ELLA01-1: A study to determine the utility of TP53 mutations as a prognostic biomarker in adenoid cystic carcinoma.

6585 Background: TP53 mutations are reported in 5% of patients with adenoid cystic carcinoma (ACC). Whilst TP53 mutations are associated with adverse clinical outcomes across multiple tumour types, their prognostic significance in ACC is unknown. We sought to determine the utility of TP53 mutations as a prognostic biomarker in a prospective cohort of ACC patients. Methods: From April 2017 to September 2019, 146 patients with ACC were prospectively recruited to an ethically approved study. DNA was extracted from archival FFPE samples and underwent targeted next generation sequencing (Qiagen GeneRead DNAseq Targeted Panel V2 n = 134; Foundation Medicine; n = 12). Clinical, pathological and outcome data were collected on all patients and Kaplan-Meier survival analysis was performed to test for survival differences between TP53 mutated and wild-type ACC. Results: 146 ACC patients (mean age 48 years, range 16-79) underwent DNA extraction and next generation sequencing for TP53 mutations. The primary site was major salivary gland in 47% and minor salivary gland in 48% (other 5%). Analysis was successful in 122/146 patients (84%). Recurrent or metastatic disease was present in 94% (115/122) at study entry. TP53 alterations were identified in 9% (11/122), most frequently within the DNA binding domain (9/11). Non-pulmonary visceral metastases were seen more frequently in TP53 wild-type than in TP53 mutated ACC (44% vs. 10%; p = 0.042), and other clinical parameters were balanced between groups. During follow-up from diagnosis (median follow up 6.6 years), death occurred in 45% of patients with TP53 mutation and in 23% with TP53 wild-type ACC (p = ns). In TP53 mutated ACC, median overall survival was significantly shorter (5.3 vs. 16.3 years), and 10-year survival rate significantly lower (42% vs. 82%) than TP53 wild-type ACC (log-rank p = 0.013). Conclusions: In this cohort of patients with ACC, TP53 mutations were seen with a higher frequency than previously reported. This may be explained by the high frequency of recurrent or metastatic disease at study entry. TP53 mutation was associated with a statistically significant reduction in overall survival in patients with recurrent and metastatic ACC. These findings suggest that stratifying by TP53 status may be of clinical value to inform follow-up strategy in addition to established clinical, pathological and genomic biomarkers.

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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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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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Backtracking Subclonal Mutations Of TP53 In Myelodysplasia (MDS) With Del(5q) With Next-Generation Sequencing (NGS)

Blood ◽

10.1182/blood.v122.21.520.520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 520-520

Author(s):

Laurence Lodé ◽

Audrey Ménard ◽

Marion Loirat ◽

Maxime Halliez ◽

Steven Richebourg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Lower Limit ◽

