo-refractoriness. Although a coexistence of these mutations was reported in CLL, a molecular basis of this phenomenon has not been described yet. To clarify this issue, we performed a detailed analysis of CLL patients with parallel NOTCH1 and TP53 mutations including single cancer cell examination. Methods TP53 mutations were determined based on FASAY analysis coupled with direct sequencing. In a collected cohort of 111 TP53 -mutated patients a presence of hot spot c.7544_7545delCT NOTCH1 mutation was assessed using direct gDNA sequencing. In NOTCH1 -TP53 -mutated patients with available material, the mutations' coexistence was tested in single flow-sorted CD19+ cells (cancer cell proportion > 80 %) using multiplex PCR followed by direct sequencing. Results The NOTCH1 mutation was detected in 19/111 (17 %) of the TP53 -mutated patients. Eleven of the NOTCH1-TP53 -mutated patients carried single TP53 mutation; multiple TP53 mutations were detected in 8 of them. Based on gDNA sequencing, the NOTCH1 and TP53 mutation coexistence in the same cancer cells was evident in 4/19 of the NOTCH1-TP53-mutated patients, as at least one of the gene mutations occurred in 100 % of the DNA. In the remaining 15 NOTCH1-TP53 -mutated patients the clonal composition was not possible to assess using sequencing data only and therefore a single cell analysis was performed in 8 of them with available material. Remarkably, irrespective of the mutation proportion, in all of these patients the NOTCH1 mutation was always present together with at least one of the detected TP53 mutations. Considering both the DNA sequencing and single cell analysis data, the 12 patientswith proven NOTCH1-TP53 mutation coexistence might be stratified into three groups with different clonal composition: i) patients with NOTCH1 and single TP53 mutations showing a comparable mutation proportion (n = 3), in which both gene mutations were always detected in the same cells and never occurred separately; ii) patients with either NOTCH1 or TP53 mutation predominance (n = 6), in which the predominant mutation was present separately as well as in combination with the coexisting mutation(s) in individual cells; iii) patients with NOTCH1 and multiple TP53 mutations showing different mutation proportion (n = 3), in which NOTCH1 mutation was present together with one of the detected TP53 mutations in the same cells, while the other TP53 mutations occurred separately. In two of the NOTCH1-TP53 -mutated patients who received intensive chemo-immunotherapy, the consecutive samples were available for single cell analysis. In one of these patients only single TP53 mutation was detected at first time point. In relapse after rituximab-dexamethasone treatment the clone carrying the original TP53 mutation expanded in parallel with another NOTCH1-TP53-mutated clone. Different situation was noticed in the second patient, in which the NOTCH1-TP53-mutated clone detected at first time point diminished after alemtuzumab treatment, while another TP53-mutated-NOTCH1-wild-type clone expanded in relapse. Conclusion We have shown that in NOTCH1-TP53 -mutated patients the mutations often coexist in the same CLL cells. These patients exhibit a considerable clonal heterogeneity that may be further influenced by chemo-immunotherapy. This study was supported by IGA NT/13493 and NT/13519, MUNI/A/1180/2014, CZ.1.05/1.1.00/02.0068. Disclosures Mayer: Janssen: Research Funding.

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Genomic Mechanisms of 17p / TP53 Loss in Primary “ultra High-risk” and Refractory Chronic Lymphocytic Leukemia: Results from the CLL2O Trial

Blood ◽

10.1182/blood.v124.21.2184.2184 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2184-2184

Author(s):

Veronica Teleanu ◽

Jennifer Edelmann ◽

Claudia Haferlach ◽

Stefan Ibach ◽

Eugen Tausch ◽

...

