Prognostic Significance of Caspase 8 Associated Protein 2 (CASP8AP2) in Childhood B cell Acute Lymphoblastic Leukemia

Prognostic Significance of Caspase 8 Associated Protein 2 (CASP8AP2) in Childhood B cell Acute Lymphoblastic Leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prognostic Significance of Caspase 8 Associated Protein 2 (CASP8AP2) in Childhood B cell Acute Lymphoblastic Leukemia Omar Arafah, Marihame Ashraf, Ahmed Mustafa Abd Elsalam, Sally Elfishawi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7478773/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Improved treatment of childhood acute lymphoblastic leukemia (ALL) depends on the identification of new molecular markers that can predict treatment response and clinical outcome. Examination of the expression patterns of a set of genes at the RNA level is one of these new modalities. The prognostic significance of caspase-8-associated protein 2 (CASP8AP2), an apoptosis-related gene, in pediatric ALL is controversial. Methods A prospective study of 70 newly diagnosed ALL patients who were treated in the National Cancer Institute (NCI), Cairo University during the period from 1st of October 2019 till the end of September 2023, to measure the CASP8AP2 expression level in bone marrow samples at the time of diagnosis and at the end of induction therapy using real-time quantitative PCR, and to assess its relation with different prevalent prognostic variables and disease outcome. All cases were followed up till end of December 2024. Results Higher initial CASP8AP2 gene expression was associated with hyperdiploid karyotyping (p = 0.009), molecularly favorable mutations (p = 0.002), early induction response (p = 0.025), and low-risk patients (p < 0.001) and had a significant impact on lowering events in the first 3 years of follow-up (p = 0.001). Meanwhile, examining post-induction gene levels failed to show similar results. Conclusions Higher initial CASP8AP2 gene expression was associated with favorable impact on event-free survival in pediatric ALL patients. Post-induction levels did not show similar correlation. Future larger studies are needed to confirm the favorable association and to search for other possibly related prognostic factors to further refine risk stratification. Childhood acute lymphoblastic leukemia CASP8AP2 gene Apoptosis Chemotherapeutic sensitivity Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common cancer in children, comprising around 25% of all childhood malignancies [ 1 ]. In the past two decades, the increased cure rates for pediatric ALL have been one of the major achievements in cancer therapy, with the current 5-year survival rate increasing to about 90% in children younger than 15 years, mainly in developed countries [ 2 ]. Great advances have been made in risk stratification, more precise response monitoring, treatment plan modification and prediction of prognosis in pediatric ALL [ 3 ]. Yet, there are certain groups of patients whose response to treatment and durable remissions are not explained by the routinely used criteria for risk assignment and response assessment tools. Therefore, new prognostic markers should be explored as relapses remain the main obstacle against curing childhood ALL [ 4 ]. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the mitochondrial death pathway (the intrinsic pathway) and the death receptor–mediated pathway (the extrinsic pathway) [ 5 ]. Defects in the apoptotic process disrupt the delicate balance between cell proliferation and cell death. Studies have shown that leukemic cells adopt these mechanisms to resist chemotherapeutic agents [ 6 ]. CASP8AP2, a gene found on chromosome 6q15–16.1, encodes the enzyme needed to help apoptosis in response to chemotherapy [ 7 , 8 ], as the CASP8AP2 protein interacts with caspase-8 death-effector domain and plays an essential regulatory role in Fas and tumor necrosis factor–mediated apoptosis [ 9 ]. CASP8AP2 has been the focus of interest in many research projects. Its prognostic significance in pediatric ALL had been extensively studied before, with inconsistent results [ 21 , 23 , 25 , 27 ]. Caspases are subdivided into three groups according to structure and function. Group I (inflammatory caspases-1, -4, -5, -11, and − 12), Group II (initiators caspases-2, -8, -9, and − 10), and Group III (caspases-3, -6, and − 7) [ 10 ] In current study, we aimed to assess the impact of the level of CASP8AP2 gene expression pre- and post-induction therapy on the outcome in pediatric ALL and to analyze its associations with early response to treatment and other prevalent prognostic factors. MATERIALS AND METHODS This prospective cohort study was conducted on 70 newly diagnosed ALL pediatric patients who were treated at the pediatric oncology department, National Cancer Institute (NCI), Cairo University – Egypt from 1st of October 2019 till the end of September 2023. Medical records were reviewed for epidemiological data (age groups, sex), date of presentation, clinical data, initial investigations. Bone marrow aspirates (BMA) were assessed for immunophenotyping by flow cytometry, ALL-associated fusion gene transcripts by fluorescence in situ hybridization (FISH), karyotyping (G-banding), along with cerebrospinal fluid (CSF) examination. Risk assignment was done according to St. Jude’s risk stratification [ 11 ], and treatment data were recorded (e.g: date and type of chemotherapy received). Data of relapse were also collected, including timing of relapse (early or late), type of relapse (hematological, central nervous system [CNS], or combined), salvage chemotherapy given (e.g: timing and number of cycles), and response to salvage therapy. Survival analysis data were collected (date of diagnosis, date of relapse, and date of death/last follow-up). Eligibility Criteria All included patients in the study aged between 1 and 18 years old, confirmed to have ALL by immunophenotyping, risk assigned and treated according to St. Jude total XV protocol [ 11 ], and had a sufficient bone marrow (BM) sample for total RNA/microRNA (miRNA) extraction as identified through the clinical pathology department data registry. Exclusion criteria: Patients who were included prospectively and died before the end of induction and patients without sufficient material for total RNA extraction at end of induction. MRD Assessment At the time of diagnosis, peripheral blood or bone marrow samples were taken; samples were examined the same day [ 12 ]. A wide range of monoclonal antibodies, including CD45, CD19, CD10, CD22, CD34, CD79A, CD1PE, CD7, CD2, CD4, CD8, CD16, CD56, CD99, TDT, CD33, CD13, CD11B, CD15, NG2, and CD117, were acquired from Beckman Coulter in Miami, USA, for the purpose of lineage assignment. The monoclonal antibodies used were from Dako (Cytomation, Denmark) and included MPO, CD3, Kappa, Lambda, CD5, cytoplasmic µ, CD20, and MHC class II. 50 µL of a sample with an adjusted cell count of approximately 1 × 10⁶ cells per tube was incubated with monoclonal antibodies according to the manufacturer’s instructions for 30 minutes at room temperature in the dark, after which the sample was lysed, and excess antibodies were removed with Phosphate Buffered Saline (PBS) [ 13 ]. In 500 mL of PBS, the cells were suspended. The analysis was carried out using a Navious cytometer. Firstly, the blast population was selected on the basis of forward scatter versus side scatter and then on the basis of CD45 versus side scatter. The cut-off value for surface monoclonal antibodies was 20%, while that for cytoplasmic antibodies was 10% [ 14 ]. Utilizing a Navious cytometer (Beckman Coulter, Miami, Florida), flow cytometry (FCM) was performed to evaluate MRD. Using INTA Prep permeabilization reagent from Beckman Coulter, intracellular staining was performed by fixing cells with reagent 1 (formaldehyde fixation reagent), washing, inducing permeability with reagent 2 (using saponin for permeability), and lysing any remaining erythrocytes. For the detection of MRD, the following monoclonal antibodies were used in four color combinations. BCP-ALL: TdT/CD10/CD19/CD45; CD10/CD20/CD19/CD45; CD34/CD38/CD19/CD45; CD34/CD22/CD19/CD45; CD19/CD34/CD45; CD10/CD20/CD22/CD45. Since this combination was one of the highest incidence marker expressions and helped distinguish hematogones from residual blasts in ALL instances after induction, it was identified at diagnosis and post-induction following morphological remission for MRD tracking. Cutoff values were deemed to be > 20% for surface monoclonal antibodies, whereas cytoplasmic values were ≥ 10%. The median number of events measured in the assay was 1,250,000 (range: 750,000–1,600,000). The detection limit (LOD) is 0.002% or one in every 50,000 cells. The lower limit of quantification (LLOQ) is 0.01% (1–10,000). Sequential gating was employed. The minimum target sensitivity for quantifying MRD was defined as the ability to detect 30 clustered MRD events in 3 × 10⁵ total cellular events (0.01%). The cutoff point for MRD1 (D15) was < 10⁻³ (0.1%), and for MRD at any time point, it was < 10⁻⁴ (0.01%) [ 15 ] at D42 (End of induction), week 17, week 48 and end of treatment in patients who ended chemotherapy. CASP8AP2 Gene Expression Analysis Nucleic Acid Extraction Under strict aseptic conditions, bone marrow samples drawn in two EDTA tubes containing 3 mL each were utilized to extract RNA. Extraction of total cellular RNA from blood was done using QIAamp RNA Blood Mini Kit for total RNA purification (Qiagen, Hilden, Germany). Concentration and purity of RNA was checked by measuring the absorbance at 260 nm (A260) using the Nanodrop spectrophotometer. Analysis of CASP8AP2 gene expression Following RNA extraction, reverse transcription was performed, and first-strand cDNA was prepared using 1 µg of total RNA using Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). Quantitative RT-PCR assays were performed using the SLAN®-48P Real-Time PCR System Hongshi system (Shanghai Hongshi Medical Technology Co., Ltd.) with Taqman Universal Mastermix (Thermo