Prognostic Factors, FLT-3 Mutations, and Treatment Outcomes with Pediatric-Inspired Protocols in Adolescent and Young Adults and Adult Patients with Acute Lymphoblastic Leukemia

Abstract

Introduction

The treatment protocols of adolescent and young adult (AYA) patients with acute lymphoblastic leukemia (ALL) have evolved, with the advent of pediatric-based regimens, measurable residual disease monitoring, and mutation analysis. Among the latter, previous reports have identified FLT-3 mutations in up to 5% of pediatric patients; however, the full clinical significance of these mutations in the non-pediatric population is still uncertain.

Methods

Our cohort includes AYA patients with ALL treated with the NY-II and BFM protocols at different time periods, allowing analysis of prognostic factors and survival outcomes. Additionally, we analyzed DNA samples for FLT-3 mutations, focusing on the potential prognostic implications and treatment responses within our cohort.

Results

No significant differences were found in overall survival or progression-free survival between the two treatment protocols. However, a higher rate of hematopoietic stem-cell transplantation was noted in the NY-II patients. Older age and high WBC count at presentation were identified as adverse prognostic factors using multivariate analysis. FLT-3 mutations were identified in 4 patients (5%) of the cohort, with only 1 patient having FLT-3 internal tandem duplication mutation and 3 patients having FLT-3-tyrosine kinase domain mutations.

Conclusions

The low rate and variability of FLT-3 mutations in an Israeli cohort precludes broad conclusions regarding their prognostic significance. In our cohort, age and WBC count but not treatment protocol or FLT-3 mutations influenced survival.

Introduction

Acute lymphoblastic leukemia (ALL) is an aggressive neoplasm encompassing both B-cell and T-cell forms (B-ALL and T-ALL, respectively). The treatment of ALL is evolving with several major leaps that significantly improved the outcome in the non-pediatric population. Among these, the advent of aggressive pediatric-inspired treatment protocols in the adolescent and young adult (AYA) population, better risk stratification, based on identified prognostic factors, understanding of the molecular and cytogenetic landscape, and the use of measurable residual disease (MRD) monitoring to guide treatment, have all improved outcomes [1–3].

In the current study, we retrospectively analyzed our cohort of AYA and adult ALL patients treated over a decade allowing us to review some of the abovementioned factors. Two pediatric-inspired protocols were utilized during the study period. The NY-II (Memorial Sloan-Kettering New York-II) protocol was historically used primarily until 2014 and the Berlin-Frankfurt-Münster (BFM) protocol used more frequently in subsequent years.

The BFM and NY-II protocols both utilize long-term, multiphase, chemotherapy regimens with intensive induction phases followed by consolidation, reinduction, and maintenance phases, employing a similar array of chemotherapeutic agents, with slight differences in dosages and specific drugs. Induction phases include high-dose chemotherapy combination of vincristine, prednisone, and anthracycline. Once remission is achieved, reinduction and consolidation phases are followed involving cyclophosphamide, high-dose methotrexate, cytarabine, and L-asparaginase. Patients who are not designated for an allogenic bone marrow (BM) transplant proceed to a long-term maintenance phase with oral chemotherapy with drugs like methotrexate, 6-mercaptopurine, and vincristine. The main advantage of the BFM protocol is its implementation on minimal residual disease (MRD) monitoring at defined time points to adjust treatment intensity according to patient risk categories – a strategy not prioritized in the earlier NY-II protocol.

Overall, the BFM protocol is more widely adopted today due to its systematic use of MRD monitoring and risk-adapted therapy, which aims to improve outcomes while managing toxicity effectively. Full versions of protocols can be found in previous publications [4–7]. The comparison of the two protocols provides insights on the use of MRD monitoring and the significance of identified prognostic factors.

In addition, we analyzed all the patients for FMS-like tyrosine kinase 3 (FLT-3) gene mutations. FLT-3 is a class III receptor tyrosine kinase predominantly expressed in hematopoietic stem cells and progenitor cells, playing a critical role in the development of myeloid and lymphoid lineages [8, 9]. Activation of FLT-3 by its ligand promotes cell survival, proliferation, and differentiation via key signaling pathways such as PI3K, RAS, and STAT5 [10]. Mutations in FLT-3, including internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, are found in approximately 30% of acute myeloid leukemia (AML) cases. These mutations, particularly FLT-3-ITD, are known to cause constitutive activation of the kinase, leading to adverse clinical outcomes and a poorer prognosis [11]. Analysis for FLT-3 mutations is routinely performed in all newly diagnosed and relapsed cases of AML, influencing clinical decision-making and allowing the addition of FLT-3 inhibitors [12]. Midostaurin, a first-generation FLT-3 inhibitor, and gilteritinib, a more specific second-generation inhibitor, have demonstrated substantial benefits in improving survival rates among patients with FLT-3-mutated AML compared to conventional therapies [13–15].

