Mutations of the TP53 Gene Occur in 13.4% of Acute Myeloid Leukemia and Are Strongly Associated with a Complex Aberrant Karyotype.

Abstract The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others. Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p<0.001). They occur frequently in particular in the subgroup showing a typical pattern of chromosomal deletions (5q, 7q, 17p). TP53 mutations might explain in part the chemoresistance of AML with complex aberrant karyotype. In addition to cytogenetics a rapid diagnostic screening for TP53 mutations could be a valuable tool to identify a subgroup of AML with poor prognosis. This would allow the early assignment of patients to alternative treatment strategies using also options targeting the TP53 pathway.

Download Full-text

In CLL with Complex Aberrant Karyotype Distinct Entities Can Be Deciphered by Molecular Genetic and Cytogenetic Parameters

Blood ◽

10.1182/blood.v112.11.3138.3138 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3138-3138 ◽

Cited By ~ 1

Author(s):

Frank Dicker ◽

Susanne Schnittger ◽

Torsten Haferlach ◽

Wolfgang Kern ◽

Claudia Haferlach

Keyword(s):

High Risk ◽

Lower Risk ◽

Tp53 Mutation ◽

Risk Group ◽

Molecular Genetic ◽

Tp53 Gene ◽

High Risk Group ◽

Complex Karyotype ◽

Tp53 Mutations ◽

The Poor

Abstract Metaphase cytogenetics have recently defined important prognostic subgroups in CLL. In addition to the poor prognosis FISH markers del(17p) and del(11q), the presence of translocations and a complex aberrant karyotype, defined by chromosome banding analyses, have been associated with shorter overall survival in a retrospective analysis. Thus far, a complex aberrant karyotype is defined by a number of three or more chromosomal aberrations, therefore, we aimed at characterizing an unselected CLL cohort of 92 patients with complex aberrant karyotype in more detail with molecular genetic, cytogenetic, and immunophenotypic parameters. Median age at diagnosis was 62.5 years (range: 33.4–83.3 years), the male/female ratio was 2.8. An unmutated IgVH status (<= 2% mutations) was detected in 51 (60.7%) of 84 analyzed samples. A positive CD38 expression (>= 30% CD38 positive cells) was detected in 55 (64%) of 86 samples and TP53 gene mutations by denaturing high performance liquid chromatography of exons 4–9 of TP53 in 29 (36.7%) of 79 samples. As the main approach for prognostication in CLL uses FISH for del(17p), del(11q), +12, del(13q) sole and normal, we related our cohort with complex aberrant karyotype into these different FISH categories. The poor prognosis markers del(17p) and del(11q) were detected with frequencies of 39.1% (n=36) and 22.8% (n=21), respectively, accounting for almost two third of all samples (n=57, 61.9%). An overlap between del(17p) and del(11q) was detected in 5 of the 36 del(17p) cases. The intermediate risk FISH marker +12 and low risk FISH markers del(13q) as sole abnormality and “normal karyotype” appeared with an incidence of 8.7% (n=8), 21.7% (n=20) and 7.6% (n=7), respectively. The high incidence of del(11q) and del(17p) in complex karyotype seems likely, as genes implicated in sensing DNA damage and in regulating apoptosis, ATM and TP53, are candidate genes in these deleted regions. As TP53 mutations have been suggested as independent poor prognostic markers, we also added TP53 gene mutation analysis to the FISH stratification. Del(17p) was associated with TP53 mutation in 26 (86.7%) of 30 analyzed cases, whereas the three residual TP53 mutations were associated with del(11q) (n=1) and del(13q) as sole abnormality (n=2). Therefore, we chose to merge samples with high risk features within the complex karyotype into one group (n=59), i.e., samples with del(17p), del(11q) or TP53 mutation, and compared these samples to the remaining samples (n=33). Effectively, the high risk group compared to the other cohort was significantly associated with an unmutated IgVH (p=0.02, Fisher’s exact test) and with an increased, median amount of cytogenetic aberrations (4.9 vs. 3.7 aberrations, p=0.005, t-test). However, no significant difference between the two groups regarding a CD38 positive status was detected (p=0.257). The prognostic impact of high risk features (del(17p), del(11q) and TP53 mutation) within the group defined above vs the lower risk group was analyzed with log-rank statistics with respect to time from diagnosis of CLL to initial treatment (TTT). 34 patients from the high risk group and 18 patients from the low risk group were available for analysis. The high risk features within the complex karyotype were significantly associated with a higher risk of early treatment in log-rank statistics with a median TTT of 12.2 month in the higher risk group compared to 70.1 month in the lower risk group (p=0.005). In conclusion, based on poor risk cytogenetic and molecular genetic features within the group of CLL with complex aberrant karyotype, we characterized patients with a higher risk of early treatment initiation. This group includes samples with del(17p), del(11q) and TP53 mutations.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Clinical Impact of TP53 Gene Mutations in Diffuse Large B-Cell Lymphoma (DLBCL): An International DLBCL Rituxan-CHOP Consortium Program Study.

