https://orcid.org/0000-0002-7069-0022
ABSTRACT
Background
AML is a complex and heterogeneous disease. The KRAS gene is one of the important genes in the pathogenesis of acute myeloid leukemia (AML). Mutant RAS can promote oncogenesis via different mechanisms including oncogenic transcription, cell cycle progression, cellular survival, growth, metabolism, and cell migration. Therefore, it is important to identify the genomic landscape of AML. The aim of the study is to identify KRAS variants in AML and their association with clinic pathological criteria and possible effects on prognosis using NGS.
Method
Hotspot mutations in the KRAS gene were studied using Ion S5 next-generation sequencing system. Bone marrow samples of newly diagnosed AML patients were collected to identify hotspot mutations in the KRAS gene. DNA amplicons were subjected to sequencing and were analyzed using ion torrent software. Patients were classified according to the FAB classification system. Patients are also classified according to the cytogenetic groups and the ELN risk stratification system.
Results
KRAS mutations were detected in exon 2, 3, whereas no mutations in KRAS exon 1. Interestingly, Novel mutations were detected in KRAS in AML Egyptian patients. Also, there was no statistically significant association of RAS mutations with different clinical and prognostic parameters. However, KRAS mutant patients tended to have increased PB WBC counts, percentage of PB, and bone marrow blasts.
Conclusion
NGS is considered a useful tool to identify KRAS variants that could be useful for risk stratification and tailored therapy in AML patients
1. Introduction
AML is a dynamic and complex disease characterized by multiple somatically acquired driver mutations, coexisting competitive clones, and disease evolution over time [Citation1]. Many of these mutations have prognostic value and are considered targets for tailored therapies.
Coexisting mutations may alter the prognostic information derived from a particular mutation, indicating that a complete understanding of the AML genomic landscape will be required to provide optimal individualized prognostication [Citation2].
Also, in relapsed AML, identifying the patient’s mutational AML profile is important to evaluate whether patients are eligible for additional investigations or alternative treatment options [Citation2].
There has been a great understanding of the mutational landscape of AML, which is mainly based on advances in sequencing techniques [Citation3,Citation4]. DNA sequencing technology has identified recurring AML gene mutations that contribute to the pathogenesis of AML, which may be missed by conventional cytogenetic analysis [Citation2]. Moreover, it was found that about 50% of AML patients have normal karyotyping [Citation5]. For these patients, NGS can be used to detect either recurrent or rare mutations, with most of those patients harboring more than one mutation even within defined AML entities [Citation6].
NRAS, HRAS, and KRAS genes encode four RAS proteins [Citation7]. The most common genetic abnormalities of the RAS family in AML are mutations in the NRAS gene [Citation8], followed by KRAS mutations, while HRAS mutations are uncommon [Citation9]. AML patients with mutant RAS genes benefit more from cytarabine-containing chemotherapy regimens than AML patients with wild-type RAS [Citation10,Citation11].
From this point of view, the current study was conducted to study the clinical effects of targeted gene sequencing for the hotspot mutations in the KRAS gene using NGS as well as its relations with other clinico-pathological criteria.
2. Methods
2.1. Samples
The study was conducted on bone marrow samples obtained from twenty-four denovo AML patients before the initiation of therapy between December 2021 and August 2022 after approval of the Ethics Committee. AML Patients aged <18 years, patients with Acute promyelocytic leukemia, secondary AML, and patients with other coexisting hematological malignancies were excluded from the study.
All AML patients at the time of diagnosis were subjected to comprehensive lab investigations including complete blood count (CBC), BM aspiration with morphological examination, flow cytometric immunophenotyping and cytogenetic analysis using the initial BM samples. The diagnosis of AML was based on the French-American-British classification (FAB) classification system. The characteristics of AML patients are summarized in (,). AML patients received chemotherapy and were followed up to categorize them into those who achieved complete remission (which is characterized by having less than 5% of blast cells in the BM, ≥1 × 109/L neutrophils, and ≥100 × 109/L platelets in the PB) and those who didn’t achieve complete remission. AML patients were also followed up to know who developed relapse and they were also categorized into those who survived or died.
Table 1. Clinical and molecular baseline characteristics of the patients (n = 24).
DNA was extracted from BM samples that were used for initial diagnosis followed by library preparation and NGS for identification of the hotspot mutations in the KRAS gene.
DNA extraction: Following the manufacturer’s instructions, DNA was extracted from BM samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Germany). The concentration of DNA was measured using Qubit™ 1X dsDNA HS (High Sensitivity) Assay with Qubit™ 4 Fluorometer (ThermoFisher Scientific, USA) according to the manufacturer’s protocol.
