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

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

Blood ◽

10.1182/blood.v108.11.2087.2087 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2087-2087

Author(s):

Hannes Herholz ◽

Claudia Schoch ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tp53 Mutation ◽

Direct Sequencing ◽

Lymphocytic Leukemia ◽

Chromosomal Translocations ◽

Entire Group ◽

Unbalanced Translocation ◽

Tp53 Mutations ◽

Cytogenetic Aberrations ◽

Balanced Translocations

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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P04.16 TP53 mutations in codon 273 is predictive of overall survival in astrocytoma patients

Neuro-Oncology ◽

10.1093/neuonc/noz126.111 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii32-iii32

Author(s):

H Noor ◽

R Rapkins ◽

K McDonald

Keyword(s):

Overall Survival ◽

Tp53 Mutation ◽

Progression Free Survival ◽

Low Grade Glioma ◽

Low Grade ◽

Wild Type ◽

Mutation Status ◽

Free Survival ◽

Tp53 Mutations ◽

Fresh Frozen

Abstract BACKGROUND Tumour Protein 53 (TP53) is a tumour suppressor gene that is mutated in at least 50% of human malignancies. The prevalence of TP53 mutation is much higher in astrocytomas with reports of up to 75% TP53 mutant cases. Rare cases of TP53 mutation also exist in oligodendroglial tumours (10–13%). P53 pathway is therefore an important factor in low-grade glioma tumorigenesis. Although the prognostic impact of TP53 mutations has been studied previously, no concrete concordance were reached between the studies. In this study, we investigated the prognostic effects of TP53 mutation in astrocytoma and oligodendroglioma. MATERIAL AND METHODS A cohort of 65 matched primary and recurrent fresh frozen tumours were sequenced to identify hotspot exons of TP53 mutation. Exons 1 to 10 were sequenced and pathogenic mutations were mostly predominant between Exons 4 and 8. The cohort was further expanded with 78 low grade glioma fresh frozen tissues and hotspot exons were sequenced. Selecting only the primary tumour from 65 matched tumours, a total of 50 Astrocytoma cases and 51 oligodendroglioma cases were analysed for prognostic effects of TP53. Only pathogenic TP53 mutations confirmed through COSMIC and NCBI databases were included in the over survival and progression-free survival analysis. RESULTS 62% (31/50) of astrocytomas and 16% (8/51) of oligodendrogliomas harboured pathogenic TP53 mutations. Pathogenic hotspot mutations in codon 273 (c.817 C>T and c.818 G>A) was prevalent in astrocytoma with 58% (18/31) of tumours with these mutations. TP53 mutation status was maintained between primary and recurrent tumours in 93% of cases. In astrocytoma, overall survival of TP53 mutant patients was longer compared to TP53 wild-type patients (p<0.01) but was not significant after adjusting for age, gender, grade and IDH1 mutation status. In contrast, astrocytoma patients with specific TP53 mutation in codon 273 showed significantly better survival compared to other TP53 mutant and TP53 wild-type patients combined (p<0.01) in our multivariate analysis. Time to first recurrence (progression-free survival) of TP53 mutant patients was significantly longer than TP53 wild-type patients (p<0.01) after adjustments were made, while TP53 mutation in codon 273 was not prognostic for progression-free survival. In oligodendroglioma patients, TP53 mutations did not significantly affect overall survival and progression-free survival. CONCLUSION In agreement with others, TP53 mutation is more prevalent in Astrocytoma and mutations in codon 273 are significantly associated with longer survival.

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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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Mutation Status and Immunoglobulin Gene Rearrangements in Patients from Northwest and Central Region of Spain with Chronic Lymphocytic Leukemia

BioMed Research International ◽

10.1155/2014/257517 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

I. González-Gascón y Marín ◽

J. A. Hernández ◽

A. Martín ◽

M. Alcoceba ◽

M. E. Sarasquete ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Central Region ◽

Lymphocytic Leukemia ◽

Gene Rearrangements ◽

Immunoglobulin Gene ◽

Mutation Status ◽

Almost All ◽

Time To First Treatment ◽

Immunoglobulin Gene Rearrangements

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

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Autoimmune Cytopenia in Chronic Lymphocytic Leukemia: Effect on Outcome and Survival, a Population Based Analysis in British Columbia, Canada

Blood ◽

10.1182/blood.v124.21.1945.1945 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1945-1945 ◽

Cited By ~ 1

Author(s):

Alaa A Alzaki ◽

Alina S Gerrie ◽

Tanya L. Gillan ◽

Steven Huang ◽

Miriam Ahmed ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Population Based ◽