Fisher Scientific, USA) and inventoried Taqman assay (Applied Biosystems, Life Technologies) for CASP8AP2 mRNA (Hs01594281_m1) and ABL1 (Hs01104728_m1) as the housekeeping gene. A total of 10 pediatric control samples were used for gene expression analysis. These were gathered from normal pediatric bone marrow donor samples supplied as screening samples before donation. Data from the amplification plot was obtained and analyzed to determine the relative expression of CASP8AP2 gene among patients and control samples and then compare cycle threshold (CT) of the target genes to the CT of the housekeeping gene. To determine the relative expression, we used the comparative cycle threshold (2 − ΔΔCT) method where the data were represented as the fold change in gene expression compared to an endogenous reference gene. In our study, the endogenous reference gene was ABL1. ΔCT data (CT target gene – CT endogenous reference gene) was used [ 16 ]. Statistical Methods Cut-off for gene expression was estimated at the maximum sensitivity and specificity using the Youden’s Index of the Receiver Operating Characteristics (ROC) curve coordinates. The cut-off values for CASP8AP2 gene expression were used to designate groups at diagnosis, where groups below cutoff threshold were designated low expressors and groups showing gene expression greater or equal to the threshold, were designated high expressors. The association between high and low expression levels and different variables was tested using Pearson’s Chi-squared test and Fisher’s exact test. To compare CASP8AP2 expression levels between different variables, we employed the Wilcoxon and Kruskal–Wallis rank sum tests. For survival analysis, the Kaplan–Meier method was used and the survival experience compared using the log-rank test. Alpha was two-sided and set at 0.05. All analyses were conducted using R version 4.1.0. A p-value less than 0.05 indicated statistical significance. Overall survival (OS) was defined as the period between the date of diagnosis and the date of death from any cause or the date of the patient’s last follow-up. Event-free survival (EFS) was defined as the period between the date of diagnosis and the date of relapse or to date of last follow-up for patients without events. RESULTS Initial patients’ characteristics are summarized in Table 1 Table 1. Clinical characteristics and initial laboratory investigations of the whole cohort (N = 70) CASP8AP2 gene expression initially and post induction and its relations to different variables A ROC curve (receiver operating characteristic curve) was used to define levels of low and high expression of CASP8AP2 gene (Table 2) . Figure 1 Table 2. CASP8AP2 gene expression cut-off in the initial and post-induction BMA Patients with high initial gene expression levels were more likely to be associated with hyperdiploid karyotyping (p = 0.007), molecular favorable mutations (t(12;21) and negative mutations) (p = 0.038), good early (Day 15) and end-of-induction (Day 42) morphological and MRD response (p = 0.019 and p = 0.029, respectively), and low-risk stratification (p < 0.001). Meanwhile, the post-induction high gene expression levels failed to maintain the same statistically significant correlation with the same favorable variables (Table 3). Table 3. Relation of initial patients’ characteristics and treatment response to the initial and post-induction BMA CASP8AP2 gene expression level Moreover, we calculated the high and low CASP8AP2 gene expression levels within each variable including the above proven associated favorable ones. Significantly high pre-induction CASP8AP2 gene levels compared to lesser low levels were mostly encountered in patients with hyperdiploid karyotyping (p = 0.009), molecular favorable (p = 0.002), early induction response (p = 0.025), and low-risk patients (p < 0.001). This difference in expression levels within each variable did not persist in post-induction gene levels (Table 4) Table 4. Relation of different CASP8AP2 gene expression cut-off levels in the initial pre-induction and post-induction BMA to the different prognostic features Survival outcome compared to gene expression levels and cause specific mortalities: Median follow-up time was 1.5 years (95% CI 1.3 to 1.8). Survival outcome was re-analyzed after dividing patients by ROC curve into high and low gene expression levels. Within the first three years of follow-up, high gene expression levels in the pre-induction BMA had been associated with better survival in terms of fewer events (p-value <0.001) Figure 2. This contrasts with post induction BMA gene level which did not correlate with lesser events (p-value 0.49 ) Figure 3. Out of the 70 patients included in the study, there were 13 mortality cases (19% of the whole cohort). Five patients (39%) died in the first year, seven (54%) in the second year, and one (7%) in the third year after induction therapy, respectively. In 4 out of the 5 patients who died in the first year, deaths occurred because of septic complications during maintenance therapy and all of them exhibited a high initial gene expression level. Only one patient had encountered early isolated hematological relapse and died from septic complications following intensive salvage chemotherapy. This patient had a low initial gene expression level. Among the seven patients who died during the second year, two died from septic complications during maintenance therapy, and one of them had a low initial gene expression level. The remaining five patients experienced relapse: two died from septic complications after receiving intensive salvage chemotherapy, and three died from refractory disease (palliative cases). Of the five relapsed cases, four exhibited a low initial gene expression level. Only one patient died during the third year, following septic complications after intensive salvage chemotherapy for an isolated hematological relapse. This patient exhibited a low initial gene expression level. To nullify the possible theoretical confounding effects of known prognostic factors that have been previously shown to influence survival outcomes, either favorable or unfavorable, survival outcome was re-analyzed during the first year of follow-up in relation to the level of gene expression at two points (initial and post-induction), within the same risk variable subgroups. Within patients younger than 10 years old, showing good early induction response and low-risk patients, those with high initial CASP8AP2 gene expression levels showed the lowest events (p=0.001, <0.001, <0.001 respectively). This was not found in high post induction CASP8AP2 gene expression levels within the same groups (Table 5) Multivariable Analysis To assess whether the effect of gene expression level alone, independent of other prognostic variables, was associated with the outcome; subgroup analysis was performed within homogeneous risk groups. Among patients ≤10 years, with early induction response and low-risk classification, those with high initial CASP8AP2 gene expression had the lowest number of events (p = 0.001, <0.001, <0.001, respectively). These associations were not maintained with post-induction gene expression. Multivariable Cox regression yielded an EFS hazard ratio (HR) of 0.33 (95% CI: 0.08–1.45, p = 0.14) for high initial CASP8AP2 expression. The model was overfitted with an observed: expected ratio = 4.9 at 3 years and EFS was significantly correlated with last follow MRD. Caution is warranted in interpretation due to small sample size, event count, and wide error margins (Table 6 and Table 7). DISCUSSION Outcomes of pediatric ALL have improved remarkably during the last five decades. Such improvements were made possible by the incorporation of new diagnostic technologies, the effective administration of conventional chemotherapeutic agents, and the provision of better supportive care [17]. Despite this success, accurately predicting relapse is still a challenge [18], emphasizing the need to explore and study further potential target effectors more deeply than before, trying to find out their possible roles and contribution. Studies have identified that alterations in the baseline level of expression of genes controlling the cell cycle, DNA repair, and apoptosis may participate in disordered leukemic cell proliferation and accordingly affect the drug response and clinical outcome of leukemia patients [19]. Identification of such new gene markers is important to implement enhancements to disease classification systems and to productively target disease with novel therapies [20]. Many studies have reported the importance of the initial gene level of CASP8AP2, an apoptosis-related gene, for the optimal response to chemotherapy and maintaining continuous complete remission in pediatric ALL, as concluded by Flotho et al., Jiao et al., Jin et al., Juarez-Velazquez et al., Liu et al., and Remke et al. [21–27]. However, others, like Kang et al. [28] and Yang et al. [29], failed to prove similar relationships. In this study, we started first to prospectively investigate whether the initial CASP8AP2 gene expression level has a certain predilection to different epidemiological, laboratory, molecular, response, and risk grouping factors. Patients with hyperdiploid karyotyping (p = 0.007), molecularly favorable mutations (p = 0.038), good morphological and molecular responses on day 15 and day 42 of induction therapy (p = 0.019 and p = 0.029, respectively), and low-risk stratification (p < 0.001) were positively associated with higher initial gene expression levels. Based on our results, the post-induction gene expression levels failed to maintain the initial statistically significant correlation with the above favorable variables as was expected based on the presumed mechanism of action. Analysis of these associations represents a double-edged sword. While the conclusion supports the link between higher gene levels and the known favorable variables and consequently a better response; it also raises the possibility that other favorable confounding