Several previous studies in pediatric ALL have reported a rate of approximately 5% of FLT-3 mutations [16–18]. A recent report included 342 pediatric patients with ALL and observed FLT-3-ITD mutations in 4% and point mutations in up to 3%, with no clear correlation with age, initial WBC counts, MRD, CNS involvement, or relapse [19]. Armstrong et al. [20] have reported 71 pediatric patients with ALL with a high rate of activating FLT-3 mutations in hyperdiploid cases, with no identified FLT-3-ITD mutations. Reports in adult ALL patients are limited. A cohort that included both pediatric and adult patients found FLT-3 mutations in 3% of the pediatric population and no identifiable mutations in the adult cohort [21]. Recent studies indicate that high FLT-3 expression may be a negative prognostic factor in ALL [22]. Additionally, FLT-3 inhibitors were shown to induce cytotoxicity in ALL cells in vitro [23]. Zhao et al. [8] recently compared FLT-3 mutations in pediatric de novo ALL and AML patients. They concluded that in AML, FLT-3 mutations correlate with poorer prognosis that was not shown in FLT-3-mutated ALL patients. Given the latter findings and relatively the paucity of data regarding FLT-3 mutations in the non-pediatric population, we aim to gain further insight on their prevalence and clinical significance, focusing on AYA age-group.

Materials and Methods

Study Design

This was a retrospective study including data collected from patient files and using DNA-archived samples. All work was performed at the hematology department laboratories under the authorization of the local Hadassah internal review board (HMO0700-21). The retrospective cohort included patients above the age of 15 years, diagnosed with ALL, and treated at Hadassah-Hebrew University Medical Center between 2004 and 2022. The AYA group is defined as ages between 15 and 39 years [24]. Exclusion criteria were inconclusive diagnosis of ALL, mixed phenotype leukemia, and lack of DNA sample for analysis. DNA samples of all eligible patients were analyzed for FLT-3 mutations. Both BM and/or peripheral blood (PB) samples were used for leukemia-derived DNA extraction.

Patients’ Characteristics

Medical records of patients with available and sufficient samples for analysis were reviewed, regardless of their FLT-3 mutation status, to gain further insights regarding prognostic factors, comparison of the outcomes of the pediatric-based NYII and BFM regimens and to provide a control FLT-3 negative group for matched analysis. Data collected included initial demographic data (age at diagnosis, ethnicity, gender, performance status), disease characteristics (laboratory results such as white blood cell count, extramedullary (EM) involvement (including spleen, thymus, and lymph nodes), central nervous involvement, cytogenetic abnormalities, and concurrent molecular data. Therapeutic details include chemotherapy protocol and response to treatment: complete remission, progression-free survival (PFS), and overall survival (OS).

Patient Samples

DNA is routinely extracted from BM and PB samples of all patients and kept at −20°C in the hematology laboratory for molecular diagnosis. DNA aliquots were assessed for quality and quantity, allowing further analysis for FLT-3 mutation.

Detection of FLT-3 Mutations

Total WBCs were extracted from BM and PB samples: red blood cells were lysed with red blood cell lysis buffer. Cell pellet was subjected for DNA extraction with Maxwell^® RSC Blood DNA Kit using Maxwell^® RSC Instrument.

Detection of FLT-3-ITD Mutation

DNA fragment encompassing exons 11–12 of FLT-3 gene was amplified by polymerase chain reaction (PCR) with GoTaq Green Master Mix (Promega) and primers as previously described [25]. Forward primer was labeled by FAM fluorescent at 5′-position that allowed visualization and analysis of PCR products by GeneScan Fragment Analysis. Briefly, 10 ng of genomic DNA was amplified by 30 cycles of denaturation at 95° for 1 min, annealing at 60° for 30 s, and extension of 72° for 30 s after initial denaturation of 95° for 2 min. 2 µL of PCR products were run at ABI310 fragment analyzer (Applied Biosystems). The amplicons with a size greater than 328bp (presenting wild type allele) were interpreted as positive for ITD mutation. Positive, negative, and non-template controls were included in each set of PCR reactions.