Clonal Evolution ◽

Applied Science ◽

Tp53 Mutations ◽

Variant Detection ◽

Serial Samples ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Landscape analyses of mutational patterns have shown that virtually all myelodysplastic syndromes (MDS) harbor somatic mutations in >80% of cases. These molecular alterations provide useful clonality markers with a potential for early diagnosis of MDS when only cytopenia without marked dysplasia is observed. These markers have been proposed as future prognostic tools to guide therapeutic strategies (Bejar et al., 2011; Itzykson et al, 2013; Mufti et al 2013). Mutational analysis is finally a good way to track disease complexity by deciphering oligoclonality in MDS and better understand clonal evolution. Alterations in the TP53 gene are the most common cause of tumor escape from apoptosis. The aim of this study was to identify TP53 mutations in consecutive samples of lower-risk MDS (IPSS ≤1) with del(5q)obtained at follow-up or progression after sequential classical treatments. Next-generation sequencing (NGS) was used to backtrack the mutant clone(s) identified in late samples. The study was performed both by conventional Sanger sequencing and NGS on a GS Junior Instrument (Roche Applied Science, Mannheim, Germany). For each sample, eight exons (4-11) were amplified from 320 ng of DNA with preconfigured primer plates provided within the IRON II study network. PCR reactions were performed using the FastStart High Fidelity PCR System kit (Roche Applied Science). After double purification with Agencourt AMPure XP beads (Beckman Coulter, Miami, FL), exon-specific amplicon pools were generated and quantified using the Quant-iT™ Broad-Range PicoGreen DNA Assay Kit (Invitrogen, Carlsbad, CA). Emulsion PCR was performed with GS Junior emPCR Reagents (Lib-A) (Roche Applied Science) using 5 x 106 beads at a copy per bead ratio of 0.6. Finally, a fraction of 5-7% enriched beads was loaded on GS Junior Titanium sequencing PicoTiterPlate kit (Roche Applied Science). Data were analyzed for sequence alignment and variant detection using the GS Junior Sequencer and GS Amplicon Variant Analyzer softwares, versions 2.7 and 2.9 (Roche Applied Science). The results were further processed using the Sequence Pilot software version 4.0.1 (JSI Medical Systems, Kippenheim, Germany). The sensitivity of variant detection was set to a lower limit of >1% for bidirectional reads. This threshold was chosen according to a recent study investigating the assay's lower limit of detection (Grossmann et al., 2013), thus underlining the strength of NGS to identify subclones at a low frequency, not detectable by conventional Sanger analysis. A total of 89 DNA samples were extracted from the cytogenetics pellets of a cohort of 40 MDS with del(5q). TP53 mutation analysis was performed on 40 initial and 49 follow-up or progression samples including serial samples for 23 subjects. The depth of coverage was at least 500X and up to 8,444X per amplicon. Of those samples obtained and analysed at time of last follow-up or progression, 14 (61%) had TP53 mutations, mostly in the DNA-binding domain. Performing backtracking on previously collected serial samples, TP53 mutations were retrieved by NGS in 43% of initial samples (n=6), which is different from what was previously described by Jädersten et al (2011). A complete scenario of clonal evolution was retrieved in 11 cases, evidenced by TP53 mutations and/or cytogenetics. These were always consecutive to treatment with lenalidomide, yet 6 of the 12 cases without clonal evolution were also consecutive lenalidomide. Figure 1 provides the example of a complete follow-up including nine time points. More correlation with treatment will be provided. Although lenalidomide remains the treatment of choice for MDS with del(5q), resistant subclones may survive and culminate even following therapy initiation. This theory was recently suggested by Landau et al. in CLL (2013) and our test results support this. Early detection of emerging subclones could lead to initiation of alternative treatment, and we thus propose that a monitoring of TP53 alleles is performed annually after the onset of therapy for MDS using NGS. Figure 1. Figure 1. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.

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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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Detection of TP53 Mutations in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). a Comparison Between a Functional Method (FASAY) and Next Generation Sequencing (NGS)

Blood ◽

10.1182/blood.v124.21.3266.3266 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3266-3266

Author(s):

Cécile Bally ◽

Aline Renneville ◽

Lionel Adès ◽

Claude Preudhomme ◽

Hugues de Thé ◽

...

Keyword(s):