Keyword(s):

High Risk ◽

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Tp53 Mutation ◽

Lymphocytic Leukemia ◽

Chromosome 17 ◽

Tp53 Mutations ◽

Ultra High Risk ◽

Treatment Naïve ◽

Donor Chromosome

Abstract Background: Unraveling the cytogenetic background helped to decipher the molecular basis of many hematologic cancers and to develop specific therapies. Recently, using chromosome banding analysis (CBA), jumping translocations were identified as a cause of 17p loss in multiple myeloma, providing new insights into the origin of clonal evolution and copy number alterations (CNA) (Sawyer et al, Blood 2014). In chronic lymphocytic leukemia (CLL) the genomic mechanisms leading to 17p loss are not fully understood. Aims: Characterization of underlying mechanisms of 17p loss using CBA and correlation with other clinicobiological features in “ultra high-risk” CLL. Methods: Samples from 112 patients (pts.) with refractory and/or 17p- CLL enrolled in the multicenter CLL2O trial were screened for CNAs by Affymetrix 6.0 SNP array analysis of CD19 sorted CLL cells and for chromosomal abnormalities by CBA using CpG oligonucleotide and interleukin-2 stimulation. Results: Considering both CBA and SNP data, 728 aberrations resulted in a mean of 6.5/case. 89 (79%) pts. had 17p deletion and 83 (74%) TP53 mutation. Regarding the origin of 17p/TP53 loss, 6 distinct types of rearrangements could be delineated: 1) whole arm translocations (WAT) 2) jumping translocations (JT) 3) dicentric chromosomes (DC) 4) cytogenetically balanced translocations (CBT) 5) other unbalanced translocations and 6) interstitial 17p deletions. WAT were identified in 33/112 (30%) cases and 30/33 (91%) involved chromosome 17 leading to 17p loss. Chromosomes involved ≥ 2 times in an unbalanced WAT were der(17;18)(q10;q10) (8, 24%), der(8;17)(q10;q10) (5, 15%), der(15;17)(q10;q10) (4, 12%), i(17)(q10) (4, 12 %), der(17;22)(q10;q10) (2, 6%). JT were identified in 11 (10 %) cases, 6 showing jumping WAT with 17q as donor chromosome, 1 case with breakpoints located in the pericentromeric regions of chromosome 17p11 (donor chromosome) and the receptor chromosomes 4p14 and 16p11. In 4 cases, initially a WAT involving 17q occurred and subsequently the partner chromosome “jumped off” leaving a 17p deletion behind. DC were detected in 19 pts., 8 with breakpoint in 17p11, 7/8 with TP53 mutation. Of note, all cases had the breakpoint on chromosome 17 in 17p11 indicating a fragile site affecting the pericentromeric region. Interestingly, of a total of 382 translocations observed by CBA, only 32 were CBT and except for those involving the IGH and IGK/L loci (n=6) all were random. 17p involvement in CBT was detected in 4 cases, 3 had TP53 deletion and all were TP53 mutated. Of the unbalanced translocations, der(17)t(8;17) was identified in 5 pts. simultaneously generating 8q gain. Nevertheless, breakpoints on chromosome 17p covered cytobands 17p11-13 and on chromosome 8, 8q11-22, one case having the breakpoint telomeric to the TP53 locus and no TP53 mutation, pointing to other putative candidate genes on 17p. In 36/112 (32%) cases, 17p deletion was induced by random rearrangements. Interstitial 17p deletions were identified in only 9/112 (8 %) cases. According to the inclusion criteria of the trial, 36/112 (32%) pts. had 17p deletion and were treatment-naïve while 76/112 (68%) were relapsed or refractory to fludarabine or bendamustine based therapy, 53/76 (70%) having a 17p deletion. Treatment naïve pts. had a mean of 7.36 aberrations/case and pretreated pts. 6.09/case. Focusing on WAT and JT, 18/33 (54%) pts. with WAT and 7/11 (63%) pts. with JT were pretreated whereas 57/78 (73%) pts. in the other cytogenetic subgroups had prior therapy exposure. Considering other genomic features, WAT and JT occurred almost exclusively within complex karyotypes (≥3 chromosomal aberrations), 31/33 WAT and 10/11 JT, were IGHV unmutated, 30/33 WAT and 11/11 JT and harbored TP53mutations, 29/33 WAT and 10/11 JT. Conclusions: “Ultra high-risk” CLL pts. are characterized by a high genomic complexity as compared to standard risk treatment-naïve CLL pts. (CLL8 trial with 1.8 CNAs/case). Previous genotoxic therapy had no influence on the total number of aberrations or the underlying mechanism, suggesting an intrinsic genomic instability of the tumor cells with TP53 alterations. WAT and JT emerged as nonrandom aberrations involved in 17p loss. Given the strong association of TP53 deletion with TP53 mutations of the remaining allele, one may speculate that TP53 mutations precedes TP53 deletion by disrupting the normal DNA repair mechanisms permitting incorrect recombinations. Disclosures Stilgenbauer: Amgen: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding.

