TP53 Expression and Mutational Analysis in Hematological Malignancy in Jeddah, Saudi Arabia

Background: Tumor protein 53 (TP53) is a tumor-suppressor gene and plays an essential role in apoptosis, cell cycle arrest, genomic stability, and DNA repair. Although it is the most often mutated gene in human cancer, it has respectively low frequency in hematological malignancy but is significantly linked with complex karyotype, poor prognosis, and chemotherapeutic response. Nevertheless, the prevalence and prognostic role of TP53 mutations in hematological malignancy in Saudi patients are not well reported. We, therefore, aim to assess the frequency of TP53 mutations in hematological malignancies in Saudi Arabia. Method: 20 different hematological malignancy samples were tested using fluorescence in situ hybridization (FISH) technique for TP53 deletion detection and next-generation sequencing (NGS) targeted panel was applied on 10 samples for mutations identification specifically TP53 mutation. Results: TP53 deletion was detected in 6 of 20 samples by FISH. Most of the 6 patients with TP53 deletion had acute lymphoblastic leukemia (ALL), and majority of them were child. NGS result revealed one heterozygous missense mutation in exon 5 of the TP53 gene (c. G9963A, p.H175R). Conclusion: To the best of our knowledge, the TP53 mutation is novel variant, and the first time we are reporting their association with myelodysplastic syndromic individual with complex karyotype. This study recommends further analysis of genomic mutations on bigger cohorts, utilizing high throughput technologies.


Introduction
TP53 is the most frequently mutated gene in most human cancers, with a frequency of 50% [1,2]. Alterations consist of mutations and deletions and are generally related to advanced disease stages, inadequate therapy response, and poor prognosis [3][4][5][6][7]. The transcription component TP53 has a central regulatory role in numerous signaling pathways, including cell cycle arrest, apoptosis, and DNA repair [8,9]. Owing to its essential function

Next-Generation Sequencing Analysis
To detect the variants in P53 and other target genes, panel sequencing analysis was performed for 10 selected cases, according to the variability in disease diagnosis (AML, ALL, MDS, and NHL) and chromosomal abnormalities detected by karyotype and FISH. ClearSeq AML HS panel (G9963A, Agilent Technologies, Santa Clara, CA, USA), targeting 48 exons among 20 myeloid leukemia-associated genes, was used to investigate the mutational hotspot regions of TP53 (ENST00000269305, exons [5][6][7][8] and other panel genes (Table 1). Genomic DNA was extracted from the patient's bone marrow using QIAamp DNA blood Mini kit (51,104, QIAGEN, Hilden, Germany) as per the manufacturer's instructions and quality was assessed by a NanoDrop2000c (5538, Thermo Scientific, Waltham, MA, USA). Purity was determined by absorbance ratio (A260/A280 = 1.7-1.9). DNA was digested and denatured to generate different fragments or target regions using the HaloPlex HS Target Enrichment System kit, (G9963A, Agilent Technologies, Santa Clara, CA, USA). Fragmented target DNA was hybridized with a library probe (HaloPlex HS probes), followed by streptavidin ligation and barcode target capturing and amplification of enriched fragment. The template library was denatured and diluted to 20 pM before next-generation sequencing using MiSeq (Illumina, San Diego, CA, USA) platform and ClearSeq AML HS panel according to manufacturer's protocol.

Clinical Characteristics of Patients
A total of 20 hematological malignancies patients (10 males and 10 females) with a median age of 43 years (range 5-69 years) were included in present study (Table 2). Most cases were of AML (45%) and ALL (45%) categories followed by non-Hodgkin's lymphoma (5%) and MDS (5%).

Cytogenetic and FISH Results
The cytogenetic results showed complex karyotype in four cases (20%) and single chromosomal abnormality in two cases (10%) while no chromosomal abnormalities were detected in remaining cases (70%) (Table 2, Figure 1) FISH results, based on the analysis of 200 interphase cells, were variable as normal signaling for TP53 found in seven, partial deletion for TP53 (11-45% of interphase cells) found in seven, mixed signaling of TP53 (15% normal cells, 45% cells with deletion in TP53, and 40% cells had three signals for TP53) found in one case and extra signals for TP53 (three signals in 20 and 30% without deletion of TP53) were present in two cases ( Figure 2). The TP53 deletion was detected in five ALL (55%), one AML and one MDS patient while TP53 amplification was detected in one NHL case only. There was no gender association with the TP53 deletion. However, TP53 deletions were detected in 62% (5/8) child samples compared to 16% (2/12) adult patient (Table 3).  [20] deletion of TP53) were present in two cases ( Figure 2). The TP53 deletion was detected in five ALL (55%), one AML and one MDS patient while TP53 amplification was detected in one NHL case only. There was no gender association with the TP53 deletion. However, TP53 deletions were detected in 62% (5/8) child samples compared to 16% (2/12) adult patient (

Next-Generation Sequencing Analysis
Sequencing analysis revealed a heterozygous missense mutation (c. G9963A, p.H175R) in the TP53 gene in MDS patient where substitution of T to C resulted in a change of amino acid from histidine to arginine at codon 175 ( Table 4).

Next-Generation Sequencing Analysis
Sequencing analysis revealed a heterozygous missense mutation (c. G9963A, p.H175R) in the TP53 gene in MDS patient where substitution of T to C resulted in a change of amino acid from histidine to arginine at codon 175 (Table 4).

Correlation of TP53 Mutation with Cytogenetic & FISH Results
In assessing the relationship of TP53 mutation to cytogenetic and FISH results, the mutation was observed in MDS patients with a complex karyotype. Interestingly, the FISH result for this patient showed a gain of TP53 gene in 60 cells out of 200 investigated cells.