Next Generation Sequencing ◽

Lower Risk ◽

Sanger Sequencing ◽

Tp53 Mutation ◽

P53 Protein ◽

Next Generation ◽

Tp53 Mutations ◽

The Cost ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background TP53 mutations inactivating p53 protein, often associated with loss of the remaining TP53 allele through 17p deletion, are major prognostic factors in many hematological malignancies, including CLL, myeloma, AML and MDS. In AML and MDS, they are usually associated with complex karyotype (including del 17p) and very poor prognosis (Blood 1991, 78(7):1652-7 , Bejar, NEJM 2011), including after allogeneic SCT (Middeke JM, Blood 2014) but they are also seen in lower risk MDS with isolated del 5q, where they confer resistance to Lenalidomide (Jadersten, JCO 2011). The advent of Next Generation Sequencing (NGS) techniques has improved the detection of such mutations, by allowing the identification of small mutated clones. Other detection methods may prove interesting, especially functional methods like FASAY ( Functional Assay of Separated Allele in Yeast) , an easy and sensitive method that detects TP53 mutations by assessing the p53 function as transcription factor (Flaman et al, PNAS 1995). We compared the detection of TP53 mutations in MDS and AML by FASAY and NGS approaches. Methods The 84 patients analyzed included 10 AML, 10 higher risk MDS, and 64 lower risk MDS with del 5q. RNA and DNA were extracted from marrow mononuclear cells. TP53 mutations were detected on RNA by FASAY where, after amplification of the TP53 mRNA, the PCR product is co transfected with an open gap repair plasmid leading, by homologous recombination, to p53 protein expression in the yeast. The yeast strain used is dependent on p53 functionality for growth and color and detection of more than 10% of small red yeast colonies defines a non-functional FASAY result. All non-functional FASAY were confirmed by the split versions of the test and TP53 defects were characterized by Sanger sequencing. The detection limit is around 10% in our hands (Manie E, Cancer Res 2009). In parallel, TP53 mutations were detected on DNA by NGS using the IRON II plate design and pyrosequencing on a GS Junior System (Roche). (Kohlmann, Leukemia 2011).FASAY (+Sanger sequencing) and NGS were performed in two different labs. Results By FASAY, 47 patients (56%) had a functional p53 and 37 cases (44%) a non-functional p53 and a mutation was confirmed by Sanger in all non functional cases. By NGS analysis, no TP53 mutation was found in 47 cases (56%) and a mutation was detected in 37 cases (44%). In the 37 mutated cases by NGS, the median proportion of mutated allele was 35% (range 3 to 99%), including a median of 72%, 35%, 25 % in AML, higher risk MDS and lower risk MDS with del 5q, respectively. The mutated clone size was lower than 10% in only 2 patients who both had lower risk MDS with del 5q (3 and 6%, respectively). A perfect correlation between FASAY and NGS was found in 80 (95.5%) cases. The 4 discordant cases included a mutation detected only by FASAY in 2 cases, and only by NGS in 2 cases. Undetected mutations by NGS were insertions of intronic sequences (intron 9) not explored by the technique used. These insertions resulted in non-functional protein well detected by FASAY which analyses the global cDNA sequence including splicing defects. Undetected mutations by FASAY were mutations in which the percentage of mutated alleles was less than 10% (3% and 6 % respectively). Finally, while the cost of NGS analysis for TP53 mutation is around 200 euros when performed alone (and around 2000 euros when combined to analysis of the 30 main other genes involved in MDS and AML), the cost of the FASAY technique is around 20 euros (prices including reagents only). Conclusion The FASAY technique is a cheap method, that in spite of a sensitivity of only 10%, was able to detect 98% of TP53 mutations detected by NGS. In fact those mutations appear to involve generally relatively large clones in MDS and AML. FASAY could also detect 2 atypical intronic mutations overlooked by NGS. Demonstrating in such difficult cases that the resulting p53 protein is non functional and therefore probably has pathophysiological significance, is an advantage of FASAY .The combination of the 2 methods, and especially the combination of DNA and RNA analysis, may be useful in such cases. Disclosures No relevant conflicts of interest to declare.

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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.

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TP53 Mutated MDS Patients Respond Equally to Hypomethylating Agents but Have Significantly Shorter Response Duration Compared to Patients with Wild Type TP53

Blood ◽

10.1182/blood.v126.23.1681.1681 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1681-1681 ◽

Cited By ~ 2

Author(s):