Blood ◽

10.1182/blood.v114.22.967.967 ◽

2009 ◽

Vol 114 (22) ◽

pp. 967-967

Author(s):

Ken H. Young ◽

Nancy Patten ◽

Sim Truong ◽

Jens Eickhoff ◽

Gabrielle L. Rocque ◽

...

Keyword(s):

Dna Binding ◽

Clinical Outcome ◽

B Cell ◽

Median Survival ◽

Tp53 Mutation ◽

Tp53 Gene ◽

Chop Regimen ◽

Tp53 Mutations ◽

Dna Binding Domains ◽

Binding Domains

Abstract Abstract 967 Mutations of the TP53 tumor suppressor gene are associated with a poor clinical outcome in DLBCL patients treated with CHOP. The impact of TP53 mutations on clinical outcome of DLBCL patients treated with Rituxan-CHOP has not been comprehensively analyzed. The purpose of this study was to analyze the frequency and type of TP53 mutations in Rituxan-CHOP treated DLBCL patients from twenty-two medical centers, and to correlate these with clinical outcome. TP53 mutations were identified in 138/604 (22.7%) Rituxan-CHOP treated DLBCL cases and included missense (n=133), nonsense (n=16), splice site (n=9) and frameshift (n=1) mutations. The presence of any TP53 mutation correlated with poor overall survival (OS) with a median OS of 50 months in the TP53 mutation group versus 69 months in the wild-type group (wt-TP53, P=0.0042). Seventy-three of 138 cases (53%) had mutations in the DNA binding domains of the TP53 gene, which were found to be the most important predictor of poor OS (P=0.0044). In contrast, mutations in the non-DNA binding domains did not correlate with poor OS (P=0.157). Overexpression of p53 protein significantly correlated with only TP53 missense mutations (P=0.002), but not with other types of TP53 mutations, while TP53 deletion did not correlate with mutation or OS. In comparison to our previous series of patients treated only with CHOP, Rituxan-CHOP regimen improved OS in both wt-TP53 and TP53 mutated groups. The 5-year survival rate was 42% in patients with any TP53 mutation (median survival=50 months) and 41% in patients with the DNA-binding domain mutations (median survival=49 months) compared to 52% for those with wt-TP53 (median survival=69 months). The complete remission rate was 51% in patients with any TP53 mutation and 44% in patients with the DNA-binding domain mutations, compared to 77% for those with wt-TP53. However, the clinical outcome and treatment response to the Rituxan-CHOP varied in patients with mutations in different regions of the DNA-binding domains. Patients with mutations in the DNA minor binding groove motif (Loop L3, 17% of all mutations) had significantly decreased median OS (17 months) when compared to patients with Loop L2 (16% of all mutations) or loop-sheet-helix motifs (Loop L1-S10-H2, 20% of all mutations) with median OS of 49 and 50 months, respectively. In contrast to our previous CHOP series study, median survival was significantly improved for Rituxan-CHOP treated DLBCL patients with mutations in the loop-sheet-helix motifs (43 months). Multivariate analysis confirmed that TP53 mutations and activated B-cell-like (ABC)/germinal center B-cell-like (GCB) subtype classification were independent predictors of OS with a hazard ratio of 0.69 (GCB vs ABC, 95% CI 0.49-0.98) and 1.60 (TP53 vs wt-TP53, 95% CI 1.10-2.31), respectively. Similar to our previous CHOP study, the TP53 mutation profile, regardless of location, was found to stratify GCB-DLBCL, but not ABC-DLBCL, into molecularly distinct subsets with different clinical outcomes in Rituxan-CHOP treated DLBCL patients. This study demonstrates the importance of TP53 mutational profile for predicting clinical outcome. Elucidation of the roles of specific TP53 domain mutations, as documented in our study, will help in refining prognostic models for DLBCL patients treated with either the CHOP or Rituxan-CHOP regimen. These findings also provide the rationale and strategies for p53 targeted therapeutic intervention in DLBCL patients. Disclosures: Kahl: Milllennium: Consultancy, Research Funding; Cephalon: Consultancy, Research Funding.