variables may be responsible for the positive outcomes observed in these patients. Concluding a definitive positive effect of high CASP8AP2 gene level on survival is not easy due to the confounding effect of other known favorable risk variables. Thus, a ROC curve was proposed to define levels of low and high expressions of CASP8AP2 gene. The ROC coordinates were estimated using the empirical trapezoidal method (Delong) with the {pROC} package v1.18.5. Survival data were then re-examined and as in Flohr et al., fewer events in the first 3 years of follow-up were observed with initial higher gene expression [21]. The pre-induction higher expression levels were as expected encountered in hyperdiploid, molecularly favorable, early induction responder and low risk patients, but similarly these higher expressions did not persist with the same risk variables in post induction levels. Losing the initial higher gene expression favorable effect on survival post-induction chemotherapy and its associations with favorable variables is not well understood and recalls the previous argument about the independent effect of the CASP8AP2 gene. Whether this is related to decreased blast cell number carrying the CASP8AP2 gene because of chemotherapy needs to be confirmed in future studies. To nullify the possible theoretical confounding effect of some known previously proven favorable or unfavorable prognostic factors and to confirm if the level of gene expression independently can be related to the effect on survival, the incidence of events that occurred during the first year of follow up in high and low CASP8AP2 gene expression level was re-examined at two points (initially and post-induction) within the same risk variable groups. As expected, within patients younger than 10 years, early induction response, and in low-risk patients, those with high initial CASP8AP2 gene expression levels showed the lowest events, but this again was not found in high post-induction gene level expressions in any comparable way. This emphasizes that the post-induction gene level has no prognostic value in pediatric patients with ALL. Moreover, within the same older age, standard/high risk patients and those not in remission, the higher initial CASP8AP2 gene level did not positively impact the survival in those groups, denoting that other factors might counteract its favorable effect in a significant way. High CASP8AP2 gene expression is noticed in rapidly proliferating leukemic cells allowing better response to chemotherapeutic agents [30]. This is the best encountered scenario before induction of disease remission and this could partially explain why its level and effect get lower after induction of remission, as almost few to non-proliferating leukemic cells are still present. CONCLUSION Current study showed that higher initial CASP8AP2 gene expression is associated with many known favorable risk factors and has a significant impact on lowering events in the first 3 years of follow-up. On the contrary, examining post-induction gene levels failed to maintain similar statistical results. Adding an initial CASP8AP2 gene-level investigation to the routine diagnostic panel on a larger scale could help prove its independence. Based on our results, measuring post-induction levels is not helpful for the time being and is not recommended. Further future studies investigating a possible role of CASP8AP2 gene expression levels and relapse are recommended. Declarations INFORMED CONSENT TO PARTICIPATE: Informed consent was obtained from patients’ parents or legal guardians involved in the study. They had read and understood all the necessary information regarding the study (Study aim, methods, benefits and possible risk if any, and expected results). They agreed to use and protect their data and gave their consent to participate completely voluntarily. They were informed that they can freely withdraw from the study at any time without reasoning and without affecting their future care. The study used archived data and did not affect patient well-being in any way. Patients’ confidentiality was always maintained in accordance with National Cancer Institute, Cairo University policies. Data collection and presentation were anonymous and both privacy and confidentiality were protected to the maximal possible standard. Consent to publish: Authors confirm that study participants provided informed consent for publication of this research. DATA AVAILABILITY STATEMENT CLINICAL TRIAL NUMBER: Not applicable CONFLICTS OF INTEREST Authors have no conflicts of interest to declare. FUNDING This study was not supported by any sponsor or funder. AUTHORS’ CONTRIBUTIONS MH and MA were involved in the conception of the study. OA, AA, and MA were involved in data curation. MH, AA, and OA were involved in formal analysis. OA and MA were involved in data analysis. SF was involved in the study methodology. MA provided the software. SF was involved in data validation. MH, OA, AA, and MA were involved in the writing of the original draft. MH, OA, AA, and SF were involved in putting the design of the study, writing, reviewing, and editing of the manuscript. OA and MA have confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript. INFORMED CONSENT: Informed consent was obtained from patients’ parents or legal guardian in the case of children under 18 years of age was obtained. ETHICAL APPROVAL: This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of National Cancer Institute, Cairo University – Egypt (NCI) IRB approval number (2110-407-018) in October 2022. Informed consent was obtained from all subjects involved in the study. The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request and the excel sheet was attached as a supplementary material. References Kakaje A, Alhalabi MM, Ghareeb A, Karam B, Mansour B, Zahra B, et al. Rates and trends of childhood acute lymphoblastic leukaemia: an epidemiology study. Sci Rep. 2020;10(1):6756. https://doi.org/10.1038/s41598-020-63528-0 National Cancer Institute: NCCR*Explorer: An interactive website for NCCR cancer statistics. Bethesda, MD: National Cancer Institute. Available online. Last accessed February 25, 2025 Moorman AV, Robinson H, Schwab C, Richer H, Hancock J, Mitchell CD, et al. Risk-directed treatment intensification significantly reduces the risk of relapse among children and adolescents with acute lymphoblastic leukemia and intrachromosomal amplification of chromosome 21: a comparison of the MRC ALL97 and UKALL2003 trials. J Clin Oncol. 2013;31(27):3389–96. https://doi.org/10.1200/JCO.2013.48.9397 Demidowicz E, Pogorzala M, Łęcka M, Zołnowska H, Marjańska A, Kubicka M, et al. Outcome of pediatric acute lymphoblastic leukemia: Sixty years of progress. Anticancer Res. 2019;39(9):5203–7. https://doi.org/10.21873/anticanres.13717 Jostins P, Schaue-Obsthof K, Schwerk C. Loss of caspases inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis. J Biol Chem. 2006;281(18):12475–84. https://doi.org/10.1074/jbc.M50989200 Debatin KM, Stahnke K, Fulda S. Apoptosis in hematological disorders. Semin Cancer Biol. 2003;13(2):149–58. https://doi.org/10.1016/S1044-579X(02)00132-1 Morgan R, Källi A, Morsi El-Kadi AS. FLASH, a component of the FAS–CAPS8AP8 apoptotic pathway, is directly regulated by hDex1 in the notochord. Dev Biol. 2004;265(1):105–12. https://doi.org/10.1016/j.ydbio.2003.09.030 Yang YC, Burch BD, Yan Y, Marzluff WF, Dominski Z. FLASH: a proapoptotic protein involved in activation of caspase-8, is essential for 3′-end processing of histone pre-mRNAs. Mol Cell. 2009;36(2):267–78. https://doi.org/10.1016/j.molcel.2009.08.010 Gregers J, Christensen IJ, Dalhoff K, Lausen B, Schrøder H, Rosthøj S, et al. The association of reduced folate carrier 80A: G polymorphism outcome in childhood acute lymphoblastic leukemia interacts with chromosome 21 copy number. Blood. 2011;115(23):4671–7. https://doi.org/10.1182/blood-2010-01-259585 Liu Y, Yuan J. Caspases in apoptosis and beyond. Oncogene. 2008; 27(48):6194–206. https://doi.org/10.1038/onc.2008.297 Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med. 2004;350(15):1535–48. https://doi.org/10.1056/NEJMra023001 Gohar S, Shloom I, Assem M, Attia I, Yassin D, Salem S, et al. Clinical significance of minimal residual disease in peripheral blood on day 8 induction in childhood B-precursor acute lymphoblastic leukemia: report from Children’s Cancer Hospital in Egypt. 2015;33:115. suppl.2:e1008–e21008. https://doi.org/10.1200/jco.2015.33.15_suppl.e21008 Sarma M, Mahmoud H, Abdelhamid T, El-Sharkawy N, ElNahas Y, El Gammal M, et al. The prognostic significance of minimal residual disease in adult Egyptian patients with precursor acute lymphoblastic leukemia. J Egypt Natl Cancer Inst. 2013;25:135–42. https://doi.org/10.1016/j.jnci.2013.05.004 Adnan Awad S, Kamel MM, Ayoub MA, Kamel AM, Elnoshokaty EH, El Hifnawi N. Immunophenotypic characterization of genotype subgroups in Egyptian pediatric patients with B-cell acute lymphoblastic leukemia. Clin Lymphoma Myeloma Leukemia. 2016;16:519–524. https://doi.org/10.1016/j.clml.2016.02.023 Farweez BAT, Kassim NA, Abdelfattah MF, Hassan NM, Hassan DEA, El-Sharkawy M. Clinical impact of early minimal residual disease detection at day 15 in precursor B-childhood acute lymphoblastic leukemia: an Egyptian experience. Egypt J Med Hum Genet. 2020;21(1):28. https://doi.org/10.1186/s43042-020-00065-5 Basu S, Pereira AF, Pineiro DH, Wang H, Valencia-Jimenez A, Siegfried BD, et al. Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber). Sci Rep. 2019;9(1):10374. https://doi.org/10.1038/s41598-019-47020-y Toft N, Birgens H, Abrahamsson J, Griškevičius L, Hallböök H, Heyman M, et al. Results of NOPHO ALL2008 treatment for patients aged 1–45 years with acute lymphoblastic leukemia. Leukemia. 2018;32(3):606–15. https://doi.org/10.1038/leu.2017.265 Hunger SP, Raetz EA. How I treat relapsed acute lymphoblastic leukemia in the pediatric population. Blood. 