Library preparation: Using the Ion AmpliSeqTM Library Kit Plus (TermoFisher Scientific, USA) according to the manufacturer’s instructions, DNA libraries were constructed from 10 ng genomic DNA per sample. The amplicon adaptors were ligated and purified using the Ion XpressTM Barcode Adapters Kit (Thermo Fisher Scientific, USA) and the Agencourt TMAM PureTM XP Reagent (Beckman Coulter, USA) respectively. Barcodes were used to guarantee that every single sample had a unique ID.
The final amplicon libraries were quantified using the Ion Library TaqMan™ Quantification kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and were equalized to 100 pM.
2.2. Ion torrent sequencing
Using the Ion One TouchTM 2 System (Thermo Fisher Scientific, USA), enriched, template-positive Ion SphereTM Particles (ISPs) were initially prepared using the Ion 520TM and Ion 530TM Kit-OT2 (Thermo Fisher Scientific, USA) according to the manufacturer’s procedure. Next, the enriched, template-positive ISPs were loaded onto the Ion 520TM Chip and sequenced using the Ion S5TM next-generation sequencing technology (ThermoFisher Scientific, USA)
2.3. Data analysis and bioinformatics
Torrent Suite™ Software (ThermoFisher Scientific, USA) was used to plan and monitor sequencing runs. Ion Reporter™ Software (ThermoFisher Scientific, USA) was used for the annotation of single-nucleotide, insertions, deletions, and splice site alterations. Sequencing runs were planned and monitored using Torrent Suite™ Software (ThermoFisher Scientific, USA). Ion Reporter™ Software (ThermoFisher Scientific, USA) was utilized to annotate single-nucleotide variations, insertions, deletions, and splice site mutations. KRAS variants were with a mean depth coverage of 1787.9 ± 413.1. Allelic frequency ranged from 6.24% to 100% ().
Table 2. Analysis of KRAS gene variants.
2.4. Statistical analysis of the data
IBM SPSS software package version 20.0 (IBM Corp, Armonk, NY) was used for analysis. To confirm the normality of the distribution of Quantitative data. Chi-square analysis is used to compare groups when analyzing categorical variables. For chi-square, Fisher’s Exact or Monte Carlo adjustment is used if more than 20% of the cells have an expected count of less than 5. Student t-test to compare two groups for quantitative variables with normal distribution. To compare the two groups, the Mann–Whitney test for quantitative variables with abnormal distribution. The significance of the results was judged significant at the 5% level.[Citation12]
3. Results
In our study, KRAS was recurrently mutated in AML patients. A total of 69 genetic variants were found in the KRAS gene. According to the type of variants, SNVs were the most common type of variant encountered in most cases (98.6%) followed by Indels (1.4%) ().
Regarding the site of mutation, 28 variants (40.6%) were detected in exonic sites and 41 variants (59.4%) were detected in non-coding regions: intronic, UTR, and splice sites.
Of the exonic variants, the most common type of mutations were the Synonymous variants (34.8%) followed by missense variants (5.8%) ().
Regarding clinical significance using the Clinvar database, benign variants were the most commonly detected variants (76.8%) followed by variants of not reported clinical significance (17.4%) and then pathogenic variants (5.8%) ().
KRAS gene mutations were most commonly observed in exon 2, 3, while no mutation was detected in exon 1. All Kras pathogenic mutations were missense point mutations affecting Kras codons 12, 13, and 61; G12 mutations predominated over Q61 mutations (G12: 12.5%; Q61: 4.2%) ().
Table 3. KRAS variants
The analysis of KRAS pathogenic variants in patients revealed the presence of pathogenic missense variant c.35 G>A KRAS, located in exon 2 (G12D) in two AML patients. A missense pathogenic variant c.34 G>T located in exon 2 (G12C) was detected. Also, one patient had a pathogenic missense variant c.182A>T in exon 3 (Q61L). A Missense benign mutation c.483 G > A in exon 5 with amino acid replacement (p. Arg161=) was also detected in 20 AML patients (83.3) ().
When studying the relationship between KRAS gene mutations and different clinical criteria and prognosis, we exclude intronic and synonymous variants. We found that patients with KRAS pathogenic mutations tended to have higher WBC count, higher initial PB count, higher initial BM blast count and lower hemoglobin level, and platelet counts than patients with KRAS benign mutations. However, these relations were not statistically significant. KRAS-mutated patients were most commonly associated with AML with monocytic lineage (50% M4 and 25% M5b. However, this association didn’t reach the clinically significant level ().
Table 4a. Comparison between Non mutated with Mutated cases according to different parameters for KRAS (n = 24).