Download Full-text

Mutations of the TP53 Gene Play an Important Role in the Pathogenesis of Acute Myeloid Leukemia with Complex Aberrant Karyotype.

Blood ◽

10.1182/blood.v104.11.63.63 ◽

2004 ◽

Vol 104 (11) ◽

pp. 63-63 ◽

Cited By ~ 9

Author(s):

Claudia Schoch ◽

Frank Dicker ◽

Alexander Kohlmann ◽

Sonja Rauhut ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Tp53 Mutation ◽

Direct Sequencing ◽

Tp53 Gene ◽

Single Amino Acid ◽

Comparative Genomic ◽

Fish Analysis ◽

Missense Mutations ◽

Acute Myeloid

Abstract Acute myeloid leukemia with complex aberrant karyotype is associated with the most unfavorable prognosis of all AML subtypes. Clinical behavior, complexity of karyotype abnormalities and the exponential increase of incidence with age shows similarities to the most common solid tumors. In the latter ones TP53 gene is the most frequently mutated gene identified so far. Therefore, we addressed the role of TP53 in AML with complex aberrant karyotype. In a first step we analyzed 360 AML cases with complex aberrant karyotype using conventional cytogenetics and 24 color FISH to resolve the chromosomal rearrangements in detail. In combination with data from comparative genomic hybridization for a subset of these patients (n=49) we identified 10 genomic regions frequently lost (5q14q33, 7q32q35, 12p13, 13q14, 16q22q24, 17p13, 18q21q22) or gained (11q23q25, 1p33p36, 8q22q24). In all 350 cases interphase FISH with a TP53 probe was performed. In 210 patients (60%) a loss of one TP53 allele was observed. 34 cases (22 with and 12 without TP53 loss) were further evaluated for p53 mutations using the Affymetrix p53 GeneChip assay. All mutations detected were verified by direct sequencing. TP53 mutations were detected in 21 of the 22 cases showing a loss of one TP53 allele (95%), while 9 of 12 cases (75%) without a TP53 loss showed a TP53 mutation on the microarray. Two mutations occurred in introns 4 and 7, while all others were located in exons (e4: n=2, e5: n=10, e6: n=3, e7: n=5, e8: n=8, e9: n=1). 22 mutations were missense mutations resulting in the substitution of a single amino-acid, while 4 were nonsense mutations. One small deletion and one insertion were detected. Four cases without mutations detected by microarray screening and 15 additional cases (9 with and 11 without TP53 loss detected with FISH) were further analyzed by DHPLC (WAVE, Transgenomics). In two of the four cases in which no mutation was detectable on the microarray mutations were found with DHPLC, which are most likely larger deletions not detectable with the p53 GeneChip assay. Furthermore, in 11 of the 15 additional cases a mutation was detected by DHPLC. Thus, in total 43 of 49 cases (88%) showed a TP53 mutation (27/30 (90%) with loss of one TP53 allele and 16/19 (84%) without loss of one TP53 allele). Three of the 6 cases in which no TP53 mutation was detected showed loss of one TP53 allele in FISH analysis. Taken together, only in 3 of 49 cases (6%) no alteration of TP53 was detected. In one of these cases an increased MDM2 expression was found using gene expression microarrays (U133A), another mechanism of inactiving TP53 function. In conclusion, the loss of normal TP53 function by loss of one allele and/or point mutation plays an important role in the pathogenesis of AML with complex aberrant karyotype and may be a major reason for chemoresistance in this prognostically most unfavorable AML subtype. TP53 alterations are detectable in more than 90% of cases with complex aberrant karyotype and are very rare in other AML subtypes. Therefore, we suggest to include the TP53 status in the definition of AML with complex aberrant karyotype.