Koichi Takahashi ◽

Hagop M. Kantarjian ◽

Keyur Patel ◽

Carlos E Bueso-Ramos ◽

Tapan Kadia ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Tp53 Mutation ◽

Prognostic Impact ◽

Hypomethylating Agents ◽

Complex Karyotype ◽

Wild Type ◽

Tp53 Mutations ◽

Duration Of Response

Abstract Background: Prognostic impact of TP53 mutations has been well described in MDS. However little is known about predictive impact on response to hypomethylating agents (HMA). Aim: To determine predictive impact of TP53 mutation on response to frontline HMA therapy in MDS. Methods: Bone marrow samples from 168 patients with untreated MDS were screened for TP53 mutation by next generation sequencing platform. All patients were treated with upfront 5-azacitidine or decitabine based HMA therapy. 13 of them had longitudinal follow up of TP53 mutation after HMA therapy. Results: 38 patients (23%) had TP53 mutations. At baseline, TP53 mutated patients were significantly more neutropenic (P = 0.02), thrombocytopenic (P = 0.008), and had higher bone marrow blast (P = 0.006). TP53 mutation was significantly associated with complex karyotype (P < 0.001), monosomal karyotype (P < 0.001), and del 17p/-17 (P < 0.001). There was a trend toward mutual exclusivity between splicing pathway gene mutations and TP53 mutation (P = 0.07). Complete response (CR) and overall response (OR) to HMA therapy was observed in 34% and 45% of TP53 mutated patients, respectively, and there was no statistical difference from wild type patients (P = 0.38 and P = 0.13). Time to achieve response was also similar between TP53 mutated and wild type patients (P = 0.2). However, TP53 mutated patients had significantly shorter CR duration compared to wild type patients (6.3 months versus 28.5 months, P = 0.001). 11 out of 13 patients who had longitudinal follow up of TP53 mutation were found to have the same persistent TP53 mutation when they lost response to HMA therapy. TP53 mutated patients had worse overall survival (P <0.001) and prognostic impact of TP53 mutation was significant after adjusting for complex karyotype or IPSS-R risk. Conclusion: MDS patients with TP53 mutations equally respond well to HMA therapy compared to WT patients. However, duration of response is significantly shorter than WT patients, which translates into worse overall survival. Longitudinal follow up showed persistence of the same TP53 clone after HMA therapy. Novel therapeutic strategy to improve duration of response in TP53 mutated MDS is urgently needed. Disclosures DiNardo: Novartis: Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding.

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Next Generation Sequencing-Based Mutational Profile of Therapy-Related AML and De Novo AML: Results from a Single Pediatric Institution in Argentina

Blood ◽

10.1182/blood-2018-99-118408 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1508-1508

Author(s):

Patricia Laura Rubio ◽

Maria Sara Felice ◽

Matias Juanes ◽

Gabriel Veneruzzo ◽

Francisco Martin Garcia ◽

...

Keyword(s):

