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

Blood ◽  
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.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 967-967
Author(s):  
Ken H. Young ◽  
Nancy Patten ◽  
Sim Truong ◽  
Jens Eickhoff ◽  
Gabrielle L. Rocque ◽  
...  
Keyword(s):  
Dna Binding ◽  
Clinical Outcome ◽  
B Cell ◽  
Median Survival ◽  
Tp53 Mutation ◽  
Tp53 Gene ◽  
Chop Regimen ◽  
Tp53 Mutations ◽  
Dna Binding Domains ◽  
Binding Domains

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

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 484-484 ◽  
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.

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 ◽  
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.

Expression of PD-L1 in colorectal cancer that lack mutations in RAS or TP53 genes.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14500-e14500 ◽  
Author(s):  
Maher Albitar ◽  
Sucha Sudarsanam ◽  
Wanlong Ma ◽  
Shiping Jiang ◽  
Wayne Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumor Cells ◽  
Tp53 Mutation ◽  
Continuous Variable ◽  
Colorectal Cancers ◽  
Wild Type ◽  
Tissue Samples ◽  
Mutation Status ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

e14500 Background: PD-L1 expression as detected by immunohistochemistry (IHC) is significantly lower in colorectal cancers (CRC) when compared with lung cancer or other types of cancer. We explored if mutations in the RAS/RAF gene family, TP53 or PIK3CA can define a subgroup of CRC that express PD-L1. Methods: Tissue samples collected from 107 patients with CRC were studied for the expression of PD-L1 using clone SP142. The same samples were also tested for mutations in NRAS, KRAS, HRAS, BRAF, TP53, and PIK3CA using Next Generation Sequencing (NGS). Results: Of the 107 CRC samples only 15 (14%) showed PD-L1 positive tumor cells (≥1%) and 8 of the 15 (7.5% of total) had PD-L1 in ≤5% of tumor cells. Detected mutations in these samples were as follows: TP53 65%, KRAS 49.5%, PI3KCA 22.5%, NRAS 5%, HRAS 1%, and BRAF 17%. There was no correlation between PD-L1 expression and mutation status in any of the RAS/RAF genes. There was also no correlation between TP53 mutation and PD-L1 expression. This was true irrespective if PD-L1 expression is considered as a continuous variable or when cut-off points of 5%, 20%, or 50% were used. However, patients without any mutation in RAS or TP53 had significantly (P = 0.005) more expression of PD-L1 when cut-off point of 5% is used. This remained true if PD-L1 expression is considered as a continuous variable (P = 0.04). There was no correlation between PIK3CA and PD-L1 expression. Conclusions: PD-L1 expression is significantly more common in CRC that lack mutations in RAS or TP53. PD-L1 expression is detected in 31% of patients with wild-type RAS/TP53 as compared with 12% in patients with RAS/TP53 mutations (P = 0.04). If a cut-off point of 5% is used, 31% of RAS/TP53-wild-type CRC were positive for PD-L1, while only 6% of RAS/TP53- mutant CRC were positive for PD-L1 (P = 0.005). This suggests that in CRC without RAS/TP53 mutation, the PD-L1 may play a more important role in oncogenesis. Exploring immunotherapy in this group of CRC patients might be justified.

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

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 6585-6585
Author(s):  
Robert Metcalf ◽  
Khaireah Mubarak ◽  
Becky Bola ◽  
Samuel Rack ◽  
David Morgan ◽  
...  
Keyword(s):  
Overall Survival ◽  
Salivary Gland ◽  
Next Generation Sequencing ◽  
Tp53 Mutation ◽  
Prognostic Biomarker ◽  
Wild Type ◽  
Tp53 Mutations ◽  
Adenoid Cystic ◽  
Generation Sequencing

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.