Download Full-text

P53 Functional Assessment and Correlation With 17p Deletion and/Or TP53 Mutation Status In Chronic Lymphocytic Leukemia (CLL). A Preliminary Report Of The ICLL001 Bomp Trial On Behalf Of The French CLL Intergroup (GCFLLC/MW - GOELAMS)

Blood ◽

10.1182/blood.v122.21.4173.4173 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4173-4173

Author(s):

Magali Le Garff-Tavernier ◽

Lauren Veronese ◽

Florence Nguyen-Khac ◽

Marie-Sarah Dilhuydy ◽

Patricia Combes ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Gene Disruption ◽

Tp53 Mutation ◽

Mutation Screening ◽

Tp53 Gene ◽

Lymphocytic Leukemia ◽

Functional Assay ◽

Advisory Committees ◽

Group 5

Abstract Despite improvement in treatment strategies, virtually all chronic lymphocytic leukemia (CLL) patients will relapse and experience tumor resistance. The 17p deletion resulting in loss of the TP53 gene, found in up to 20-40% of relapsing patients, is strongly associated with impaired response to genotoxic agents, reduced progression free survival and poor overall survival. The 17p deletion usually coincides with TP53 mutation, leading to the impairment of the p53-associated pathway. In addition, sole TP53 mutations (without 17p deletion) appear also associated with poor outcome in prospective trials. However, TP53 mutation screening is time consuming, can be not exhaustive, and the respective impact of different patterns of TP53 gene impairment on p53 function and prognostic remains unclear. We previously developed a functional assay to detect p53 dysfunction (Le Garff-Tavernier, 2011) and we aim to validate this analysis on a large prospective trial. Clinical and laboratory data were collected from CLL patients (pts) enrolled in the ICLL001 – BOMP phase II trial of the French CLL intergroup (NCT01612988) evaluating a prephase of ofatumumab (300 mg) followed by 6 monthly courses of BOMP including bendamustine (70 mg/m2 d1-2), ofatumumab 1000 mg TD (d1 and d15 on 1st and 2nd courses) and high dose methylprednisolone (1 g/m2 d1-3) in fit patients with relapsing CLL and IWCLL treatment criteria. In addition to conventional screening, we focused on p53 evaluation. FISH analysis for 17p deletion was done with a 10% cut-off for positive result, TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11) and the p53 functional status in CLL cells was determined by a flow cytometry assay based on induction of p53 and p21 protein expression after etoposide and nutlin-3a exposition. Data from the first 55 enrolled pts are available. Sex ratio M/F was 3.3 and median age was 63.8 yrs (44.6-76.4). CLL diagnostic had been done 7,2 (1,9-16,8) years before inclusion. All patients had according to IWCLL criteria an active disease of Binet stage of A (11%), B (57%) and C (32%) respectively. Patients had been previously pretreated with a median of 1 (1-3) lines, including FCR (or FCR-like) in 51 (93%) pts and 22 (42%) pts had experienced high-risk relapses within 24 months post-FCR, with 7 (13%) pts being fludarabine refractory (less than PR after fludarabine regiment and/or response lasting less than 6 months). IGVH gene status was unmutated in 90%, elevated β2-microglobulin (>4) was found in 52%. Karyotypes were complex (≥ 3 abnormalities) in 18/46 (39%) successful cases. Using FISH, we found 15/55 (27%) del17p (median of positive cells 71%, range 10-98), 6/55 (11%) tri12, 18/55 (33%) del11q, 35/55 (64%) del13q. Results of p53 functional assay was available for 52 pts with the following results: normal in 31 pts and abnormal in 21 pts including type A (n=4), type B (n=13) and type C (n=4) dysfunction. Mutation screening was available in 55 pts. No mutation were detected in 38 pts, one significant mutation was detected in 14 pts within exon 5 (n=1), exon 6 (n=2), exon 7 (n=2), exon 8 (n=6), exon 10 (n=1) and intronic splice site (n=2) ; 3 pts had 2 mutations within exons 7 and 8 (n=1), exons 7 and 10 (n=1), exons 5 and 7 (n=1). Among the 52 pts with available functional results we found the 7 following groups (Table). In this study, the sensitivity and specificity of the p53 functional test to detect patient with 17p deletion and/or TP53 mutation was 89.5% (66.9 –99.7) and 87.9% (71.8 – 96.6) respectively. Response to p53/21 functional assay 17p deletion TP53 mutation n % Group 1 Normal No No 29 56 Group 2 Abnormal Yes Yes 13 25 Group 3 Abnormal Yes No 1 2 Group 4 Abnormal No Yes 3 5.5 Group 5 Abnormal No No 4 7.5 Group 6 Normal Yes No 1 2 Group 7 Normal No Yes 1 2 This study shows that an in vitro p53 functional analysis can predict with an acceptable sensitivity the presence of TP53 gene disruption and could be useful to identify pts with TP53 mutation without 17p deletion. Interestingly, this functional assay coupled with cytogenetic and mutational screening could reveal 3 sub-groups of pts with potential clinical consequences: i) normal p53 function despite a del17p deletion (group 6) ii) normal p53 function despite a TP53 mutation (group 7) and in contrast iii) abnormal p53 function without any TP53 gene disruption (group 5) allowing to describe alternative alterations of p53 pathway. Disclosures: Dilhuydy: Roche: Honoraria. Leblond:Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Feugier:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Tournilhac:MUNDIPHARMA: Consultancy, travel funding Other; GSK: Consultancy, travel funding, travel funding Other; Celgene: Consultancy, teaching, teaching Other.