2023;16(16):1803–12. https://doi.org/10.1182/blood.2019010043 Cathpole D, Lai A, Guo D, Chen QR, Khan J. Gene expression in fresh state segregate patients with childhood acute lymphoblastic leukemia: an independent validation study identifies that endoglin is associated with patient outcome. Leuk Res. 2007;31(3):221–7. https://doi.org/10.1016/j.leukres.2007.04.021 McBride H, Aouacheria A, Widlak P, Viegas L, Ciech N, Kasner M, et al. The role of inhibition of apoptosis in acute leukemias and myelodysplastic syndrome. Front. 2019;12:192. https://doi.org/10.3389/fonc.2019.00192 Flohr C, Coustan-Smith E, Pei D, Ivamoto S, Song G, Cheng C, et al. Genes contributing to minimal residual disease in childhood acute lymphoblastic leukemia: prognostic significance of CASP8AP2. Blood. 2006;108(13):007–15. https://doi.org/10.1182/blood-2006-01-0322 Coustan-Smith E, Pei D, Cheng C, Song G, Pui CH, et al. A set of genes that regulate cell proliferation predicts treatment outcome in childhood acute lymphoblastic leukemia. Blood. 2007;110(4):1271–7. https://doi.org/10.1182/blood-2007-01-064878 Jiao Y, Cui L, Gao C, Li W, Zhao X, Lu S, et al. CASP8AP2 is a promising diagnostic biomarker in pediatric acute lymphoblastic leukemia. Leukemia Res. 2013;26(3):167–71. https://doi.org/10.1016/j.leukres.2011.05.023 Jin JF, Wang CJ, Cui L, Liu FF, Wang KL, Li WJ, et al. Interaction of E2F3a and CASP8AP2 regulates histone expression and chemosensitivity of leukemic cells. J Pediatr Hematol Oncol. 2023;45(3):e339–44. https://doi.org/10.1097/MPH.0000000000002558 Juarez-Velazquez MR, Salas-Labadia C, Reyes-Leon A, Navarrete-Meneses MP, Fuentes-Panana EM, Perez-Ver P. Genetic markers in the prognosis of childhood acute lymphoblastic leukemia. In: Mejia-Arangure JM, editor. Clinical epidemiology of acute lymphoblastic leukemia – from the molecules to the clinic [Internet]. InTech; 2013 [cited 2022 Feb 5]. Available from: http://www.intechopen.com/books/clinical-epidemiology-of-acute-lymphoblastic-leukemia-from-the-molecules-to-the-clinic/genetic-markers-in-the-prognosis-of-childhood-acute-lymphoblastic-leukemia Liu FF, Wang KL, Deng LP, Liu X, Wu M yuan, Wang TY, et al. Transcription factor E2F3a regulates CASP8AP2 transcription and enhances sensitivity to chemotherapeutic drugs in acute lymphoblastic leukemia. Cancer Cell Int. 2018;18(1):1–9. https://doi.org/10.1186/s12935-018-0531-1 Remke M, Pfister S, Kox C, Toedt G, Becker N, Benner A, et al. High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-β and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response. Blood. 2009;114(5):1053–62. https://doi.org/10.1182/blood-2008-10-186536 Kang H, Chen IM, Wilson CS, Bedrick EJ, Harvey RC, Atlas SR, et al. Gene expression classifiers for relapse-free survival and minimal residual disease improve risk classification and outcome prediction in pediatric B-precursor acute lymphoblastic leukemia. Blood. 2010;115(7):1394–405. https://doi.org/10.1182/blood-2009-05-218560 Yang YL, Lin SR, Chen JS, Lin SW, Yu SL, Chen HY, et al. Expression and prognostic significance of the apoptotic genes BCL2L13, Livin, and CASP8AP2 in childhood acute lymphoblastic leukemia. Leuk Res. 2010;34(1):18–23. https://doi.org/10.1016/j.leukres.2009.07.023 Ehrhardt H, Wachter F, Maurer M, Stahnke K, Jeremias I. Important role of caspase-8 for chemosensitivity of ALL cells. Clin Cancer Res. 2011;17(24):7605–13. https://doi.org/10.1158/1078-0432.CCR-11-0513 Tables Tables 1 to 7 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 13 Nov, 2025 Reviews received at journal 04 Nov, 2025 Reviewers agreed at journal 04 Nov, 2025 Reviews received at journal 03 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviewers invited by journal 30 Oct, 2025 Submission checks completed at journal 22 Oct, 2025 First submitted to journal 22 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7478773","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":537466104,"identity":"8f1e722e-659d-43b3-8958-96991e38865a","order_by":0,"name":"Omar Arafah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFAD5gNsDB/ArARitbAlsDHOIFkLMw8xWvjFDj/7wPDHLp+/jfnZY5uawwz87DkGDB9+4dYiOTvNeAZjW7LljGNs5sY5xw4zSPa8MWCc2Ydbi8HtBGMGxgZmA4b7DWbSuQ2HGQxu5Bgw8/bg05L+mYHhT72B/DH2b9KWQC32IC1/8WrJMQZ6/bCBwTEeM2lGkC0SQC0MP/D5JaeYIbHtuIHhMZ4yyZ5j6TwSZ54VHOxtwK2FXzp9M8OHP9UGcsfYt0n8qLGW429P3vjgxx/cWsAgAYkNjpoDjG0EtGABhGwZBaNgFIyCkQQAIwNMHtoxDJ4AAAAASUVORK5CYII=","orcid":"","institution":"National Cancer Institute, Cairo University","correspondingAuthor":true,"prefix":"","firstName":"Omar","middleName":"","lastName":"Arafah","suffix":""},{"id":537466105,"identity":"b6ee4d4a-3040-483a-b1fc-7db6b38ac0bb","order_by":1,"name":"Marihame Ashraf","email":"","orcid":"","institution":"National Cancer Institute, Cairo University","correspondingAuthor":false,"prefix":"","firstName":"Marihame","middleName":"","lastName":"Ashraf","suffix":""},{"id":537466106,"identity":"7ae78ab2-5caf-4f35-9b47-b45c33b255c8","order_by":2,"name":"Ahmed Mustafa Abd Elsalam","email":"","orcid":"","institution":"National Cancer Institute, Cairo University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"Mustafa Abd","lastName":"Elsalam","suffix":""},{"id":537466107,"identity":"a092514b-1499-41f6-a983-63a5bcd9429c","order_by":3,"name":"Sally Elfishawi","email":"","orcid":"","institution":"National Cancer Institute, Cairo University","correspondingAuthor":false,"prefix":"","firstName":"Sally","middleName":"","lastName":"Elfishawi","suffix":""},{"id":537466108,"identity":"c9a22813-c9e7-4029-b76b-785ae224ef8b","order_by":4,"name":"Mahmoud Hammad","email":"","orcid":"","institution":"National Cancer Institute, Cairo University","correspondingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Hammad","suffix":""}],"badges":[],"createdAt":"2025-08-28 09:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7478773/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7478773/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94844899,"identity":"347af32f-e48c-4979-90ac-974a43b1211d","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":479844,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript2210.docx","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/eef0a21b50577ebb9ff665da.docx"},{"id":94984911,"identity":"6b433510-beba-4135-b514-160e29a3ee84","added_by":"auto","created_at":"2025-11-03 06:56:54","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7167,"visible":true,"origin":"","legend":"","description":"","filename":"285a42bc3c3c494e88832954152fc1f4.json","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/ee994a62e67dc923eb11d3a7.json"},{"id":94985211,"identity":"7b6433cd-71fb-465a-bcd7-666ef31ecb8f","added_by":"auto","created_at":"2025-11-03 06:57:41","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155191,"visible":true,"origin":"","legend":"","description":"","filename":"285a42bc3c3c494e88832954152fc1f41enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/63898ed98cd56c80cbd50dc0.xml"},{"id":94985461,"identity":"e4944c88-f347-4165-add5-225dc52566d3","added_by":"auto","created_at":"2025-11-03 06:58:12","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182341,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/073dd67fa0cc5a29640aae42.jpeg"},{"id":94844901,"identity":"12b525c6-4a7f-46be-a8b1-4cb7012d5165","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229348,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/96e247ca6743efae0d52878a.jpeg"},{"id":94844902,"identity":"ef7dcb81-d4fa-40fd-9ba3-abad3926521c","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":228028,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/f0dfa2d0616a7106579e10cb.jpeg"},{"id":94844905,"identity":"742cfbd3-d40b-47f1-b057-fc07ebfb0c1f","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34049,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/8953a802697a4a1a7cdd8ccb.png"},{"id":94844906,"identity":"1c70f596-2882-41b5-8680-b471c1ee2a3a","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34509,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/adf266029f798716dc730179.png"},{"id":94844907,"identity":"9f5fb881-076e-47c3-8de2-136949bcce2f","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35544,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/7b82b8311142ce690b7ef88d.png"},{"id":94844910,"identity":"23eb8638-61a1-4e1b-9ce1-9d983df35727","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157588,"visible":true,"origin":"","legend":"","description":"","filename":"285a42bc3c3c494e88832954152fc1f41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/7d3726c3afaf9f98df09e090.xml"},{"id":94844909,"identity":"066e734b-0882-405e-ba20-d95d89db5ce8","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166653,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/f14dc439aced2689406d0b4f.html"},{"id":94844895,"identity":"519b4df5-86df-4a27-89ce-d817197eef9b","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC curve for low and high CASP8AP2 gene expression levels\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/138f889d0b6d632c458c28cd.png"},{"id":94984901,"identity":"7bfe4ece-7faf-4d94-8622-f67bde34da16","added_by":"auto","created_at":"2025-11-03 06:56:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvent-free and Overall survival analysis according to the expression level of CASP8AP2 gene in pre-induction BMA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/867122ec350f2bf6fac753b0.png"},{"id":94985367,"identity":"c30c114f-626a-418f-b177-4666bf6f2941","added_by":"auto","created_at":"2025-11-03 06:58:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvent-free and Overall survival analysis according to the expression level of CASP8AP2 gene in post-induction BMA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/01b2a6991153c91e5a877a5d.png"},{"id":94990324,"identity":"af133f1f-a83a-46e9-9ce9-0d89a3778e13","added_by":"auto","created_at":"2025-11-03 07:16:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1024866,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/7e178e12-20b9-4336-9739-9a3fad700262.pdf"},{"id":94844896,"identity":"306f6bef-bc1a-461e-98f7-376394b5a65a","added_by":"auto","created_at":"2025-10-31 10:06:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36717,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7478773/v1/b095aec13661f23c52654e56.