Next Generation Sequencing ◽

De Novo ◽

Normal Karyotype ◽

Next Generation ◽

Wild Type ◽

Tp53 Mutations ◽

Classical Genetic ◽

Genetic Features ◽

De Novo Aml ◽

Generation Sequencing

Abstract Introduction Therapy-related acute myeloid leukemia (t-AML) and de novo AML (dnAML) are a group of aggressive diseases with poor outcome, in spite of recent advance in therapy. However, the alterations in different genes seems to be associated with this dismal prognosis. Recently, different groups have shown that t-AML in adult patients has a mutational profile distinct from dnAML. TP53 mutations were mostly described in t-AML. Next-generation sequencing (NGS) is a fast and accurate method used to demonstrate the presence of multiple alterations in several genes. The comparison of t-AML and dn-AML mutational profiles could be useful for improving our understanding of the molecular genetics events and in the outcome of each group as well. Objectives: 1-To describe the incidence of pathogenic variants in t-AML and dn-AML using a customized NGS panel, 2-to study correlations of these mutations with classical genetic features in each group of AMLs, 3-to compare the mutational profile of both diseases, and 4-to evaluate the prognostic impact of these mutations. Patients and Methods A total of 136 available DNA samples from non-APL dn-AML and 11 t-AML pediatric patients were retrospectively analyzed. The samples were sequenced on a Miseq (Illumina) platform, using a customized amplicon-based NGS panel (Illumina) including 65 genes. A total of 172,360 bp of DNA target regions were covered with 1,207 amplicons with a mean coverage of 1,308. Sequences obtained were analyzed by in-house bioinformatics pipeline, with particular analysis for NPM1, CEBPA and FLT3 genes, using Pindel and ITDseek algorithms. Results At least one pathogenic variant was detected in 100 out of 136 (74%) dn-AML analyzed cases, and 11 out of 11 (100%) t-AML patients. Overall, 317 mutations were found in 49 genes with a mean of 3.2 variants per patient for dn-AML, and 53 mutations in 26 genes with a mean of 4.8 variants per patient for t-AML. The most frequently mutated genes were: BCORL1 (5.4%, p=ns), FLT3 (5.0%, p=ns), KMT2A (5.0%, p=ns), BCOR (4.7%, p=0.0126), RB1 (4.4%, p=ns) for dn-AML, and KDM6A (15.1%, p<0.00001), BCOR (9.4%), CUX1 (9.4%, p=0.0020), BCORL1 (5.7%), FLT3 (5.7%) for t-AML. TP53 mutations were only detected in 5 dnAML cases. In the group of patients with dnAML, the distribution of classical genetic abnormalities was: KMT2A/11q23 (24%), RUNX1-RUNX1T1 (22%), Normal karyotype (19%), CBFB-MYH11 (9%). The most frequent mutations in normal karyotype were: FLT3 (38.1%, p=0.0019), CEBPA (27.3%, p=0.0001), NPM1 (28.6%, p<0.00001) and WT1 (23.8%, p=0.0001). In the group of t-AML, the genetic features were: KMT2A/11q23 (45%, p=ns) and Normal karyotype (9%, p=ns). CBFA2T2 mutation was statistically associated with the presence of RUNX1-RUNX1T1 in dn-AML (p=0.0002). No other significant association between variants and genetic groups was observed. Complete remission was achieved in 92% of cases in dn-AML and 72% in t-AML (median follow-up: 21 and 5 months respectively). Leukemia-free survival probability (LFSp) was 60(5)% [High-Risk: 55(6)%; Low-Risk: 71(10)%] for dn-AML and 13(11)% for t-AML (p=0.0008). In dn-AML group, LFSp(SE) of patients with at least one deleterious variant was 60(6)%, whereas those without any variant showed LFSp(SE): 58(10)(p=ns). In the normal karyotype group, the genotype NPM1/CEBPA-mutated plus FLT3/WT1-wild-type disclosed a LFSp(SE) of 86(13)% while remaining patients showed a LFSp(SE) of 42(14)% (p=ns). Conclusions We describe the incidence of mutations detected by NGS in a series of pediatric t-AML and dn-AML in our country. In spite of the limited number of patients, the mutation profile of t-AML seems to be different from the de novo counterpart. BCOR mutations were significantly associated with dn-AML cases, while KDM6A and CUX1 variants with t-AML patients. In contrast with adult reports, non TP53 mutations were observed in our group of t-AML patients. There was a significantly higher incidence of mutations in FLT3, CEBPA, NPM1 and WT1 within the normal karyotype group of dn-AML. The combined genotype NPM1/CEBPA-mutated plus FLT3/WT1-wild-type showed a trend to a better LFSp in dn-AML. The molecular genetic profile characterization of these heterogeneous diseases allows expansion of our understanding about the leukemogenic mechanisms involved. Disclosures No relevant conflicts of interest to declare.

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Clinical Impact of TP53 Mutations in Patients with MDS and Isolated Deletion 5(q) Treated with Lenalidomid: Results from the German Prospective Le-Mon-5 Trial

Blood ◽

10.1182/blood.v124.21.1920.1920 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1920-1920

Author(s):

Maximilian Mossner ◽

Johann Christoph Jann ◽

Evi Launiger-Lörsch ◽

Daniel Nowak ◽

Uwe Platzbecker ◽

...

Keyword(s):

Next Generation Sequencing ◽

Research Funding ◽

Cytogenetic Response ◽

Detection Sensitivity ◽

Next Generation ◽

Tp53 Mutations ◽

Transfusion Independence ◽

Blast Count ◽

Generation Sequencing

Abstract Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q). Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of < 5% in the bone marrow, a platelet count > 50.000/µl and absolute neutrophil counts of > 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment. Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. Disclosures Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

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Comparison the Distinct Clinical Outcome with TP53 Mutation Status in MDS and AML :an Retrospective Study

Blood ◽

10.1182/blood-2019-124622 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5405-5405

Author(s):

Xinrong Xiang ◽

Yang Ou ◽

Yu Wu

Keyword(s):