Lymphocytic Leukemia ◽

Time Interval ◽

Poor Prognostic Factor ◽

Significant Difference ◽

Positive Direct ◽

Time To First Treatment

Abstract Background: Among Chronic Lymphocytic Leukemia (CLL) patients (pts), 4-10% are diagnosed with autoimmune cytopenias (AC) at some point during the course of their disease. This is less common than cytopenias related to bone marrow infiltration (10-20%). Infiltrative cytopenias (IC) are clearly a poor prognostic factor. However, the effect of AC on survival and prognosis of CLL pts remains understudied. Objectives: To determine the prevalence of AC and IC among CLL pts and their effect on overall survival (OS) and time to first treatment (TTFT) compared to patients without cytopenia. Furthermore, the effect of different treatment modalities including chemotherapy and chemo-immunotherapy on the disease course was evaluated in patients with AC. Methods: A population-based retrospective analysis through an electronic search of pts within the Providence Health Care CLL database between 1978 and 2013 was carried out. The diagnostic criteria for autoimmune hemolytic anemia (AIHA) were positive direct antiglobulin test and laboratory evidence of hemolysis, for immune thrombocytopenia (ITP) the exclusion of other etiologies of thrombocytopenia and for pure red cell aplasia (PRCA) anemia with low reticulocyte count and bone marrow evidence of decreased erythropoiesis. Infiltrative cytopenia diagnosis was confirmed by bone marrow biopsy based on lymphocyte percentage and cellularity. Anemia was defined as hemoglobin <100 g/L. Thrombocytopenia was defined as platelets <100 x 109/L. Baseline features of pts with AC and IC were compared using Chi-squared analysis for categorical and the Kruskal-Wallis test for continuous variables. Overall survival was calculated from the date of initial treatment to the date of death from any cause. Time to first treatment (TTFT) was defined as the time interval between the date of diagnosis and date of first CLL treatment. Survival analysis was performed by the Kaplan–Meier method using IBM SPSS statistics for windows. Results: Among 754 pts with CLL, 80 (10.6%) developed cytopenias (anemia and thrombocytopenia). Of those, 50 (6.6%) had IC and 30 (4%) had AC. There was no significant difference between the 2 groups in terms of age, gender, hemoglobin, platelets, LDH, WBC and lymphocyte count at diagnosis. The time to development of cytopenias for the IC and AC groups was similar with median of 3 and 4 years (yrs) from diagnosis, respectively. Within the AC group 16 pts had AIHA, 8 had ITP, 5 had both (Evan's Syndrome) and 1 had PRCA. The median OS was 12.2 yrs (5.9–18.3) and 13 yrs (1.6-24.3) for IC and AC, respectively (p=0.260). However, when compared to CLL pts without cytopenias (median not reached), the AC group had worse OS (p< 0.005) (Fig 1). For the IC and AC groups, the median TTFT was 6.5 yrs (4.5-8.5) and 8.2 yrs (4.1–12.3), respectively (p=0.191). For the CLL pts without cytopenias TTFT was 8.1 yrs (2-12.2), similar to the AC group (p=0.88) (Fig 2). For AC pts, the OS was not significantly different based on treatment received: alkylator based therapy vs. chemo-immunotherapy (p= 0.885). The effect of concomitant hypo-gammaglobulinemia on OS and treatment outcome was studied.Of the 30 pts with AC, 26 had a serum protein electrophoresis done. Of those, 10 (38.5%) had normal results and 16 (61.5%) had low gammaglobulin levels (IgG< 6 g/L); the mean OS was 18.1 yrs and 15.7 yrs respectively (median not reached), (P=0.433). Conclusion: The prognosis of pts with autoimmune and infiltrative cytopenias was similar.However, CLL pts with AC had worse OS compared to those without cytopenias. There was no significant difference in TTFT between AC and IC or when compared to CLL pts without cytopenias. For the AC group, neither treatment with chemotherapy vs. chemo-immunotherapy nor having concomitant hypo-gammaglobulinemia had an effect on outcome. To our knowledge, there are limited population based studies addressing the importance of determining the etiology of cytopenias in CLL pts and the effect of AC on survival. CLL immune complications need to be studied further especially in the context of novel agents and their effects on immune reconstitution. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Very Poor Outcome and Chemoresistance of Acute Myeloid Leukemia Patients with TP53 Mutations: Correlation with Complex Karyotype and Clinical Outcome

Blood ◽

10.1182/blood.v124.21.484.484 ◽

2014 ◽

Vol 124 (21) ◽

pp. 484-484 ◽

Cited By ~ 2

Author(s):

Cristina Papayannidis ◽

Anna Ferrari ◽

Stefania Paolini ◽

Carmen Baldazzi ◽

Chiara Sartor ◽

...

Keyword(s):

Overall Survival ◽

Clinical Outcome ◽

Mutation Rate ◽

Sanger Sequencing ◽

Tp53 Mutation ◽

Complex Karyotype ◽

Wild Type ◽

Cytogenetic Abnormalities ◽

Tp53 Mutations ◽

Poor Outcome

Abstract Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious.. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p<0.0001) and relapse free-survival (RFS) (p < 0.0001). (Figure 1) Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001). Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher's exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures No relevant conflicts of interest to declare.