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prognostic Significance of Caspase 8 Associated Protein 2 (CASP8AP2) in Childhood B cell Acute Lymphoblastic Leukemia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAcute lymphoblastic leukemia (ALL) is the most common cancer in children, comprising around 25% of all childhood malignancies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the past two decades, the increased cure rates for pediatric ALL have been one of the major achievements in cancer therapy, with the current 5-year survival rate increasing to about 90% in children younger than 15 years, mainly in developed countries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGreat advances have been made in risk stratification, more precise response monitoring, treatment plan modification and prediction of prognosis in pediatric ALL [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Yet, there are certain groups of patients whose response to treatment and durable remissions are not explained by the routinely used criteria for risk assignment and response assessment tools. Therefore, new prognostic markers should be explored as relapses remain the main obstacle against curing childhood ALL [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the mitochondrial death pathway (the intrinsic pathway) and the death receptor\u0026ndash;mediated pathway (the extrinsic pathway) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Defects in the apoptotic process disrupt the delicate balance between cell proliferation and cell death. Studies have shown that leukemic cells adopt these mechanisms to resist chemotherapeutic agents [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCASP8AP2, a gene found on chromosome 6q15\u0026ndash;16.1, encodes the enzyme needed to help apoptosis in response to chemotherapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], as the CASP8AP2 protein interacts with caspase-8 death-effector domain and plays an essential regulatory role in Fas and tumor necrosis factor\u0026ndash;mediated apoptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CASP8AP2 has been the focus of interest in many research projects. Its prognostic significance in pediatric ALL had been extensively studied before, with inconsistent results [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCaspases are subdivided into three groups according to structure and function. Group I (inflammatory caspases-1, -4, -5, -11, and \u0026minus;\u0026thinsp;12), Group II (initiators caspases-2, -8, -9, and \u0026minus;\u0026thinsp;10), and Group III (caspases-3, -6, and \u0026minus;\u0026thinsp;7) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn current study, we aimed to assess the impact of the level of CASP8AP2 gene expression pre- and post-induction therapy on the outcome in pediatric ALL and to analyze its associations with early response to treatment and other prevalent prognostic factors.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eThis prospective cohort study was conducted on 70 newly diagnosed ALL pediatric patients who were treated at the pediatric oncology department, National Cancer Institute (NCI), Cairo University \u0026ndash; Egypt from 1st of October 2019 till the end of September 2023.\u003c/p\u003e\u003cp\u003eMedical records were reviewed for epidemiological data (age groups, sex), date of presentation, clinical data, initial investigations. Bone marrow aspirates (BMA) were assessed for immunophenotyping by flow cytometry, ALL-associated fusion gene transcripts by fluorescence in situ hybridization (FISH), karyotyping (G-banding), along with cerebrospinal fluid (CSF) examination. Risk assignment was done according to St. Jude\u0026rsquo;s risk stratification [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and treatment data were recorded (e.g: date and type of chemotherapy received).\u003c/p\u003e\u003cp\u003eData of relapse were also collected, including timing of relapse (early or late), type of relapse (hematological, central nervous system [CNS], or combined), salvage chemotherapy given (e.g: timing and number of cycles), and response to salvage therapy.\u003c/p\u003e\u003cp\u003eSurvival analysis data were collected (date of diagnosis, date of relapse, and date of death/last follow-up).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEligibility Criteria\u003c/h2\u003e\u003cp\u003eAll included patients in the study aged between 1 and 18 years old, confirmed to have ALL by immunophenotyping, risk assigned and treated according to St. Jude total XV protocol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and had a sufficient bone marrow (BM) sample for total RNA/microRNA (miRNA) extraction as identified through the clinical pathology department data registry.\u003c/p\u003e\u003cp\u003eExclusion criteria: Patients who were included prospectively and died before the end of induction and patients without sufficient material for total RNA extraction at end of induction.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMRD Assessment\u003c/h3\u003e\n\u003cp\u003eAt the time of diagnosis, peripheral blood or bone marrow samples were taken; samples were examined the same day [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A wide range of monoclonal antibodies, including CD45, CD19, CD10, CD22, CD34, CD79A, CD1PE, CD7, CD2, CD4, CD8, CD16, CD56, CD99, TDT, CD33, CD13, CD11B, CD15, NG2, and CD117, were acquired from Beckman Coulter in Miami, USA, for the purpose of lineage assignment. The monoclonal antibodies used were from Dako (Cytomation, Denmark) and included MPO, CD3, Kappa, Lambda, CD5, cytoplasmic \u0026micro;, CD20, and MHC class II. 50 \u0026micro;L of a sample with an adjusted cell count of approximately 1 \u0026times; 10⁶ cells per tube was incubated with monoclonal antibodies according to the manufacturer\u0026rsquo;s instructions for 30 minutes at room temperature in the dark, after which the sample was lysed, and excess antibodies were removed with Phosphate Buffered Saline (PBS) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn 500 mL of PBS, the cells were suspended. The analysis was carried out using a Navious cytometer. Firstly, the blast population was selected on the basis of forward scatter versus side scatter and then on the basis of CD45 versus side scatter.\u003c/p\u003e\u003cp\u003eThe cut-off value for surface monoclonal antibodies was 20%, while that for cytoplasmic antibodies was 10% [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Utilizing a Navious cytometer (Beckman Coulter, Miami, Florida), flow cytometry (FCM) was performed to evaluate MRD.\u003c/p\u003e\u003cp\u003eUsing INTA Prep permeabilization reagent from Beckman Coulter, intracellular staining was performed by fixing cells with reagent 1 (formaldehyde fixation reagent), washing, inducing permeability with reagent 2 (using saponin for permeability), and lysing any remaining erythrocytes.\u003c/p\u003e\u003cp\u003eFor the detection of MRD, the following monoclonal antibodies were used in four color combinations. BCP-ALL: TdT/CD10/CD19/CD45; CD10/CD20/CD19/CD45; CD34/CD38/CD19/CD45; CD34/CD22/CD19/CD45; CD19/CD34/CD45; CD10/CD20/CD22/CD45. Since this combination was one of the highest incidence marker expressions and helped distinguish hematogones from residual blasts in ALL instances after induction, it was identified at diagnosis and post-induction following morphological remission for MRD tracking.\u003c/p\u003e\u003cp\u003eCutoff values were deemed to be \u0026gt;\u0026thinsp;20% for surface monoclonal antibodies, whereas cytoplasmic values were \u0026ge;\u0026thinsp;10%. The median number of events measured in the assay was 1,250,000 (range: 750,000\u0026ndash;1,600,000). The detection limit (LOD) is 0.002% or one in every 50,000 cells. The lower limit of quantification (LLOQ) is 0.01% (1\u0026ndash;10,000). Sequential gating was employed. The minimum target sensitivity for quantifying MRD was defined as the ability to detect 30 clustered MRD events in 3 \u0026times; 10⁵ total cellular events (0.01%).\u003c/p\u003e\u003cp\u003eThe cutoff point for MRD1 (D15) was \u0026lt;\u0026thinsp;10⁻\u0026sup3; (0.1%), and for MRD at any time point, it was \u0026lt;\u0026thinsp;10⁻⁴ (0.01%) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] at D42 (End of induction), week 17, week 48 and end of treatment in patients who ended chemotherapy.\u003c/p\u003e\n\u003ch3\u003eCASP8AP2 Gene Expression Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eNucleic Acid Extraction\u003c/strong\u003e\u003cp\u003eUnder strict aseptic conditions, bone marrow samples drawn in two EDTA tubes containing 3 mL each were utilized to extract RNA. Extraction of total cellular RNA from blood was done using QIAamp RNA Blood Mini Kit for total RNA purification (Qiagen, Hilden, Germany). Concentration and purity of RNA was checked by measuring the absorbance at 260 nm (A260) using the Nanodrop spectrophotometer.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAnalysis of CASP8AP2 gene expression\u003c/strong\u003e\u003cp\u003eFollowing RNA extraction, reverse transcription was performed, and first-strand cDNA was prepared using 1 \u0026micro;g of total RNA using Applied Biosystems\u0026trade; High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). Quantitative RT-PCR assays were performed using the SLAN\u0026reg;-48P Real-Time PCR System Hongshi system (Shanghai Hongshi Medical Technology Co., Ltd.) with Taqman Universal Mastermix (Thermo Fisher Scientific, USA) and inventoried Taqman assay (Applied Biosystems, Life Technologies) for CASP8AP2 mRNA (Hs01594281_m1) and ABL1 (Hs01104728_m1) as the housekeeping gene.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eA total of 10 pediatric control samples were used for gene expression analysis. These were gathered from normal pediatric bone marrow donor samples supplied as screening samples before donation.