Download Full-text

Cytogenetic and Molecular Genetic Clonal Evolution in CLL Is Associated with an Unmutated IGHV Status and Frequently Leads to a Combination of Loss of 17p and TP53 mutation

Blood ◽

10.1182/blood.v128.22.3213.3213 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3213-3213

Author(s):

Claudia Haferlach ◽

Sabine Jeromin ◽

Niroshan Nadarajah ◽

Melanie Zenger ◽

Wolfgang Kern ◽

...

Keyword(s):

Median Time ◽

Tp53 Mutation ◽

Clonal Evolution ◽

Molecular Genetic ◽

Normal Karyotype ◽

Employment Equity ◽

Mutation Status ◽

Equity Ownership ◽

Mutation Analyses

Abstract Background: The clinical course in CLL is very heterogeneous ranging from stable disease to a rather rapid progression requiring treatment. The acquisition of genetic abnormalities termed clonal evolution (CE) is likely to correlate with clinical progression and might be used to guide treatment strategies. Aim: The aim of this study was to evaluate the frequency of CE on the cytogenetic (CCE) and molecular genetic (MCE) levels and its association with the IGHV mutation status and clinical outcome. Methods: 179 CLL cases were selected on the basis that chromosome banding analysis (CBA) and mutation analyses in TP53 and SF3B1 all having been performed at least at two time points. The median age at first evaluation was 72 years (range: 46-95). The first time point of analysis was at primary diagnosis (n=131) or during course of disease but prior to any treatment (n=48). In all patients interphase FISH was performed with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), and the centromeric region of chromosome 12 and the IGHV mutation status was evaluated. A total of 465 CBA, 417 TP53 and 424 SF3B1 mutation analyses were evaluated. The median number of samples per patient was 2 (range: 2-9). The time between samples ranged from 1 month to 9.8 years (median 21 months). For all patients clinical follow-up data was available with a median follow-up of 7.4 years and 5-year overall survival (OS) of 88%. Results: At first investigation CBA revealed a normal karyotype in 31 (17%) patients. In cases with an aberrant karyotype the pattern of abnormalities was typical for CLL: del(13q); 51% (homozygous: 15%), +12: 18%, del(11q): 16%, and del(17p): 5%. A complex karyotype (≥3 abnormalities) was present in 18%. The IGHV status was unmutated (IGHV-U) in 56% of cases and TP53 and SF3B1 mutations were detected in 10% and 15%, respectively. CCE was observed in 63/179 patients (35%). The median time to CCE was 46 months (range 3-111). The most frequent abnormalities gained during CCE were loss of 17p (14/63; 22%), 13q (11/63; 18%), and 11q (10/63; 16%). Acquired loss of 17p was more frequent in SF3B1mutated CLL (19% vs 6%, p=0.04). MCE was observed in 29/179 cases (16%). TP53 and SF3B1 mutations were acquired during