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p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative toTP53 mutation

Blood ◽

10.1182/blood.v98.3.814 ◽

2001 ◽

Vol 98 (3) ◽

pp. 814-822 ◽

Cited By ~ 171

Author(s):

Andrew R. Pettitt ◽

Paul D. Sherrington ◽

Grant Stewart ◽

John C. Cawley ◽

A. Malcolm R. Taylor ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Tp53 Mutation ◽

Lymphocytic Leukemia ◽

Tp53 Mutations ◽

Sporadic Tumor ◽

Direct Demonstration ◽

Baseline Levels ◽

Induced Apoptosis ◽

Cell Chronic Lymphocytic Leukemia

Abstract The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21CIP1/WAF1 after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation.

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

Blood ◽

10.1182/blood.v110.11.490.490 ◽

2007 ◽

Vol 110 (11) ◽

pp. 490-490 ◽

Cited By ~ 1

Author(s):

Frank Dicker ◽

Hannes Herholz ◽

Susanne Schnittger ◽

Aki Nakao ◽

Nancy Patten ◽

...

Keyword(s):

Poor Prognosis ◽

Tp53 Mutation ◽

Clinical Course ◽

Direct Sequencing ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Time To Progression ◽

Single Nucleotide ◽

Tp53 Mutations ◽

The Poor

Abstract 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.

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Mutational Analysis of TP53 Gene in Chronic Lymphocytic Leukemia: Comparison of Different Methodological Approaches

Blood ◽

10.1182/blood.v120.21.3911.3911 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3911-3911

Author(s):

Barbara Kantorova ◽

Jitka Malcikova ◽

Jana Kminkova ◽

Veronika Navrkalova ◽

Barbora Dvorakova ◽

...

Keyword(s):

Functional Analysis ◽

Chronic Lymphocytic Leukemia ◽

Mutation Analysis ◽

Gene Deletion ◽

Direct Sequencing ◽

Tp53 Gene ◽

Lymphocytic Leukemia ◽

Nonsense Mutations ◽

Tp53 Mutations

Abstract Abstract 3911 Background The adverse prognostic significance of p53 aberrations (gene deletion at locus 17p13.1 and/or TP53 mutations) has been already proven in chronic lymphocytic leukemia (CLL). In contrast to the standardized examination of the gene deletion by interphase FISH, various methodologies with different detection efficiency are applied for mutation analysis. To reduce inter-laboratory variability, the European Research Initiative on CLL (ERIC) has recently released recommendations for p53 mutational testing (Pospisilova et al., 2012). However, the optimal detection methodology has not been established yet. Aim To compare molecular-biological methods for exact determination of TP53 mutational status in CLL patients. Methodology The analyzed cohort included 100 high-risk CLL patients with unfavorable disease prognosis represented by unmutated IgVH gene status, 17p and 11q deletions and/or chemotherapy resistence. Mutational screening of TP53 gene was performed in all patients by the combination of the following methods: (1) direct Sanger sequencing (DNA and/or cDNA), (2) denaturing high-performance liquid chromatography (DHPLC; Varian), (3) functional analysis (FASAY), (4) CLL custom resequencing microarray (Affymetrix), (5) Roche AmpliChip p53 Test (Roche Molecular Systems). In the selected samples, the presence of mutations was confirmed by ultra-deep next generation sequencing (NGS; GS Junior System, Roche). Results The parallel p53 analysis using all five above mentioned detection techniques revealed totally 66 mutations in 47/100 patients. The predominant proportion of the identified alterations was represented by prognostically adverse missense substitutions (67%), mainly localized in p53 DNA-binding domain (5–8 exons). Other clinically relevant sequencing variants included frameshift mutations (15%), splice-site mutations (8%), nonsense mutations (6%) and in-frame deletions (4%). Although the used detection methods reached comparable sensitivity (with the exception of direct sequencing), some inconsistent results were observed. In comparison with DNA-based methodologies, the FASAY failed in recognition of nonsense mutations leading to RNA degradation (nonsense-mediated decay phenomenon). On the other hand, the technical aspects of chip arrays have not facilitated the proper determination of deletions and insertions. From this perspective, DHPLC in connection with direct sequencing enabled the most specific recognition of the present gene alterations. Using this methodic combination, 57/66 mutations covering all mutation types were clearly identified. Nevertheless, for the correct evaluation of the biological importance and the clinical consequences of the detected mutations, the DNA screening should be supplemented with functional analysis. Conclusion The heterogeneous biological properties of TP53 mutations require sensitive and specific detection methodology. Although many different methods are currently used for mutation analysis, each of them has some advantages and shortcommings. The combination of DNA testing with functional analysis offers the most efficient tool for improved prediction of the disease course and the response of patients to therapy. Disclosures: No relevant conflicts of interest to declare.