\u003c/p\u003e\u003cp\u003eData from the amplification plot was obtained and analyzed to determine the relative expression of CASP8AP2 gene among patients and control samples and then compare cycle threshold (CT) of the target genes to the CT of the housekeeping gene. To determine the relative expression, we used the comparative cycle threshold (2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT) method where the data were represented as the fold change in gene expression compared to an endogenous reference gene.\u003c/p\u003e\u003cp\u003eIn our study, the endogenous reference gene was ABL1. ΔCT data (CT target gene \u0026ndash; CT endogenous reference gene) was used [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStatistical Methods\u003c/h3\u003e\n\u003cp\u003eCut-off for gene expression was estimated at the maximum sensitivity and specificity using the Youden\u0026rsquo;s Index of the Receiver Operating Characteristics (ROC) curve coordinates. The cut-off values for CASP8AP2 gene expression were used to designate groups at diagnosis, where groups below cutoff threshold were designated low expressors and groups showing gene expression greater or equal to the threshold, were designated high expressors. The association between high and low expression levels and different variables was tested using Pearson\u0026rsquo;s Chi-squared test and Fisher\u0026rsquo;s exact test. To compare CASP8AP2 expression levels between different variables, we employed the Wilcoxon and Kruskal\u0026ndash;Wallis rank sum tests.\u003c/p\u003e\u003cp\u003eFor survival analysis, the Kaplan\u0026ndash;Meier method was used and the survival experience compared using the log-rank test. Alpha was two-sided and set at 0.05. All analyses were conducted using R version 4.1.0. A p-value less than 0.05 indicated statistical significance.\u003c/p\u003e\u003cp\u003eOverall survival (OS) was defined as the period between the date of diagnosis and the date of death from any cause or the date of the patient\u0026rsquo;s last follow-up.\u003c/p\u003e\u003cp\u003eEvent-free survival (EFS) was defined as the period between the date of diagnosis and the date of relapse or to date of last follow-up for patients without events.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eInitial patients\u0026rsquo; characteristics are summarized in \u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Clinical characteristics and initial laboratory investigations of the whole cohort\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(N = 70)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCASP8AP2 gene expression initially and post induction and its relations to different variables\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA ROC curve (receiver operating characteristic curve) was used to define levels of low and high expression of CASP8AP2 gene \u003cstrong\u003e(Table 2)\u003c/strong\u003e. \u003cstrong\u003eFigure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. CASP8AP2 gene expression cut-off in the initial and post-induction BMA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients with high initial gene expression levels were more likely to be associated with hyperdiploid karyotyping (p = 0.007), molecular favorable mutations (t(12;21) and negative mutations) (p = 0.038), good early (Day 15) and end-of-induction (Day 42) morphological and MRD response (p = 0.019 and p = 0.029, respectively), and low-risk stratification (p \u0026lt; 0.001). Meanwhile, the post-induction high gene expression levels failed to maintain the same statistically significant correlation with the same favorable variables \u003cstrong\u003e(Table 3).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eTable 3. Relation of initial patients\u0026rsquo; characteristics and treatment response to the initial and post-induction BMA CASP8AP2 gene expression level\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMoreover, we calculated the high and low CASP8AP2 gene expression levels within each variable including the above proven associated favorable ones. Significantly high pre-induction CASP8AP2 gene levels compared to lesser low levels were mostly encountered in patients with hyperdiploid karyotyping (p = 0.009), molecular favorable (p = 0.002), early induction response (p = 0.025), and low-risk patients (p \u0026lt; 0.001). This difference in expression levels within each variable did not persist in post-induction gene levels \u003cstrong\u003e(Table 4)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRelation of different CASP8AP2 gene expression cut-off levels in the initial pre-induction and post-induction BMA to the different prognostic features\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival outcome compared to gene expression levels and cause specific mortalities:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedian follow-up time was 1.5 years (95% CI 1.3 to 1.8). Survival outcome was re-analyzed after dividing patients by ROC curve into high and low gene expression levels. Within the first three years of follow-up, high gene expression levels in the pre-induction BMA had been associated with better survival in terms of fewer events (p-value \u0026lt;0.001)\u003cstrong\u003e\u0026nbsp;Figure 2.\u0026nbsp;\u003c/strong\u003eThis contrasts with post induction BMA gene level which did not correlate with lesser events (p-value 0.49\u003cstrong\u003e) Figure 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOut of the 70 patients included in the study, there were 13 mortality cases (19% of the whole cohort). Five patients (39%) died in the first year, seven (54%) in the second year, and one (7%) in the third year after induction therapy, respectively.\u003c/p\u003e\n\u003cp\u003eIn 4 out of the 5 patients who died in the first year, deaths occurred because of septic complications during maintenance therapy and all of them exhibited a high initial gene expression level. Only one patient had encountered early isolated hematological relapse and died from septic complications following intensive salvage chemotherapy. This patient had a low initial gene expression level.\u003c/p\u003e\n\u003cp\u003eAmong the seven patients who died during the second year, two died from septic complications during maintenance therapy, and one of them had a low initial gene expression level. The remaining five patients experienced relapse: two died from septic complications after receiving intensive salvage chemotherapy, and three died from refractory disease (palliative cases). Of the five relapsed cases, four exhibited a low initial gene expression level.\u003c/p\u003e\n\u003cp\u003eOnly one patient died during the third year, following septic complications after intensive salvage chemotherapy for an isolated hematological relapse. This patient exhibited a low initial gene expression level.\u003c/p\u003e\n\u003cp\u003eTo nullify the possible theoretical confounding effects of known prognostic factors that have been previously shown to influence survival outcomes, either favorable or unfavorable, survival outcome was re-analyzed during the first year of follow-up in relation to the level of gene expression at two points (initial and post-induction), within the same risk variable subgroups. Within patients younger than 10 years old, showing good early induction response and low-risk patients, those with high initial CASP8AP2 gene expression levels showed the lowest events (p=0.001, \u0026lt;0.001, \u0026lt;0.001 respectively). This was not found in high post induction CASP8AP2 gene expression levels within the same groups \u003cstrong\u003e(Table 5)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultivariable Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether the effect of gene expression level alone, independent of other prognostic variables, was associated with the outcome; subgroup analysis was performed within homogeneous risk groups. Among patients \u0026le;10 years, with early induction response and low-risk classification, those with high initial CASP8AP2 gene expression had the lowest number of events (p = 0.001, \u0026lt;0.001, \u0026lt;0.001, respectively). These associations were not maintained with post-induction gene expression.\u003c/p\u003e\n\u003cp\u003eMultivariable Cox regression yielded an EFS hazard ratio (HR) of 0.33 (95% CI: 0.08\u0026ndash;1.45, p = 0.14) for high initial CASP8AP2 expression. The model was overfitted with an observed: expected ratio = 4.9 at 3 years and EFS was significantly correlated with last follow MRD. Caution is warranted in interpretation due to small sample size, event count, and wide error margins \u003cstrong\u003e(Table 6 and Table 7).\u003c/strong\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOutcomes of pediatric ALL have improved remarkably during the last five decades. Such improvements were made possible by the incorporation of new diagnostic technologies, the effective administration of conventional chemotherapeutic agents, and the provision of better supportive care [17]. Despite this success, accurately predicting relapse is still a challenge [18], emphasizing the need to explore and study further potential target effectors more deeply than before, trying to find out their possible roles and contribution.\u003c/p\u003e\n\u003cp\u003eStudies have identified that alterations in the baseline level of expression of genes controlling the cell cycle, DNA repair, and apoptosis may participate in disordered leukemic cell proliferation and accordingly affect the drug response and clinical outcome of leukemia patients [19]. Identification of such new gene markers is important to implement enhancements to disease classification systems and to productively target disease with novel therapies [20].\u003c/p\u003e\n\u003cp\u003eMany studies have reported the importance of the initial gene level of CASP8AP2, an apoptosis-related gene, for the optimal response to chemotherapy and maintaining continuous complete remission in pediatric ALL, as concluded by Flotho et al., Jiao et al., Jin et al., Juarez-Velazquez et al., Liu et al., and Remke et al. [21\u0026ndash;27]. However, others, like Kang et al. [28] and Yang et al. [29], failed to prove similar relationships.