Detection of Activation Mutations in TKD of FLT-3 Protein

DNA samples were screened for point mutations within the activation loop of TKD (FLT-3/TKD mutations). Mutations within codons 835 and 836 in exon 17 of FLT-3 gene were detected by allele-specific PCR real time. Allele-specific PCR real time was performed with StepOnePlus ABI instrument (Applied Biosystems). Reverse allele-specific primers were adapted from Scholl et al. [26] Common forward primer and probe were designed and used in all reactions. A common reverse primer was designed for amplification of wild-type allele and control for DNA quality and quantity. Each mutated and wild-type allele was tested in separate PCR runs. Positive, negative, and non-template controls were included in each set of PCR reactions. A threshold of 0.05% was set for all reactions. Primer sequences are provided in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000543861).

Statistical Analysis

Clinical data and results were tested for the association between two categorical variables by the chi-square test or Fisher’s exact test. The comparison of a quantitative variable between two independent groups was performed by using the two-sample t test. The comparison of quantitative variables between two independent groups was carried out by applying either the two-sample t test or the Mann-Whitney (M-W) nonparametric test. Nonparametric tests were used for variables, which were not normally distributed. The effect of categorical variables on OS as well as PFS was assessed by using the Kaplan-Meier survival analysis with the log-rank test for the comparison of survival curves. The effect of quantitative variables on OS as well as PFS was assessed by using the Cox regression model. This model was also applied as the multivariate model for survival. Propensity-score matching was used for creating pairs of patients treated by different protocols. Patients were matched according to demographic, laboratory, and clinical variables. The McNemar test was used for comparing categorical outcomes between the paired patients. All tests applied were two tailed, and a p value of 0.05 or less was considered statistically significant.

Results

Patient’s Characteristics

The cohort included 74 patients from Israel, of which 58 were male (78.4%). The median age was 27 years (range: 16–76 years). Out of 74 patients, 18 (24%) were of African descent and 56 (76%) were of European descent. Forty patients had B-cell ALL (54.1%) and 34 T-cell ALL (45.9%). Patients’ baseline characteristics are summarized in Table 1. The median white blood cell (WBC) count at diagnosis was 12.4 × 10⁹/L (1.3–400 × 10⁹/L). Four patients had t(9;22), i.e., Philadelphia chromosome (PH+)-positive ALL (5.5%). Thirty-five patients (47%) had EM involvement at diagnosis, while only 4 (5.4%) had central nervous system (CNS) involvement.

Table 1.

Patient baseline characteristics

Characteristics	Values
Age	27 (16–76)
Gender
Male	58
Female	16
Ethnicity
African descent	18
European descent	56
WBC, 103/µL	12.4 (1.3–400)
WHO (subtypes)
B-ALL	40 (54)
T-ALL	34 (45.9)
PH+	4 (5.4)
Cytogenetic abnormalities	(31/74)
Standard risk	23
High risk	8
EM lesions	35 (47.2)
CNS involvement	4 (5.4)
Induction outcome (out of 72)
CR	63 (85%)
Partial remission	5 (6.7%)
Refractory	6 (8%)
Relapse	16 (21.6%)
Early relapse <1 yr from induction	7 (9.5%)
Late relapse >1 yr from induction	9 (12.2%)
HSCT	28 (39.4)
Years of follow-up (median)	3.02 (0.04–17.79)

WBC, white blood cell; ALL, acute lymphoblastic leukemia; PH, Philadelphia chromosome; CNS, central nervous system; CR, complete remission; HSCT, hematopoietic stem-cell transplantation.