the course of the disease in 23 (14%) and 7 (5%) cases, respectively. The median time to MCE was 61 months (range 1.5-109). Of note, in 2 cases with TP53 deletion a TP53 mutation was acquired and in 2 cases with TP53 mutation a TP53 deletion was acquired. In 12 CLL both a TP53 deletion and a TP53 mutation were acquired (table). CCE and MCE were significantly associated with IGHV-U (p=0.003; p<0.001) and with each other (p<0.001). In more detail, in 71% of cases with CCE and 90% of cases with MCE an IGHV-U was present. Thus, CCE and MCE were less frequent in IGHVmut CLL (23% and 4%). In 30% of CLL with CCE also MCE occurred. In addition CCE was associated with an aberrant karyotype at first investigation (p<0.001). CCE occurred in only 3% of CLL with a normal karyotype but in 42% of CLL with an aberrant karyotype. Time to treatment was significantly shorter in patients with CCE, MCE and both compared to the respective patients without (2.1 vs 5.5 yrs, p=0.004; 1.8 vs 4.8 yrs, p=0.07; 2.2 vs 5.3 yrs; p=0.04). While no impact of CCE on OS was observed in patients with a mutated IGHV status, in patients with an unmutated IGHV status a tendency to shorter OS was observed in cases with CCE compared to those without (7 year OS: 67% vs 83%; p=0.2). No impact on OS was observed for MCE. This may be due to rather short follow up after CE. However, if CCE and MCE resulted in CLL harboring both TP53 deletion and TP53 mutation 5 year OS was significantly shorter than in CLL with neither TP53 deletion nor TP53 mutation (75% vs 91%, p=0.03). Conclusions: 1) We observed CCE in 35% and MCE in 16% of CLL. 2) The pattern of cytogenetic abnormalities acquired during the course of the disease is similar to the pattern observed in CLL at diagnosis, however the frequency varies with del(17p) being the most frequently gained in CE. 3) CCE and MCE were highly correlated to IGHV-U. 4) In 25% of CLL with CCE and MCE CE resulted in the co-occurrence of TP53 deletion and TP53 mutation, which was associated with a significantly shorter OS emphasizing the necessity to reevaluate the TP53 status during the course of the disease to guide treatment. 5) The frequency and impact of CE needs to be further studied in unselected patient cohorts in which CBA and mutational analysis is performed on a regular basis. Table Table. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

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.

Download Full-text

The Response to DNA Damage in CLL Cells Is Partly Determined by the Type of TP53 Mutation and Genomic Aberrations

Blood ◽

10.1182/blood.v112.11.3119.3119 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3119-3119

Author(s):

Julia Mohr ◽

Thorsten Zenz ◽

Dirk Winkler ◽

Andreas Bühler ◽

Daniel Mertens ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Tp53 Mutation ◽