\u003c/p\u003e\n\u003cp\u003eIn this study, we started first to prospectively investigate whether the initial CASP8AP2 gene expression level has a certain predilection to different epidemiological, laboratory, molecular, response, and risk grouping factors. Patients with hyperdiploid karyotyping (p = 0.007), molecularly favorable mutations (p = 0.038), good morphological and molecular responses on day 15 and day 42 of induction therapy (p = 0.019 and p = 0.029, respectively), and low-risk stratification (p \u0026lt; 0.001) were positively associated with higher initial gene expression levels. Based on our results, the post-induction gene expression levels failed to maintain the initial statistically significant correlation with the above favorable variables as was expected based on the presumed mechanism of action.\u003c/p\u003e\n\u003cp\u003eAnalysis of these associations represents a double-edged sword. While the conclusion supports the link between higher gene levels and the known favorable variables and consequently a better response; it also raises the possibility that other favorable confounding variables may be responsible for the positive outcomes observed in these patients.\u003c/p\u003e\n\u003cp\u003eConcluding a definitive positive effect of high CASP8AP2 gene level on survival is not easy due to the confounding effect of other known favorable risk variables. Thus, a ROC curve was proposed to define levels of low and high expressions of CASP8AP2 gene.\u003cbr\u003e\u0026nbsp;The ROC coordinates were estimated using the empirical trapezoidal method (Delong) with the {pROC} package v1.18.5. Survival data were then re-examined and as in Flohr et al., fewer events in the first 3 years of follow-up were observed with initial higher gene expression [21]. The pre-induction higher expression levels were as expected encountered in hyperdiploid, molecularly favorable, early induction responder and low risk patients, but similarly these higher expressions did not persist with the same risk variables in post induction levels. Losing the initial higher gene expression favorable effect on survival post-induction chemotherapy and its associations with favorable variables is not well understood and recalls the previous argument about the independent effect of the CASP8AP2 gene. Whether this is related to decreased blast cell number carrying the CASP8AP2 gene because of chemotherapy needs to be confirmed in future studies.\u003cbr\u003e\u0026nbsp;To nullify the possible theoretical confounding effect of some known previously proven favorable or unfavorable prognostic factors and to confirm if the level of gene expression independently can be related to the effect on survival, the incidence of events that occurred during the first year of follow up in high and low CASP8AP2 gene expression level was re-examined at two points (initially and post-induction) within the same risk variable groups. As expected, within patients younger than 10 years, early induction response, and in low-risk patients, those with high initial CASP8AP2 gene expression levels showed the lowest events, but this again was not found in high post-induction gene level expressions in any comparable way.\u003cbr\u003e\u0026nbsp;This emphasizes that the post-induction gene level has no prognostic value in pediatric patients with ALL. Moreover, within the same older age, standard/high risk patients and those not in remission, the higher initial CASP8AP2 gene level did not positively impact the survival in those groups, denoting that other factors might counteract its favorable effect in a significant way.\u003c/p\u003e\n\u003cp\u003eHigh CASP8AP2 gene expression is noticed in rapidly proliferating leukemic cells allowing better response to chemotherapeutic agents [30]. This is the best encountered scenario before induction of disease remission and this could partially explain why its level and effect get lower after induction of remission, as almost few to non-proliferating leukemic cells are still present.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eCurrent study showed that higher initial CASP8AP2 gene expression is associated with many known favorable risk factors and has a significant impact on lowering events in the first 3 years of follow-up. On the contrary, examining post-induction gene levels failed to maintain similar statistical results. Adding an initial CASP8AP2 gene-level investigation to the routine diagnostic panel on a larger scale could help prove its independence. Based on our results, measuring post-induction levels is not helpful for the time being and is not recommended. Further future studies investigating a possible role of CASP8AP2 gene expression levels and relapse are recommended.\u003c/p\u003e"},{"header":"Declarations","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003e\u003cu\u003eINFORMED CONSENT TO PARTICIPATE:\u0026nbsp;\u003c/u\u003e\u003c/strong\u003eInformed consent was obtained from patients\u0026rsquo; parents or legal guardians involved in the study. They had read and understood all the necessary information regarding the study (Study aim, methods, benefits and possible risk if any, and expected results). They agreed to use and protect their data and gave their consent to participate completely voluntarily. They were informed that they can freely withdraw from the study at any time without reasoning and without affecting their future care. The study used archived data and did not affect patient well-being in any way. Patients\u0026rsquo; confidentiality was always maintained in accordance with National Cancer Institute, Cairo University policies. Data collection and presentation were anonymous and both privacy and confidentiality were protected to the maximal possible standard.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003e\u003cu\u003eConsent to publish:\u003c/u\u003e\u003c/strong\u003e Authors confirm that study participants provided informed consent for publication of this research.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLINICAL TRIAL NUMBER:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was not supported by any sponsor or funder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMH and MA were involved in the conception of the study. OA, AA, and MA were involved in data curation. MH, AA, and OA were involved in formal analysis. OA and MA were involved in data analysis. SF was involved in the study methodology. MA provided the software. SF was involved in data validation. MH, OA, AA, and MA were involved in the writing of the original draft. MH, OA, AA, and SF were involved in putting the design of the study, writing, reviewing, and editing of the manuscript. OA and MA have confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINFORMED CONSENT:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from patients\u0026rsquo; parents or legal guardian in the case of children under 18 years of age was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eETHICAL APPROVAL:\u003c/u\u003e\u003c/strong\u003e This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of National Cancer Institute, Cairo University \u0026ndash; Egypt (NCI) IRB approval number (2110-407-018) in October 2022. Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during this study are available from the corresponding author on reasonable request and the excel sheet was attached as a supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKakaje A, Alhalabi MM, Ghareeb A, Karam B, Mansour B, Zahra B, et al. Rates and trends of childhood acute lymphoblastic leukaemia: an epidemiology study. Sci Rep. 2020;10(1):6756. https://doi.org/10.1038/s41598-020-63528-0\u003c/li\u003e\n\u003cli\u003eNational Cancer Institute: NCCR*Explorer: An interactive website for NCCR cancer statistics. Bethesda, MD: National Cancer Institute. Available online. Last accessed February 25, 2025\u003c/li\u003e\n\u003cli\u003eMoorman AV, Robinson H, Schwab C, Richer H, Hancock J, Mitchell CD, et al. Risk-directed treatment intensification significantly reduces the risk of relapse among children and adolescents with acute lymphoblastic leukemia and intrachromosomal amplification of chromosome 21: a comparison of the MRC ALL97 and UKALL2003 trials. J Clin Oncol. 2013;31(27):3389\u0026ndash;96. https://doi.org/10.1200/JCO.2013.48.9397\u003c/li\u003e\n\u003cli\u003eDemidowicz E, Pogorzala M, Łęcka M, Zołnowska H, Marjańska A, Kubicka M, et al. Outcome of pediatric acute lymphoblastic leukemia: Sixty years of progress. Anticancer Res. 2019;39(9):5203\u0026ndash;7. https://doi.org/10.21873/anticanres.13717\u003c/li\u003e\n\u003cli\u003eJostins P, Schaue-Obsthof K, Schwerk C. Loss of caspases inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis. J Biol Chem. 2006;281(18):12475\u0026ndash;84. https://doi.org/10.1074/jbc.M50989200\u003c/li\u003e\n\u003cli\u003eDebatin KM, Stahnke K, Fulda S. Apoptosis in hematological disorders. Semin Cancer Biol. 2003;13(2):149\u0026ndash;58. https://doi.org/10.1016/S1044-579X(02)00132-1\u003c/li\u003e\n\u003cli\u003eMorgan R, K\u0026auml;lli A, Morsi El-Kadi AS. FLASH, a component of the FAS\u0026ndash;CAPS8AP8 apoptotic pathway, is directly regulated by hDex1 in the notochord. Dev Biol. 2004;265(1):105\u0026ndash;12. https://doi.org/10.1016/j.ydbio.2003.09.030\u003c/li\u003e\n\u003cli\u003eYang YC, Burch BD, Yan Y, Marzluff WF, Dominski Z. FLASH: a proapoptotic protein involved in activation of caspase-8, is essential for 3\u0026prime;-end processing of histone pre-mRNAs. Mol Cell. 2009;36(2):267\u0026ndash;78. https://doi.org/10.1016/j.molcel.2009.08.010\u003c/li\u003e\n\u003cli\u003eGregers J, Christensen IJ, Dalhoff K, Lausen B, Schr\u0026oslash;der H, Rosth\u0026oslash;j S, et al. The association of reduced folate carrier 80A: G polymorphism outcome in childhood acute lymphoblastic leukemia interacts with chromosome 21 copy number. Blood. 