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Clinical Impact Of Small TP53 Mutated Subclones In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.116.116 ◽

2013 ◽

Vol 122 (21) ◽

pp. 116-116

Author(s):

Davide Rossi ◽

Hossein Khiabanian ◽

Carmela Ciardullo ◽

Valeria Spina ◽

Alessio Bruscaggin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Sanger Sequencing ◽

Tp53 Mutation ◽

Lymphocytic Leukemia ◽

Genetic Defects ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Genetic Event ◽

Tp53 Mutations

Abstract Introduction TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations are currently limited to lesions that are represented in the majority of CLL cells. Next generation sequencing (NGS) allows sensitive detection of mutations harbored by a small fraction of the tumor cell population. Here we aim at assessing the frequency, evolution during disease course, and prognostic impact of small TP53 mutated subclones in newly diagnosed CLL. Methods The study was based on a consecutive series of 309 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; clonal TP53/NOTCH1/SF3B1/BIRC3 lesions: 11/11/7/5%; median follow-up: 8.1 years). TP53 mutations (exons 4-8) were screened on peripheral blood (PB) samples (tumor representation 70-98%) by amplicon-based deep-NGS (GSJ, 454 Life Sciences) (average depth: 2660). A bioinformatic algorithm was developed to call TP53 variants out of background noise. By dilution experiments, deep-NGS allowed to detect mutant allele fractions of 0.3%. TP53 variants were considered subclonal if missed by Sanger sequencing, which was performed in parallel. Subclonal TP53 variants were confirmed by duplicate deep-NGS and independently validated by allele specific PCR (AS-PCR). TP53 variant allele frequency (VAF) was corrected for tumor representation. Results Deep-NGS identified 50 subclonal TP53 mutations (VAF 0.3%-11%) in 28/309 (9%) CLL (Fig 1A). All subclonal mutations were non-silent, were missed by Sanger sequencing, and were validated by AS-PCR. The molecular spectrum of subclonal TP53 mutations (i.e. missense/truncating ratio, transition/transversion ratio, distribution across hot spot codons; p>.05; Fig. 1B-D), as well as the residual transactivational activity of mutants toward the p21 promoter (p=.872) were highly consistent with that of fully clonal TP53 mutations reported in CLL (Zenz T, Leukemia 2010). Subclonal TP53 mutations were the sole TP53 genetic event in 15/309 (4.8%) CLL, while in 13/309 (4.2%) cases subclonal TP53 mutations co-existed in the same leukemic population along with a clonal TP53 mutation or with 17p deletion. In cases (n=12) harboring more than one TP53 mutation, the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. By combining subclonal TP53 mutations, clonal TP53 mutations and 17p deletion, 50/309 (16%) CLL harbored at least one TP53 defect at diagnosis. Subclonal TP53 mutations were significantly enriched among cases presenting with advanced stage (Binet C: 26%; p=.005) and clonal TP53 abnormalities (37%; p<001). Cases harboring solely subclonal TP53 mutation showed a median overall survival (3.4 years) significantly shorter than TP53 wild type cases (10.8 years; p=.028), and similar to that of cases with clonal TP53 genetic defects (3.1 years; p=.375) (Fig. 1E). By multivariate analysis, cases harboring subclonal TP53 mutations had a significantly increased hazard of death (HR: 2.0; p=.023) after adjusting for age, disease stage, IGHV mutation status, clonal TP53 genetic defects, and lesions of NOTCH1, SF3B1 and BIRC3. Subclonal TP53 mutations showed a similar allele fraction in paired PB and lymph-node CLL cells in 3/5 assessable cases, suggesting a systemic spread of mutated subclones across disease compartments. Among cases harboring solely subclonal TP53 mutations, longitudinal deep-NGS of sequential samples documented the outgrowth of the TP53 variant to a fully clonal level in 57% (4/7) of cases. In all these cases, clonal selection was strongly associated with treatment exposure and development of a chemorefractory phenotype. Conversely, in cases managed by watch-and-wait only, the load of TP53 mutations did not increase during follow-up. Conclusions Small TP53 mutated subclones detected by deep-NGS occur in a significant fraction of newly diagnosed CLL, have the same unfavorable prognostic impact as clonal TP53 defects, and anticipate the development of a chemorefractory phenotype among CLL requiring treatment. Search of minor subclones by deep-NGS should be considered for a comprehensive assessment of TP53 disruption in CLL. D.R. and H.K equally contributed; R.F., R.R. and G.G equally contributed. Disclosures: No relevant conflicts of interest to declare.