No significant difference in mutant frequency was found between cytogenetic risk groups. Three patients who had pathogenic missense KRAS mutations were cytogenetically normal. Interestingly, one patient had 2 KRAS pathogenic missense point mutations of both codons G12 and Q61. That patient was AML M4eo with an inv (16) (p13.1q22) ().
Table 4b. Comparison between Non mutated with Mutated cases according to different parameters for KRAS (n = 24).
4. Discussion
AML is a clinically and genetically diverse and dynamic disease [Citation13]. The importance of the identification of recurring genetic mutations in AML extends beyond the choice of therapy. Evaluation of these mutations can be done in AML patients and used as a standardized method for minimal residual disease (MRD) monitoring [Citation2].
KRAS gene is considered an important oncogene to be implicated in human cancer [Citation14]. Particular tumor types are frequently associated with specific mutant Ras isoforms, with N-Ras mutations being more common in leukemias. However, the association between the type of tumor and the mutant Ras gene is not entirely specific [Citation15]. This signal specificity is explained by differences in the posttranslational modifications of RAS proteins, making them have distinct trafficking routes and hence localize to certain microdomains of the plasma membrane. Consequently, they are capable of generating specific signal outputs [Citation16]. Considering KRAS in our study, all Kras pathogenic mutations were missense point mutations affecting Kras codons 12, 13, and 61 with G12 mutations prevailing over Q61 mutations. Consistent with our findings, Emanuele et al., found that KRAS missense mutations were detected in codons 12 and 13, while no mutation was found in codon 61.[Citation17]
When mutant RAS are locked in a GTP-bound state, they can bind to and activate a variety of downstream effector proteins. This can lead to a variety of cellular consequences, such as cell proliferation, growth, survival, migration, and neoplastic transformation [Citation18]. However, the variation in the Ras dosage (the percentage of a Ras population that is GTP-bound), the stability of the active state, and the cellular and tissue context that contributes to the genetic and epigenetic environment in which Ras genes operate may be responsible for the heterogeneity in the proliferative capacity of the mutant RAS [Citation19].
The KRAS gene has a potential impact on cellular physiology. Therefore, AML patients with KRAS mutations may have severe disease patterns [Citation8]. This may be related to advanced age, higher WBCs, and raised platelet counts in KRAS-mutated patients [Citation20]. Similarly, this can explain our finding that patients with KRAS pathogenic mutations tended to have higher WBC count, higher initial PB count, higher initial BM blast count and lower hemoglobin level, and platelet counts than patients with KRAS benign mutations.
In our study, KRAS-mutated patients were most commonly associated with AML with monocytic lineage (M4 and M5). However, this association didn’t reach the clinically significant level. While Barletta et al. [Citation21] found that there were no differences in the FAB distribution between kras-positive and kras-negative AML patients.
Regarding the cytogenetic risk groups, there is no significant difference between KRAS mutation and cytogenetic risk groups. Interestingly, we had one patient who had 2 different kras pathogenic missense point mutations of both codons G12 and Q61. That patient was FAB M4eo with an inv (16) (p13.1q22). Barletta et al. [Citation21] showed an overrepresentation of KRAS mutations in patients with inv (16) [Citation21]. As our sample size is small with only one patient having inv (16), further study on this presentation would require a larger sample size.
Regarding the ELN risk groups, KRAS mutated cases were associated with the intermediate risk group. Although these associations were not statistically significant. Most of KRAS-mutated patients didn’t achieve complete remission.
Unfortunately, the percentage of patients with non-complete remission in the non mutated group was more than the percentage of those who achieved complete remission. This can be explained by the higher percentage of refractory AML cases (70.8%) than those who achieved complete remission (20.8%). There was no association between KRAS mutations and the risk of relapse. There were more KRAS mutated cases of those who died were more than who survived. The limitations we faced in studying the relationship between gene mutations and survival were the small-sized sample and the higher number of dead cases due to different causes such as sepsis, thrombosis, or disease processes.
In conclusion, AML is a heterogeneous disease with many gene mutations of known or still not reported clinical significance. We detected intronic and exonic variants in the AML Egyptian patients of not previously reported clinical significance either by the Clinvar or Cosmic databases. In the current study, one novel exonic mutation c. *5598T>C was detected in exon 6 of the KRAS gene in 6 AML patients with a depth of coverage >1500. This finding showed that missense point mutations in codons other than the common codons may also contribute to the pathophysiology of the disease. In addition, novel intronic variants of not reported clinical significance were detected in the KRAS gene: c. *5-139A>G c.-11-59A>G and c.-11C>T.
A larger sample size from various locations is needed in extended research to better understand the relationship between these newly discovered mutations and prognosis. These changes may have specific clinical importance and hence tailored therapy.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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