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

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.

scholarly journals GUCY2D Gene Loss-of-Function Mutations Responsible for Leber Congenital Amaurosis 1

2019 ◽  
Author(s):  
Feng Xue ◽  
Tianying Wei ◽  
Junhui Sun ◽  
Yuqin Luo ◽  
Yanan Huo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Guanylate Cyclase ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Loss Of Function ◽  
Wild Type ◽  
Leber Congenital Amaurosis ◽  
Cellular Location ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background: Leber congenital amaurosis (LCA) is a group of severe congenital neurodegenerative diseases. Variants in guanylate cyclase 2D (GUCY2D), which encoded guanylate cyclase protein (ROS-GC1) associate with LCA1, accounting for 6–21% of all LCA cases. Methods: In this study, one family with LCA1 was recruited from China. A combination of next-generation sequencing (NGS) and Sanger sequencing was used for disease-causing mutations screening. Additionally, immunohistochemistry and HPLC-coupled tandem mass-spectrometry (HPLC-MS/MS) were used to confirm the cellular location and catalytic activity of ROS-GC1 mutants, respectively. Results: We found three novel mutations (c.139_139delC, c.835G>A and c.2783G>A) in GUCY2D gene. The results showed that mutation c.139_139delC results in a truncated protein and destroys the structure of ROS-GC1 protein. Mutations c.835G>A and c.2783G>A exert no effects on cellular location, whereas significantly reduce the catalytic activity of ROS-GC1. Conclusions: Our findings highlight the clinical range of LCA. Moreover we used HPLC-MS/MS to analyze the concentration of 3', 5'-cyclic guanosine monophosphate (cGMP), suggesting that HPLC-MS/MS can be an effective alternative method to evaluate the catalytic activity of wild type (wt) and mutant ROS-GC1.

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

Blood ◽  
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.

scholarly journals Comparative Study of NGS Platform Ion Torrent Personal Genome Machine and Therascreen Rotor-Gene Q for the Detection of Somatic Variants in Cancer

2020 ◽  
Vol 9 (1) ◽  
pp. 4
Author(s):  
Angela Lombardi ◽  
Margherita Russo ◽  
Amalia Luce ◽  
Floriana Morgillo ◽  
Virginia Tirino ◽  
...  
Keyword(s):  
Target Genes ◽  
Personal Genome ◽  
Analysis Method ◽  
Wild Type ◽  
Specific Analysis ◽  
Current Therapies ◽  
Genomic Aberrations ◽  
Melanoma Patients ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Molecular profiling of a tumor allows the opportunity to design specific therapies which are able to interact only with cancer cells characterized by the accumulation of several genomic aberrations. This study investigates the usefulness of next-generation sequencing (NGS) and mutation-specific analysis methods for the detection of target genes for current therapies in non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and melanoma patients. We focused our attention on EGFR, BRAF, KRAS, and BRAF genes for NSCLC, melanoma, and mCRC samples, respectively. Our study demonstrated that in about 2% of analyzed cases, the two techniques did not show the same or overlapping results. Two patients affected by mCRC resulted in wild-type (WT) for BRAF and two cases with NSCLC were WT for EGFR according to PGM analysis. In contrast, these samples were mutated for the evaluated genes using the therascreen test on Rotor-Gene Q. In conclusion, our experience suggests that it would be appropriate to confirm the WT status of the genes of interest with a more sensitive analysis method to avoid the presence of a small neoplastic clone and drive the clinician to correct patient monitoring.

scholarly journals Next-Generation Sequencing in Acute Lymphoblastic Leukemia

2019 ◽  
Vol 20 (12) ◽  
pp. 2929 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Giorgina Specchia ◽  
Francesco Albano
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Age Of Onset ◽  
Lymphoblastic Leukemia ◽  
Genomic Analysis ◽  
Next Generation ◽  
Acute Leukemias ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias, and features different outcomes depending on the age of onset. Improvements in ALL genomic analysis achieved thanks to the implementation of next-generation sequencing (NGS) have led to the recent discovery of several novel molecular entities and to a deeper understanding of the existing ones. The purpose of our review is to report the most recent discoveries obtained by NGS studies for ALL diagnosis, risk stratification, and treatment planning. We also report the first efforts at NGS use for minimal residual disease (MRD) assessment, and early studies on the application of third generation sequencing in cancer research. Lastly, we consider the need for the integration of NGS analyses in clinical practice for genomic patients profiling from the personalized medicine perspective.

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