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Biallelic ATM Inactivation Significantly Reduces Survival in Chronic Lymphocytic Leukemia Patients Treated with Alkylating Agent/Purine Analogue Therapy: Results From the UKCLL4 Trial

Blood ◽

10.1182/blood.v118.21.467.467 ◽

2011 ◽

Vol 118 (21) ◽

pp. 467-467

Author(s):

Anna Skowronska ◽

Gulshanara Ahmed ◽

Ceri E Oldreive ◽

David Oscier ◽

Anton Parker ◽

...

Keyword(s):

Overall Survival ◽

Tp53 Mutation ◽

Alkylating Agents ◽

Univariate Analysis ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Purine Analogues ◽

Free Survival ◽

The Difference ◽

11Q Deletion

Abstract Abstract 467FN2 The Ataxia Telangiectasia Mutated (ATM) gene located at 11q23 plays a central role in DNA damage response. Deletion of 11q23 occurs in approximately 20–30% chronic lymphocytic leukemia (CLL) cases and in 30% of these exhibit mutations in their remaining ATM allele. A smaller proportion of CLL tumours exhibit the presence of ATM mutations in the absence of 11q deletion. We have previously shown that ATM mutations are associated with a shorter overall survival from diagnosis and treatment free survival (TFS) in an unselected CLL cohort. Furthermore, several prospective and retrospective studies, including the UKCLL4 trial, show an inferior overall survival and progression free survival after therapy (OS and PFS) in cases with an 11q deletion treated with alkylating agents and/or purine analogues. The entire ATM coding region was screened for sequence changes using high-performance liquid chromatography and sequencing and known polymorphisms excluded. Screening was performed on 238/777 cases enrolled in the UKCLL4 trial, including all available cases with 11q deletions, with the remaining cases being randomly selected. Other than significant enrichment for patients with 11q deletion (p<0.0001) and a reduction in cases with trisomy 12 (p=0.019), the cohort did not differ from the other 539 patients in the trail. ATM mutations were detected in 35/238(14.7%) cases and no cases with biallelic mutations were observed. In 224 patients 11q deletion and ATM mutation data were available; 49(22%) had an 11q deletion only, 16 (7%) an ATM mutation only, 18 (8%) an ATM mutation plus an 11q deletion and 141 (63%) exhibited both ATM alleles wild type. Apart from an association with 11q deletion (p=0.001), ATM mutations were associated with advanced stage of disease (p=0.01) but not with any other variable. Overall response rate (ORR), PFS and OS were analysed in 188 cases with no detected TP53 mutation or 17p deletion. Compared to ATM wild type cases (ORR=99/109, 91%), 11q deletion alone (ORR=28/38, 74%) and ATM mutation plus 11q deletion (ORR=8/15, 53%) were both associated with reduced ORR, independent of treatment arm (HR= 0.24; 95% CI 0.09 to 0.69; p= 0.007 and HR= 0.09; 95% CI, 0.02 to 0.36; p= 0.001 respectively), whereas ATM mutation alone (ORR=13/15, 87%) was not (p=0.46). In univariate analysis, cases with ATM mutation plus 11q deletion showed significantly reduced PFS (median, 9.2 months) compared to cases with ATM wild type (32.1 months), 11q deletion alone (17.2 months) and ATM mutation alone (37.1 months) (all p<0.003). The difference in PFS between 11q deletion alone and ATM wild type was also significant (p=0.002), whereas between ATM mutation alone and ATM wild type it was not (p=0.98). Similarly, OS for cases with ATM mutation plus 11q deletion was significantly reduced (median, 43.5 months) compared to cases with ATM wild type (89.4 months) and ATM mutation alone (88.9 months) (p=0.001 and 0.036 respectively) and again, the difference between 11q deletion alone (54.4 months) and ATM wild type was significant (p=0.001), whereas between ATM mutation alone and ATM wild type it was not (p=0.63). In multivariate analysis, including the combined ATM mutation and 11q deletion scoring, treatment arm, IGVH mutational status, age, gender and disease stage as covariates. Compared to ATM wild type cases, the presence of deletion 11q alone or of ATM mutation plus 11q deletion was independently associated with significantly increased risk of progression after therapy (Hazard ratio (HR) 1.86, 95% CI 1.23–2.75, p=0.002 and HR 4.11, 95% CI 2.33–7.27, p<0.001 respectively) where as the presence of ATM mutation alone was not. Further univariate analysis showed that while cases with TP53 mutation and 17p deletion had significantly reduced PFS (median 3.4 months) compared to cases with ATM mutation plus 11q deletion (9.2 months) and those with monoalleic TP53 abnormality (6.7 months), the difference in between cases with ATM mutation plus 11q deletion and those with monoalleic TP53 abnormality was not significant (p=0.53). Our results suggest that ATM mutations influence CLL progression under treatment with purine analogues and alkylating agents, independently of other established prognostic markers and that ATM status should be considered before such treatments. It remains to be determined whether ATM mutations have equal impact on patients' response to immunochemotherapy. Disclosures: No relevant conflicts of interest to declare.

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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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