TP53 is a major tumor suppressor which plays an important role in tumorigenesis, proliferation, and cell survival in most human cancers 38. The previous research confirmed that greater than 80% of human cancers have mutations in TP53. The current model of the IARC database (R20, July 2019) includes over 29900 somatic mutations and 9200 variations reported in SNP databases (“Database Development”, 2019). Nowadays, it is undisputed that the inactivation of the TP53 gene due to a mutation is a critical step in tumor transformation and progression1. The activity of TP53 lies in its ability to activate and suppress a broad set of target genes whose products regulate, among other things: the cell cycle arrest and apoptosis when the DNA is damaged 39.
TP53 gene might not have a specific role in developing all tumors. However, mutations of this gene have been related to a complicated karyotype, poor prognosis, and poor response to chemotherapy 40-42. There is a lack of the published data in Saudi Arabia that describe the frequency of the TP53 mutations and their relationship with cytogenetic and clinical phenotype in hematological neoplasms. Therefore, we endeavored in this study to evaluate the TP53 deletion using the FISH technique and TP53 mutations screening TP53 using NGS technology and their relationship with cytogenetics and clinical phenotype in leukemia patients.
Previous studies have shown that FISH is a powerful cytogenetic technique used to evaluate the TP53 alterations in patients with hematological malignancies 43, 44. In our study, 20 patients samples were examined, and the TP53 deletion was detected in 35% of the cases. Similarly, there were about 35% of cases with normal signaling of TP53 and two cases with extra signals in TP53.
TP53 deletion was identified in about 62.5% of all investigated child samples, whereas the deletion was detected at a lower rate (16%) of adult cases. Furthermore, the highest average of TP53 deletion has been noticed in patients with ALL (55%), which is in concordance with and even higher than what was reported by other studies (56%) 45.
TP53 changes were mainly seen in a hypodiploid subtype of ALL, mainly due to germline changes, which changed the disease manifestation to Li-Fraumeni syndrome. Therefore, it becomes important to know if the identified variant is a secondary event contributing to risk stratification and treatment response 46.
According to the analysis of 10 samples by NGS, only one (MDS patient) was harboring a TP53 mutation in exon 5. The detected mutation was a heterozygote point mutation (T to C) that changed amino acid residue from histidine to arginine at codon 175 of the TP53 gene. The mutation was found in an MDS patient who was the only case in the study. Based on our knowledge and from the search on different databases (ClinVar - NCBI”, 2020; “IARC TP53 Search”, 2020; “Search results on cosmic for H175R”, 2020), this particular mutation (H175R) we observed in our study was not reported previously in MDS or any other hematological malignancies. However, this mutation was found in lung adenocarcinoma from Korean patients47. According to cytogenetic and FISH results, the mutation was associated with a complex karyotype and TP53 gene amplification detected by FISH. This finding aligns with what was published before that TP53 mutation is associated with a complex karyotype and poor prognosis in MDS48, 49.
The reason why TP53 mutations are associated with the complex karyotype remains unclear and raises the question of whether these mutations promote and induce increasing cellular instability or whether these mutations are secondary mutations that occur only after chromosomal instability. Previous studies showed that TP53 mutations in hematological malignancies are highly prevalent in a complex karyotype and deletion of chromosome 17p. At the same time, in the other cytogenetic subgroups, they are deficient, suggesting that chromosomes instability may precede mutations in TP53 22, 50, 51. However, further studies and examination on larger cohorts are needed to assess these possibilities.
Targeted NGS in our research enabled us to discover mutations in other genes rather than TP53. The analysis revealed that TP53 mutation was associated with other genes mutations such as TET2, SRSF2, ASXL1, U2AF1, NPM1, and SETBP1. Similar co-occurrence results for these mutations with TP53 mutation in MDS were published52-54. Interestingly, among these mutations, we found exclusive mutations on ASXL1 (K1368T) and SETBP1 (V231L) that were associated mainly with TP53 mutation 55. Reported that ASXL mutations are frequently seen in MDS in association with SETBP1 mutations, inhibiting myeloid differentiation and inducing leukemic transformation 56. Furthermore, they reported that SETBP1 is a driver for ASXL1 mutation, and ASXL1 is a poor prognostic biomarker associated with short survival. Another study focused on TP53 and ASXL1 prognosis in AML and MDS reported that they are two independents factors associated with poor prognosis and short survival; nevertheless, none of the studies had reported the pathogenic significance of the particularly identified mutations on these genes, their importance on disease pathogenicity cannot be ignored and further functional validation should be done 57.
Based on our knowledge, mutation of ASXL1 (K1368T) was also not previously reported, and its pathogenicity was not assessed or examined before. On the other hand, a SETBP1 (V231L) mutation was found in Schinzel-Giedion Midface Retraction Syndrome with a mild effect, as reported by Illumina Clinical Services Laboratory (“VCV000159885.1 - ClinVar - NCBI”, 2020). Therefore, the exclusiveness of the identified mutations in this project will be considered variants with unknown significance. As for the correlation of TP53 mutations with tumor type and cytogenetic abnormalities, in AML, all patients were found with wild-type TP53 (six patients had a normal karyotype and one with a single chromosomal abnormality). In addition, one patient has TP53 deletion by FISH. This finding is consistent with other published work, which indicated that TP53 mutations are infrequent in AML without a complex karyotype, highlighting its importance as a therapeutic target through activation of the intact gene50, 58, 59.
In the Lymphoma patient, there was no TP53 mutation. Instead, the patient had a normal karyotype with a TP53 deletion based on FISH. This finding is consistent with Ahmad et al., study, which revealed that TP53 mutations in Saudi non-Hodgkin’s lymphoma are infrequent, as, from 45 patients, only one patient showed a mutation in the TP53 gene 60.
Further examination and screening on a larger cohort are highly recommended to confirm research findings. Also, the used panel covers only 4 exons from TP53, representing the exons that include the most reported hotspot mutations in the gene. That limits the study finding as there might be a chance of detecting other variants of the TP53 gene on the uncovered regions. Therefore, whole gene sequencing for TP53 is important to confirm the absence of any changes on the gene. That will support the recommendation of utilizing the activation of the Wilde type gene in controlling tumor progression. Moreover, the FISH technique remains a powerful tool for clinical diagnosis, and further screening on the clinical impact of FISH analysis for TP53 on AML and ALL manifestation is recommended.