Metaphase cytogenetic analysis was available for 31 patients (41.8%). Twenty three of 74 (31%) were considered to have cytogenetically intermediate risk features, whereas 8 of 74 (10.8%) were considered as high risk, according to the ELN classification. Forty-four patients (59.5%) were treated according to the BFM protocol [4], 25 (33.8%) were treated with the NY-II protocol [5], and 5 (6.8%) with other protocols. Twenty-eight patients (37.8%) underwent allogeneic hematopoietic stem-cell transplantation (HSCT), of which 11 in first complete remission (CR1), 14 in second CR (CR2), and 2 with active disease. For 1 patient, there were missing data as to the stage at which transplantation was performed. Of the patients that proceeded to transplant in CR1, molecular MRD testing of the IgH/TCR rearrangement was available for 6 patients, only one of whom was MRD positive at the time of transplant. Sixty-seven (90.5%) of the 74 patients achieved CR after induction therapy; 6 (8%) patients had a refractory disease on day 78 of induction. Early mortality rates during induction were low (2.7%).

Analysis of Additional Prognostic Factors

Additional prognostic factors that may affect clinical outcomes and prognosis in ALL were analyzed. In a univariate analysis, the statistically significant predictive variables for OS were older age (p = 0.05), CNS involvement (p = 0.00002 (Table 2; Fig. 1), and a higher white blood cell count at diagnosis (WBC >12,400 × 10⁹/L, p = 0.03). Age and WBC remained significant by the multivariate logistic regression analysis (Table 2). The same factors were analyzed for PFS. Of note, only a high WBC was found to be significant for PFS (p = 0.019, Table 3).

Table 2.

Prognostic predictive factors for OS

Variable	Median	HR (95% CI)	Univariate analysis (p value)	Multivariate analysis (p value)
WBC count (×109/L)	12.4	1.005 (1–1.01)	0.037	0.088
Age (years)	27	1.029 (1–1.05)	0.051	0.025
Gender (men:female)		1.47 (1.23–2.06)	0.645
CNS involvement		1.161 (0–2.556)	0.00002
EM involvement		1.42 (1.41–1.44)	0.873
B-cell ALL versus T-cell ALL		1.46 (1.46–1.46)	0.739
BFM versus NY-II protocols		0.822 (0.73–0.98)	0.140

WBC, white blood cell; ALL, acute lymphoblastic leukemia; CNS, central nervous system; EM, extramedullary.

Fig. 1.

OS by CNS involvement.

Table 3.

Prognostic predictive factors for DFS

Variable	HR (95% CI)	Univariate analysis (p value)
WBC count (×109/L)	1.008 (1.001–1.016)	0.019
Age	1.018 (0.98–1.05)	0.352
Gender (men:female)	1.23 (1.03–1.73)	0.433
EM involvement	1.15 (1.09–1.19)	0.748
B-cell ALL versus T-cell ALL	1.4 (1.35–1.46)	0.265
BFM versus NY-II protocols	0.89 (0.82–1.03)	0.465

WBC, white blood cell; ALL, acute lymphoblastic leukemia; EM, extramedullary.

FLT-3 ITD Mutational Status of 74 Patients at Diagnosis

We identified only 1 patient with FLT-3-ITD mutation (1.3%) who had B-cell ALL. Cytogenetic data were not available; his WBC at diagnosis was 13.3 × 10⁹/L, with no evidence of EM or CNS involvement. He was treated with the NY-II protocol and achieved CR. The patient did not proceed to an allogenic BM transplant and is currently in CR with a follow-up of 9 years to date.

We identified 3 patients with FLT-3 TKD mutations. No common characteristics were found. Among the three, two were females and one male. The average age at diagnosis was 48 years (26–65 years) with 2 of the patients diagnosed with B-ALL and one with T-ALL. Two of the 3 patients had leukocytosis at diagnosis (31 × 10⁹/L and 26 × 10⁹/L, respectively). Two of the three had EM involvement (66%), compared to 46% in the FLT-3 wt group, and no CNS involvement was demonstrated in any of the patients. Two patients were treated with BFM protocol; one of those was refractory. This patient received three additional lines of treatment subsequent to BFM, with no sustainable response and succumbed to his disease. The second patient achieved a CR with a negative IgH MRD but succumbed to sepsis. The third patient was treated with NY-II protocol. He achieved a CR, following relapse 4 years later, underwent an allogenic BM transplantation in CR but died from infectious complications of the procedure. Patients’ data are summarized in online supplementary Table S2.