Correlation of TP53 Mutation with Other Genes Mutations
Using targeted NGS with ClearSeq AML HS panel (Agilent Technologies), we identified a total of 91 mutations in 14 of the 20 genes analyzed in our cohort. The analysis showed that patient with TP53 mutation also had mutations in NPM1, TET2, SRSF2, ASXL1, SETBP1 (Table 5). Furthermore, among these mutations, there were two unique mutations in both ASXL1 (K1368T) and SETBP1 (V231L) that were exclusively associated with TP53 mutation and did not present in the other patients (see Table 6, Figure 3).

Structural and Functional Impact at the Protein Level
Three-dimensional structures of TP53, ASXL1 and SETBP1 were visualized, and the changes induced by specific mutations were focused (Figure 4). In p53, position Arg175 is one of the hot-spots for mutation in human cancer [38], because this residue plays an important role in maintaining the structure of the DNA-binding domain but isn't involved Silent (S1275)

Structural and Functional Impact at the Protein Level
Three-dimensional structures of TP53, ASXL1 and SETBP1 were visualized, and the changes induced by specific mutations were focused (Figure 4). In p53, position Arg175 is one of the hot-spots for mutation in human cancer [38], because this residue plays an important role in maintaining the structure of the DNA-binding domain but isn't involved in direct interaction with DNA ( Figure 5). Arginine to histidine mutation might executes its function by directly binding other transcription factors and gene promoters and transcriptionally altering their expressions by recruiting cofactors or corepressors. The mutations found in ASXL1 and SETBP1 are located on the flexible loop and are on the periphery, they might be altering their interactions with other proteins (Table 7).

Discussion
TP53 is a major tumor suppressor which plays an important role in tumorigenesis, proliferation, and cell survival in most human cancers [39]. 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 29,900 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 progression [1]. 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 [40].
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 [41][42][43]. 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 using NGS technology and their relationship with cytogenetics and clinical phenotype in leukemia patients.

Discussion
TP53 is a major tumor suppressor which plays an important role in tumorigenesis, proliferation, and cell survival in most human cancers [39]. 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 29,900 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 progression [1]. 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 [40].
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 [41][42][43]. 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 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 [44,45]. 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. This study showed that, 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%) [46]. 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 [47].
Gain of mutation for TP53 was observed in two cases; one of them was an adult male patient with AML and had a normal karyotype. The other one was an adult female with MDS and with complex karyotype. The MDS patient had an abnormality on chromosome 17, and that distribution in the chromosome structure might be associated with the extra signals detected by FISH. Therefore, to evaluate if the detected changes by FISH were originally derived from a mutation on the TP53 gene or whether the gene is intact in the positive cases by FISH, NGS sequencing using a targeted panel was performed on 10 selected samples [47].
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 patients [48]. 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 MDS [49,50].
p53 tumor suppressor homotetramer structure is composed of four identical protein chains tied together by the tetramerization domain at the center. Zinc is an essential cofactor with 1 zinc ion per subunit. A long flexible region in each chain then connects to the second stable domain: a large DNA-binding domain, rich in arginine residues that recognizes DNA's specific regulatory sites and interacts with DNA. The transactivation domain found near the end of each arm, activates the neighboring proteins involved in DNA-reading machinery. R175H is a hotspot mutation (corresponds to variant dbSNP:rs28934578). This missense variant found in Li-Fraumeni syndrome (LFS), germline mutation, in sporadic cancers and somatic mutation. This natural SNV found in the DNA-binding domain, involved in positioning other DNA-binding amino acids. Arg175 belongs to region required for interaction with HIPK1, ZNF385A, FBXO42 and AXIN1. It does not induce SNAI1 degradation but reduces interaction with ZNF385A and causes loss of susceptibility to calpain.
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 [22,51,52]. 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 published [53][54][55] Interestingly, among these mutations, we found exclusive mutations on ASXL1 (K1368T) and SETBP1 (V231L) that were associated mainly with TP53 mutation [56]. Reported that ASXL mutations are frequently seen in MDS in association with SETBP1 mutations, inhibiting myeloid differentiation and inducing leukemic transformation [57]. 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 [58].
ASXL1 codes for Polycomb group protein ASXL1, a huge 165.432 kD and length of 1541 amino acid residues. Variant K1368T isn't yet reported. As there were no experimentally determined structures, AI-based predicted Alphafold structure was used. Residue of interest 1368 is three-dimensionally located on the flexible loop at periphery. 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). SETBP1 (SET Binding Protein 1) doesn't have any experimentally determined structures, hence, AI-based predicted Alphafold structure was used. It seems to have disordered regions. SNV V231L (corresponds to variant dbSNP:rs11082414) is benign as per ClinVar. It is a DNA-binding protein, functioning as an epigenetic hub that joins group of proteins that act together on histone methylation to make chromatin more accessible and regulate gene expression (Piazza et al., 2018). Not much is known about the overall function of the SETBP1 protein and the effect of SET binding.
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 gene [51,59,60].
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 study of Ahmad et al., 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 [61]. For ALL, only one case was selected for NGS analysis for a patient with a complex karyotype and TP53 deletion according to the FISH result, and no mutation was detected in TP53. Although in a 2014 study, Stengel et al. revealed that TP53 mutations were above average in ALL with complex karyotype, the patient did not show any mutation in TP53 [22].

Conclusions
Further examination and screening on a larger cohort is highly recommended to confirm our 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. This 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 to support the recommendation of utilizing the activation of the wild-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. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.