2011;115(23):4671\u0026ndash;7. https://doi.org/10.1182/blood-2010-01-259585\u003c/li\u003e\n\u003cli\u003eLiu Y, Yuan J. Caspases in apoptosis and beyond. Oncogene. 2008; 27(48):6194\u0026ndash;206. https://doi.org/10.1038/onc.2008.297\u003c/li\u003e\n\u003cli\u003ePui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med. 2004;350(15):1535\u0026ndash;48. https://doi.org/10.1056/NEJMra023001\u003c/li\u003e\n\u003cli\u003eGohar S, Shloom I, Assem M, Attia I, Yassin D, Salem S, et al. Clinical significance of minimal residual disease in peripheral blood on day 8 induction in childhood B-precursor acute lymphoblastic leukemia: report from Children\u0026rsquo;s Cancer Hospital in Egypt. 2015;33:115. suppl.2:e1008\u0026ndash;e21008. https://doi.org/10.1200/jco.2015.33.15_suppl.e21008\u003c/li\u003e\n\u003cli\u003eSarma M, Mahmoud H, Abdelhamid T, El-Sharkawy N, ElNahas Y, El Gammal M, et al. The prognostic significance of minimal residual disease in adult Egyptian patients with precursor acute lymphoblastic leukemia. J Egypt Natl Cancer Inst. 2013;25:135\u0026ndash;42. https://doi.org/10.1016/j.jnci.2013.05.004\u003c/li\u003e\n\u003cli\u003eAdnan Awad S, Kamel MM, Ayoub MA, Kamel AM, Elnoshokaty EH, El Hifnawi N. Immunophenotypic characterization of genotype subgroups in Egyptian pediatric patients with B-cell acute lymphoblastic leukemia. Clin Lymphoma Myeloma Leukemia. 2016;16:519\u0026ndash;524. https://doi.org/10.1016/j.clml.2016.02.023\u003c/li\u003e\n\u003cli\u003eFarweez BAT, Kassim NA, Abdelfattah MF, Hassan NM, Hassan DEA, El-Sharkawy M. Clinical impact of early minimal residual disease detection at day 15 in precursor B-childhood acute lymphoblastic leukemia: an Egyptian experience. Egypt J Med Hum Genet. 2020;21(1):28. https://doi.org/10.1186/s43042-020-00065-5\u003c/li\u003e\n\u003cli\u003eBasu S, Pereira AF, Pineiro DH, Wang H, Valencia-Jimenez A, Siegfried BD, et al. Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber). Sci Rep. 2019;9(1):10374. https://doi.org/10.1038/s41598-019-47020-y\u003c/li\u003e\n\u003cli\u003eToft N, Birgens H, Abrahamsson J, Gri\u0026scaron;kevičius L, Hallb\u0026ouml;\u0026ouml;k H, Heyman M, et al. Results of NOPHO ALL2008 treatment for patients aged 1\u0026ndash;45 years with acute lymphoblastic leukemia. Leukemia. 2018;32(3):606\u0026ndash;15. https://doi.org/10.1038/leu.2017.265\u003c/li\u003e\n\u003cli\u003eHunger SP, Raetz EA. How I treat relapsed acute lymphoblastic leukemia in the pediatric population. Blood. 2023;16(16):1803\u0026ndash;12. https://doi.org/10.1182/blood.2019010043\u003c/li\u003e\n\u003cli\u003eCathpole D, Lai A, Guo D, Chen QR, Khan J. Gene expression in fresh state segregate patients with childhood acute lymphoblastic leukemia: an independent validation study identifies that endoglin is associated with patient outcome. Leuk Res. 2007;31(3):221\u0026ndash;7. https://doi.org/10.1016/j.leukres.2007.04.021\u003c/li\u003e\n\u003cli\u003eMcBride H, Aouacheria A, Widlak P, Viegas L, Ciech N, Kasner M, et al. The role of inhibition of apoptosis in acute leukemias and myelodysplastic syndrome. Front. 2019;12:192. https://doi.org/10.3389/fonc.2019.00192\u003c/li\u003e\n\u003cli\u003eFlohr C, Coustan-Smith E, Pei D, Ivamoto S, Song G, Cheng C, et al. Genes contributing to minimal residual disease in childhood acute lymphoblastic leukemia: prognostic significance of CASP8AP2. Blood. 2006;108(13):007\u0026ndash;15. https://doi.org/10.1182/blood-2006-01-0322\u003c/li\u003e\n\u003cli\u003eCoustan-Smith E, Pei D, Cheng C, Song G, Pui CH, et al. A set of genes that regulate cell proliferation predicts treatment outcome in childhood acute lymphoblastic leukemia. Blood. 2007;110(4):1271\u0026ndash;7. https://doi.org/10.1182/blood-2007-01-064878\u003c/li\u003e\n\u003cli\u003eJiao Y, Cui L, Gao C, Li W, Zhao X, Lu S, et al. CASP8AP2 is a promising diagnostic biomarker in pediatric acute lymphoblastic leukemia. Leukemia Res. 2013;26(3):167\u0026ndash;71. https://doi.org/10.1016/j.leukres.2011.05.023\u003c/li\u003e\n\u003cli\u003eJin JF, Wang CJ, Cui L, Liu FF, Wang KL, Li WJ, et al. Interaction of E2F3a and CASP8AP2 regulates histone expression and chemosensitivity of leukemic cells. J Pediatr Hematol Oncol. 2023;45(3):e339\u0026ndash;44. https://doi.org/10.1097/MPH.0000000000002558\u003c/li\u003e\n\u003cli\u003eJuarez-Velazquez MR, Salas-Labadia C, Reyes-Leon A, Navarrete-Meneses MP, Fuentes-Panana EM, Perez-Ver P. Genetic markers in the prognosis of childhood acute lymphoblastic leukemia. In: Mejia-Arangure JM, editor. Clinical epidemiology of acute lymphoblastic leukemia \u0026ndash; from the molecules to the clinic [Internet]. InTech; 2013 [cited 2022 Feb 5]. Available from: http://www.intechopen.com/books/clinical-epidemiology-of-acute-lymphoblastic-leukemia-from-the-molecules-to-the-clinic/genetic-markers-in-the-prognosis-of-childhood-acute-lymphoblastic-leukemia\u003c/li\u003e\n\u003cli\u003eLiu FF, Wang KL, Deng LP, Liu X, Wu M yuan, Wang TY, et al. Transcription factor E2F3a regulates CASP8AP2 transcription and enhances sensitivity to chemotherapeutic drugs in acute lymphoblastic leukemia. Cancer Cell Int. 2018;18(1):1\u0026ndash;9. https://doi.org/10.1186/s12935-018-0531-1\u003c/li\u003e\n\u003cli\u003eRemke M, Pfister S, Kox C, Toedt G, Becker N, Benner A, et al. High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-\u0026beta; and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response. Blood. 2009;114(5):1053\u0026ndash;62. https://doi.org/10.1182/blood-2008-10-186536\u003c/li\u003e\n\u003cli\u003eKang H, Chen IM, Wilson CS, Bedrick EJ, Harvey RC, Atlas SR, et al. Gene expression classifiers for relapse-free survival and minimal residual disease improve risk classification and outcome prediction in pediatric B-precursor acute lymphoblastic leukemia. Blood. 2010;115(7):1394\u0026ndash;405. https://doi.org/10.1182/blood-2009-05-218560\u003c/li\u003e\n\u003cli\u003eYang YL, Lin SR, Chen JS, Lin SW, Yu SL, Chen HY, et al. Expression and prognostic significance of the apoptotic genes BCL2L13, Livin, and CASP8AP2 in childhood acute lymphoblastic leukemia. Leuk Res. 2010;34(1):18\u0026ndash;23. https://doi.org/10.1016/j.leukres.2009.07.023\u003c/li\u003e\n\u003cli\u003eEhrhardt H, Wachter F, Maurer M, Stahnke K, Jeremias I. Important role of caspase-8 for chemosensitivity of ALL cells. Clin Cancer Res. 2011;17(24):7605\u0026ndash;13. https://doi.org/10.1158/1078-0432.CCR-11-0513\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 7 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Childhood acute lymphoblastic leukemia, CASP8AP2 gene, Apoptosis, Chemotherapeutic sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-7478773/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7478773/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eImproved treatment of childhood acute lymphoblastic leukemia (ALL) depends on the identification of new molecular markers that can predict treatment response and clinical outcome. Examination of the expression patterns of a set of genes at the RNA level is one of these new modalities. The prognostic significance of caspase-8-associated protein 2 (CASP8AP2), an apoptosis-related gene, in pediatric ALL is controversial.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA prospective study of 70 newly diagnosed ALL patients who were treated in the National Cancer Institute (NCI), Cairo University during the period from 1st of October 2019 till the end of September 2023, to measure the CASP8AP2 expression level in bone marrow samples at the time of diagnosis and at the end of induction therapy using real-time quantitative PCR, and to assess its relation with different prevalent prognostic variables and disease outcome. All cases were followed up till end of December 2024.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHigher initial CASP8AP2 gene expression was associated with hyperdiploid karyotyping (p\u0026thinsp;=\u0026thinsp;0.009), molecularly favorable mutations (p\u0026thinsp;=\u0026thinsp;0.002), early induction response (p\u0026thinsp;=\u0026thinsp;0.025), and low-risk patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and had a significant impact on lowering events in the first 3 years of follow-up (p\u0026thinsp;=\u0026thinsp;0.001). Meanwhile, examining post-induction gene levels failed to show similar results.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eHigher initial CASP8AP2 gene expression was associated with favorable impact on event-free survival in pediatric ALL patients. Post-induction levels did not show similar correlation. Future larger studies are needed to confirm the favorable association and to search for other possibly related prognostic factors to further refine risk stratification.\u003c/p\u003e","manuscriptTitle":"Prognostic Significance of Caspase 8 Associated Protein 2 (CASP8AP2) in Childhood B cell Acute Lymphoblastic Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 10:06:19","doi":"10.21203/rs.3.rs-7478773/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2025-11-14T04:23:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T14:25:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46308744860281172039122671531538877423","date":"2025-11-04T13:45:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T11:34:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245031717568689157925848982835284119183","date":"2025-11-01T11:54:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T11:38:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T13:53:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2025-10-22T09:28:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5df54748-2ed3-4617-8447-b498416756db","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-20T04:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 10:06:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7478773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7478773","identity":"rs-7478773","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}