Comparison of BFM and NY-II Protocols

Due to the long period of this retrospective study, the treatment regimens varied during the study-cohort years. In the early period, up to 2014 (2004–2014), the NY-II protocol was mainly used and the BFM protocol in the subsequent period. The switch from NY-II to BFM in 2014 occurred as a part of national effort for standardization. A comparison of patients’ characteristics of the two different protocols study groups is summarized in Table 4. The median age was 24 years in the NY-II cohort as compared with 32 years in the BFM cohort (p = 0.008). Of note, most of the patients (85%) who received the BFM or NYII were below the age of 39 years and are best regarded as AYA. PFS and OS did not differ between the two regimens (Fig. 2a, b). We further performed a propensity score to compare between the patients treated with the two protocols, yet no identifiable differences in OS or PFS were noted either (online suppl. Tables S3–4).

Table 4.

Comparison between BFM and NY-II protocols

Variable	BFM (n = 44)	NY-II (n = 25)	p value
WBC count, × 109/L	13.2	12.7	0.8
Age, years	24 (16–42)	32 (19–54)	0.008
Male	36 (81.8%)	19 (76%)	0.564
Female	8 (18.2%)	6 (24%)
Cytogenetics
High risk	15 (68.2%)	8 (88.9%)	0.379
Standard risk	7 (31.8%)	1 (11.1%)
CNS involvement	2 (4.5%)	2 (8%)	0.617
EM involvement	21 (47.7%)	11 (44%)	0.765
B-cell ALL	23 (52.3%)	14 (56%)	0.765
T-cell ALL	21 (47.7%)	11 (44%)
HSCT – CR 1	5 (11.3%)	5 (20%)	0.003
While in CR 2	10 (22.7%)	4 (16%)
With active disease/MRD	1 (2%)	1 (4%)

WBC, white blood cell; ALL, acute lymphoblastic leukemia; CNS, central nervous system; EM, extramedullary; MRD, minimal residual disease.

Fig. 2.

Comparison of BFM and NY-II regarding OS (a) and relapse-free survival (b).

A significantly higher number of patients underwent HSCT in the NY-II group as compared with the BFM group (10 of 25 patients [44%] vs. 16 of 44 patients [36%], respectively, p = 0.003). In the NY-II group, 5 patients (20%) were in CR1, 4 patients (16%) were in CR2, one had a primary refractory disease. In the BFM group, 5 patients (11%) were in CR1, 10 patients (22%) were in CR2, and one had a primary refractory disease.

Identification of Factors Associated with Early Relapse

Early relapse (within the first year of induction) was reported in 7 of 74 (9.4%) patients, whereas nine of 74 (12.1%) experienced a late relapse. In order to identify possible predictors for early relapse, we compared baseline and post-response characteristics of the two groups (Table 5). A trend for higher initial WBC in the early versus late relapse groups was noted (median 83 × 10⁹/L vs. 8 × 10⁹/L respectively, p = 0.08) and older age (median 35 years vs. 20 years, respectively, p = 0.08). In addition, T-ALL patients tended to have a higher early/late relapse ratio in comparison with B-ALL cell (p = 0.06). Overall, there was no statistically significant difference in OS between patients exhibiting early and late relapse (Fig. 3).

Table 5.

Comparison between early and late relapse

Variable	Early relapse (n = 7)	Late relapse (n = 9)	p value
WBC count, × 109/L	83	8	0.081
Age, years	35	20	0.079
Male	4 (57.1%)	8 (88.9%)	0.262
Female	3 (42.9%)	1 (11.1%)
Cytogenetics
High risk	0	1 (11.1%)	1
Standard risk	2 (28.5%)	5 (55.5%)
CNS involvement	0	0
EM involvement	4 (44.4%)	3 (33.3%)	0.615
B-cell ALL	1 (14.3%)	6 (66.7%)	0.06
T-cell ALL	6 (85.7%)	3 (33.3%)
HSCT	5 (71%)	9 (100%)	0.37

WBC, white blood cell; ALL, acute lymphoblastic leukemia; CNS, central nervous system; EM, extramedullary; HSCT, hematopoietic stem-cell transplantation.

Fig. 3.

OS analysis in early relapse in comparison with late relapse.

Discussion

Treatment of adult patients with ALL is highly challenging. In recent years, novel agents have changed our treatment paradigm of B-ALL [1, 27–29]. Nonetheless, better prognostication and identification of novel targets for therapy is of great value. Herein, we present an analysis of a cohort of ALL patients treated at Hadassah Medical Center between the years 2014 and 2022, aimed to evaluate prognostic factors, comparison of the two pediatric-inspired protocols NYII and BFM, and the prevalence and significance of FLT-3 mutations.