Overall Survival ◽

Dna Binding ◽

Clinical Outcome ◽

Tp53 Mutation ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

Hypomethylating Agents ◽

Tp53 Mutations

Background: The TP5 gene is by far the most frequently mutated gene in human cancer. Some studies have shown that TP53 should be used as an independent prognostic biomarker in the prognostic stratification of myelodysplastic syndromes(MDS) and acute myeloid leukemia (AML). In this study, we want to explore the effects of different TP53 mutation status on clinical outcomes in AML and MDS. Method and Results: We retrospectively collected and analyzed 51 patients with TP53 mutation from Jan 1st , 2015 to May 1st , 2019 in West China Hospital, Sichuan University, China. Among 51 patients, 27 patients were diagnosed as MDS,11 were diagnosed as AML. Then average age is 55.7(15-77). According to IPSS-R of MDS, there are 18 out of 27 patients in the high-risk and very high-risk groups, only 1 patient in the intermediate risk group and 8 patients lost follow up. 10 out of 11 AML patients were in the high risk group according to the NCCN guideline. In our analysis, the incidences of complex karyotype and additional gene mutations were 86.67% and 36.84% in MDS and AML patients. AML evolution was 40.74% in MDS patients. The median follow-up time was 16 months (4-51 months) and the median overall survival was 7.61 months (range, 2-17 months). So far, 28.95% patients are still being treated, of which 9 have been treated with hypomethylating agents. To compare the clinical outcome with different TP53 mutations status, firstly, we identified the mutation types and various mutants activity. We counted 62 TP53 mutations in 51 patients and marked their sites at the transcriptional level in Fig1 . Codons 248, 273, 175, 282, 245, and 249 are most common somatic mutations in TP53. Herein, the TP53 mutation hot spots found in 38 AML or MDS patients are clustered in the DNA-binding domain. Then we used the luciferase reporting system to test the p53 mutant activity with missense mutations in the DNA-binding domain as a transcription factor. The results in Fig2 showed that with the exception of R158H, M237V , V143M , Q136E and H178Q, almost all the other mutation types have no activity. And we demonstrated these TP53 hotspot mutations in the structure ( Figure 3). The overall survival time of patients with residual P53 mutant activity (except for the compound mutation) far exceeded the median overall survival(7.61months). For the other patients with mutants which almost completely lost the activity of p53 protein as a transcription factor, two patients were treated with hypomethylating agents followed by hematopoietic stem cell transplantation have survived for 14 and 11 months after diagnosis, respectively. As shown in Fig4 and Fig 5, we compared the OS of these two group patients with different P53 mutant activity and different function areas for 6 months and 12 months, respectively. The results showed that the patients with residual P53 activity survived better Conclusion: Patients with different TP53 mutations do have different clinical outcomes. Patients with residual P53 mutant activity do respond better to treatment, and the final clinical outcome of patients who lose P53 activity completely is poor, but hypomethylating agents and hematopoietic stem cell transplantation can also ppropriately prolong survival time. Legends to the figures Figure1: 62 TP53 mutations in 51 patients with hematological neoplasms. The TP53 mutation hot spots found in 38 AML or MDS patients are clustered in the DNA-binding domain. There were 9 patients with multiple TP53 mutations. The black label represents somatic mutation, and the red label represents germline mutation. Missense mutations account for 61.70%(29/47) of all the TP53 mutations in 38 patients. Figure 2: The p53 mutant activity with missense mutations in DNA binding domain tested by the luciferase reporting system. We compared the normalized RLU on PUMA of P53 mutant with wild type. Figure 3: The structure of the p53 core domain bound to DNA. We marked the residues affected by the TP53 hotspot mutations . The deeper the red, the higher the mutation frequency and the more the mutation clustered Figure 4 : The overall survival of 30 follow-up patients. We use a bar chart to show the clinical outcome(OS) of 30 patients with different mutation types and P53 structure affected by different p53 domains. Figure 5 : The probability of overall survival of 30 follow-up patients. They are divided in two groups: patients with residual P53 activity VS patients with inactive P53 mutant. Disclosures No relevant conflicts of interest to declare.

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