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TP53 Mutation and Survival in Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.8762 ◽

2010 ◽

Vol 28 (29) ◽

pp. 4473-4479 ◽

Cited By ~ 343

Author(s):

Thorsten Zenz ◽

Barbara Eichhorst ◽

Raymonde Busch ◽

Tina Denzel ◽

Sonja Häbe ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

High Performance ◽

Tp53 Mutation ◽

Prospective Trial ◽

Progression Free Survival ◽

Complete Response ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Tp53 Mutations ◽

The Impact

Purpose The precise prognostic impact of TP53 mutation and its incorporation into treatment algorithms in chronic lymphocytic leukemia (CLL) is unclear. We set out to define the impact of TP53 mutations in CLL. Patients and Methods We assessed TP53 mutations by denaturing high-performance liquid chromatography (exons 2 to 11) in a randomized prospective trial (n = 375) with a follow-up of 52.8 months (German CLL Study Group CLL4 trial; fludarabine [F] v F + cyclophosphamide [FC]). Results We found TP53 mutations in 8.5% of patients (28 of 328 patients). None of the patients with TP53 mutation showed a complete response. In patients with TP53 mutation, compared with patients without TP53 mutation, median progression-free survival (PFS; 23.3 v 62.2 months, respectively) and overall survival (OS; 29.2 v 84.6 months, respectively) were significantly decreased (both P < .001). TP53 mutations in the absence of 17p deletions were found in 4.5% of patients. PFS and OS for patients with 17p deletion and patients with TP53 mutation in the absence of 17p deletion were similar. Multivariate analysis identified TP53 mutation as the strongest prognostic marker regarding PFS (hazard ratio [HR] = 3.8; P < .001) and OS (HR = 7.2; P < .001). Other independent predictors of OS were IGHV mutation status (HR = 1.9), 11q deletion (HR = 1.9), 17p deletion (HR = 2.3), and FC treatment arm (HR = 0.6). Conclusion CLL with TP53 mutation carries a poor prognosis regardless of the presence of 17p deletion when treated with F-based chemotherapy. Thus, TP53 mutation analysis should be incorporated into the evaluation of patients with CLL before treatment initiation. Patients with TP53 mutation should be considered for alternative treatment approaches.

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