The cohort included 74 patients and their baseline characteristics are in line with other major series of ALL patients analyzed to date [30, 31]. There was a significantly higher percentage of patients with T-ALL (46%) as compared to other reports [32] and a relatively low percentage of Ph+ ALL patients (13%).

Established poor prognostic factors include high white blood cell (WBC) counts at presentation and advanced age at diagnosis as well as CNS involvement, male versus women, race and ethnicity (black and Hispanic vs. Caucasian and Asian), T-ALL versus B-ALL, and the evolving field of cytogenetic and genomic features [2, 18, 22, 33–35]. Some of these parameters, at least in part, have been identified as surrogate markers for other abnormalities, particularly genetic alterations. For example, older populations have a higher prevalence of high-risk molecular abnormalities [36, 37].

Factors previously known to impact survival, such as older age, CNS involvement, and a high initial WBC count, were also identified in our cohort. However, only older age and a high initial WBC count retained statistical significance in a multivariate analysis for OS. Of note, our cohort had a relatively high rate of male patients, while no selection was employed. AML studies have shown gender-related differences in outcome and mutational landscape [32]. Data in ALL are limited [2] and further study in larger cohorts is required. In our cohort, gender did not impact OS.

We identified only 1 patient (1.3%) with FLT-3-ITD mutation and 3 patients with FLT-3 TKD mutations (4%) precluding the drawing of any conclusions regarding their prognostic significance. Our findings are in accordance with previous reports demonstrating a similar rate of FLT-3 ITD mutation of 0–1.21% and FLT-3 TKD mutation of 0–1.89% [16, 17]. Two of the 3 patients with FLT-3 TKD had EM disease, a higher percentage than in the FLT-3-negative cohort. While the small numbers do not allow a clear conclusion, such correlation was not reported before.

A comparison of the two protocols (NY-II and BFM) over the two time periods revealed some insight into the significance of MRD testing. We found that although the patients treated by these two protocols were comparable in their baseline characteristic, no difference in outcomes was found comparing both OS and PFS. However, patients treated by the NY-II protocol had a higher frequency of HSCT (p = 0.003). Most probably, this difference reflects a shift in the treatment paradigm, with strictly defined MRD-guided indication for HSCT in the BFM protocol, which allowed better identification of the high-risk patients in a need of HSCT. Indeed, a higher percentage of patients were referred to HSCT in CR1 in the NY-II group versus the BFM group (45% vs. 31%, respectively).

Finally, we compared the baseline characteristics of patients with early and late relapse. Older age and high WBC count tended to have early relapses, while no other predictors for early relapse were identified. Older age is associated with reduced use of intensive chemotherapy, which has expected toxicities, and a high WBC might reflect a more aggressive disease biology, both of which can contribute to an earlier relapse rate. This finding concurs with older series assessing for risk prognostications in ALL [2, 18, 22].

Our study has several limitations. First, as this is a single-center cohort it is limited in cohort size and geographic region, especially in view of the low rate of FLT-3 mutations in ALL. Second, our mutation detection method might miss less common FLT-3 mutation involving the juxtamembrane region [16].

We report a low prevalence of FLT-3 mutations in an Israeli cohort of AYA and adult patients with ALL, precluding broad conclusions. While low in frequency, detection of FLT-3 mutations might still allow consideration of the incorporation of FLT-3 inhibitors and thus may become of clinical value. Further study in such a cohort of relapsed and refractory patients is warranted.

Statement of Ethics

This study protocol was reviewed and approved by the Hadassah Medical Center internal review board (approval No. HMO0700-21). Given the retrospective nature of the study and as all tests were done as part of routine testing in leukemia, no informed consent was required by the committee.

Conflict of Interest Statement

There is no conflict of interest to declare.

Funding Sources

There are no funding sources for this study.

Author Contributions

Conceptualization: B.N. and A.G.; data curation: U.O., N.G.E.-Z., S.A., and V.V.; writing – original draft preparation: N.E.-Z.G., A.H., and B.N.; and writing – review and editing: B.N. and M.E.G. All authors have read and agreed to the published version of the manuscript.

Funding Statement

There are no funding sources for this study.

Data Availability Statement

All the data that support the findings of this study are in the article. Further inquiries can be directed to the corresponding author.