Normal Karyotype ◽

Type B ◽

Tp53 Mutations ◽

Damage Response ◽

Dna Damage Response Pathway ◽

11Q Deletion

Abstract A central role of the DNA damage response pathway and particularly p53 has been suggested by the prognostic role of 17p and 11q deletions in CLL. In the critical regions, two prominent genes are located that are involved in the cells’ response to DNA damage. Functional assessment of p53 may be of help to more precisely define a subgroup of patients with defects in the DNA damage response pathway. The most commonly used assays include FACS measurement of induction of p53/p21 after DNA damage, induction of p53 by nutlins or the measurement of transcriptional targets of p53 by RT-PCR or MLPA. The growing evidence of the impact of sole TP53 mutations on prognosis led us to analyse the DNA damage response in CLL with special focus on cases with TP53 mutations in the absence of 17p deletion. We investigated the response to gamma-irradiation (5Gy) in a cohort of 71 CLL cases. We enriched the cohort for the bad prognostic subgroups 17p-, 11q- and TP53 mutation in the absence of 17p deletion in order to precisely dissect the DNA damage response in these subgroups. Levels of p53 and p21 before and after treatment were determined by flow cytometry. Cells were additionally stained with a CD19 antibody to differentiate between CLL cells and CD19 negative cells (T-cells). We developed a strategy to normalize the median values of the CLL cells to the T-cells. Defects in the ATM/p53 pathway can be divided into type A and type B defects that are characterized by an impaired p21 induction and a high or a low basal p53 level, respectively (Pettitt et al. 2001). Basal p53 levels in 17p-cases (n=15) were significantly higher than for cases with TP53 mutation (P=0.012), 11q deletion (P<0.01) and normal karyotype/13p deletion (P<0.01), respectively). Furthermore, induction of p21 was impaired after irradiation compared to 11q del and normal/13q del (P=0.0066 and P=0.0002). In the cases with a sole TP53 mutation (n=10) we observed higher basal p53 levels compared to 11q- and normal/13q- cases (P=0.044 and P=0.007). The p21 response was heterogeneous, which may be partly explained by the presence of the mutation in subclones. Some cases with TP53 mutation (n=3) showed low p21 induction with low basal p53 levels resembling type B defects. This finding was explained by the presence of splice site and frameshift mutations that prevent formation of p53 protein. These findings suggest that not all cases with TP53 will exhibit the classical type A pattern of expression. A heterogeneous pattern of p21 induction was observed in patients with an 11q deletion (n=23) and in patients with normal karyotype or 13q deletion (n=23), the induction of 11q deleted cases being significantly lower (P=0.0102). Still, there were 11q deleted cases that responded normally to DNA damage (9/23). These cases can be hypothesized to have retained a functional ATM copy. Similarly, some of the cases with normal karyotype or 13q deletion showed impaired p21/p53 induction (n=6) and the cause for this impairment are under investigation. This study shows the importance of 17p deletions and TP53 mutations in altering the response to DNA damage in CLL. While this assay provides some insight into the functionality of the ATM/p53 pathway and the thus potentially the treatment response, the clinical and diagnostic value is currently unclear. We observed F-refractory cases showing a rather normal in vitro response to DNA damage. Along the same lines, cases with sole TP53 mutation are partly grouped with the type B defect, suggesting that TP53 mutations cannot be correctly grouped in all cases. Since the induction of p21 was not very strong, the use of other p53 targets could help to define different groups more precisely. Our findings suggest that the type of TP53 mutation will impact the DNA damage response and potentially lead to misclassification. In how far the heterogeneous pattern of 11q cases and some cases with low risk cytogenetics will help to differentiate biological and prognostic subgroups will need to be assessed in a large independent cohort with homogenous treatment. Induction of p21 and p53 in CLL with different genetic profile. Figure Figure

Download Full-text

Next Generation Sequencing (NGS) of TP53 Gene Allows to Identify a Very High Risk Group of Philadelphia Negative Acute Lymphoblastic Leukemia (ALL) Patients

Blood ◽

10.1182/blood.v124.21.3796.3796 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3796-3796

Author(s):

Silvia Salmoiraghi ◽

Greta Ubiali ◽

Manuela Tosi ◽

Barbara Peruta ◽

Marie Lorena Guinea Montalvo ◽

...

Keyword(s):

Clinical Outcome ◽

Tp53 Mutation ◽

Lymphoblastic Leukemia ◽

Tp53 Gene ◽

Wild Type ◽

Poor Clinical Outcome ◽

Tp53 Mutations ◽

Next Generation Sequencing Ngs ◽

Roche Diagnostics ◽

Generation Sequencing

Abstract Background and Aim of the Study For both childhood and adult Acute Lymphoblastic Leukemia (ALL) patients, clinical risk factors such as age, white cell count, response to steroids, time to complete remission, as well as biologic characteristics such as immunophenotype and cytogenetic at diagnosis are important but not sufficient in predicting clinical outcome. Aberrations of TP53 play a crucial role in the molecular pathogenesis of leukemias and lymphomas in which their presence is associated to disease progression and represents a strong predictor of poor clinical outcome. In childhood ALL, hereditary and acquired TP53 mutations are involved both in the pathogenesis and progression of the disease. In adult ALL, TP53 mutations are frequent in patients negative for recurrent fusion genes and correlate with poor response to induction therapy (Chiaretti S. et al, Haematologica 2013). The aim of this study was to evaluate the impact of TP53 alterations, analyzed by Next Generation Sequencing (NGS), on the outcome of a cohort of T (n= 57) and B (n= 114) precursor, Philadelphia (Ph) negative, adult ALL patients enrolled into the NILG-ALL 09/2000 clinical trial (ClinicalTrials.gov identifier: NCT00358072, Bassan R. et al, Blood 2009) in which molecular minimal residual disease was used to guide post-remissional therapy. Patients and Study design Among the 171 patients who were investigated for TP53 mutations, 16 proved also positive for t(4;11) and 3 for t(1;19). We analyzed DNA isolated from mononuclear cells obtained from bone marrow or peripheral blood samples containing at least 30% of blasts at diagnosis. The TP53 gene was sequenced using 454 ultra-deep sequencing (Roche Diagnostics) for alterations in exons 4 to 11, following the protocol developed in the IRON-II consortium. The sequencing data were analyzed by the Roche Diagnostics GS Run Browser and GS Amplicon Variant Analyzer software. The probabilities of survival were estimated using the Kaplan Meier method. The log-rank test was used to compare survival probabilities between subgroups of patients. Results and Discussion The data obtained by NGS allowed to identify 15 coding mutations detected in the DNA binding domain region (exons 5 to 8). These alterations were observed at diagnosis in 14 patients (8%), (11 B-precursor ALL and 3 T-ALL). In 12 cases these aberrations were single nucleotide changes, in 2 cases we found a duplication (one of 4 and the other of 8 nucleotides) and in one case there was an 11 base pair DNA insertion. Remarkably, all of these DNA alterations led to missense or frame-shift mutations that introduced a premature stop codon. Moreover, they were detected with a wide range of allele burden (from 5% to 97%) pointing out that TP53 mutations can be present at diagnosis in different proportions within the leukemic clones. All patients carrying a TP53 alteration reached complete remission after induction therapy but 13 out of 14 suffered an early relapse. Frequency of relapses was significantly higher in mutated than in wild-type cases (p=0.019). Relapse DNA samples were available in 3 patients and in all of them we detected the same TP53 mutation found at diagnosis, indicating the presence of a stable mutated clone. The univariate analysis enlightens a clear relationship between TP53 mutation with an increasing age (p= 0.0003) but no correlation with other clinical features such as gender, hemoglobin, white blood count, platelets, percentage of blasts and cytogenetics at diagnosis. Moreover, patients with mutated TP53 showed a Disease Free Survival (DFS) and Overall Survival (OS) dramatically shorter than wild-type patients. The 2 years DFS was 43% in the TP53 non-mutated subjects compared to 7% in the mutated (p=0.0007). Similarly, the 2 years OS was of 50% in wild-type patients and of 7% in mutated patients (p=0.0011) (Figure 1). Conclusions In adult ALL, response to induction chemotherapy is not different in patients with a wild-type or a TP53 mutated gene, but in these latter cases the leukemia relapse rate is dramatically higher. The frequency of these mutations observed at diagnosis and the poor clinical outcome indicate the need of their identification during the diagnostic work up of adult ALL to guide treatment strategies. The use of a highly sensitive deep sequencing approach is crucial to identify also minor leukemic clones carrying TP53 mutations that may lead to the rapid emergence of a treatment resistant disease. Disclosures Kohlmann: AstraZeneca: Employment.

Download Full-text