In recent years, a higher survival rate has been achieved in children with Acute Lymphoblastic Leukemia (ALL) due to scientific advances and the impact of new chemotherapy protocols (1). In children under 15 years of age, the 5-year Overall survival (OS) rate has increased from 60% in 1975 to 90% in 2005 (1, 2).

Initial treatment of patients with newly diagnosed ALL is based on a combination of chemotherapies. The intensity of chemotherapy is based on the group risk assessment. Traditionally, risk stratification has been based on clinical factors such as age and sex, white blood cell count at diagnosis, and immunophenotypic, cytogenetic, and molecular characteristics. The objective was to select the least possible toxic treatment according to the possibility of survival (2, 3).

In addition to the risk group, the percentage of minimal residual disease (MRD) at the end of induction is related to the long-term outcome (4). The treatment response is strongly influenced by the sensitivity or resistance of leukemic cells to drugs, as well as by the pharmacodynamics and pharmacogenomics of each patient; the early response has great prognostic importance (5–7).

The MRD results at the end of induction therapy or consolidation can change the prognosis assigned by the risk classification at the beginning of the treatment and is a tool used to define post-induction therapy (8). The majority of bone marrow MRD-based survival analyses have employed the value at the end of induction or consolidation therapy (9, 10). The impact of the treatment response during the initial days of induction therapy has not been widely studied (11).

Different types of chemotherapy are known to induce cellular stress through different mechanisms, resulting in leukemic cell apoptosis (12, 13). Resistance to cell death or apoptosis is one of the factors involved in cancer development and constitutes one of the factors in chemotherapy treatment to which a patient does not respond adequately (14).

Transcription factor NF-κB is involved in diverse regulatory functions of the response to cellular stress, proliferation, differentiation, apoptosis, and tumorigenesis (15). This transcription factor is constitutively activated in different types of cancer, including ALL, and has been associated with malignant cell survival and resistance to chemotherapy (16).

Pentoxifylline (PTX) can inhibit the phosphorylation of the NF-kB inhibitor (IkBa); this phosphorylation releases NF-kB for its subsequent translocation to the nucleus (17). Our group and others have proven that PTX possesses anti-tumor properties in vivo and in vitro (18–20). Even in children with ALL, PTX increases apoptosis, during the steroid prophase, by modifying the gene expression related to cell death and apoptosis (21, 22).

Acute lymphoblastic leukemia (ALL) is the most frequent neoplasm in childhood and cure rates of over 90% have been achieved in developed countries; despite therapeutic advances in many countries, this survival rate has not yet been achieved in all cases (23). Efforts are needed to reduce treatment-related toxicity, improve early detection, and prevent treatment abandonment, among other aspects. Targeted therapy with novel drugs could be a strategy to increase treatment success in areas where the cure of patients with ALL remains low.

In this study, we evaluated the addition of PTX to standard ALL treatment during induction therapy to achieve earlier remission and investigated its direct effect on tumor cell apoptosis. Published research has shown that PTX inhibits the phosphorylation of the NF-kB inhibitor: IKB-alpha (17, 18). NF-kB is widely recognized for its role in cell survival, proliferation, and the modulation of gene expression associated with apoptotic pathways (22).

The results of this study demonstrated that the administration of PTX during induction therapy significantly increased the percentage of apoptosis in BMMC (see Figure 2 ). These findings are consistent with those of a previous study conducted by our group in children with ALL during the steroid window (21). However, the latter study only investigated the effect of PTX on apoptosis after the first 7 days of treatment with steroids (day 8), prior to the start of intravenous chemotherapy. In contrast, in the present study, PTX was added during throughout the entire induction therapy to one group compared with another group that did not receive PTX; this allowed a more extended evaluation of the effect of PTX on tumor cell apoptosis and its potential benefit as an adjunct therapy in the treatment of ALL. We found that apoptosis continued to increase significantly on days 8 and 15; nonetheless, no differences were observed on day 22. An explanation is that on day 22 (after 2 weeks of induction therapy with four drugs), the majority of patients reached remission, as observed in the study by Pui et al., in which only 92 of 478 patients persisted with an MRD of >0.1% at day 19; these results are relevant because it is known that the earlier remission is achieved, the better the prognosis (24).

Although multiple studies have demonstrated the effectiveness of PTX in inducing apoptosis in tumor cells, it is noteworthy that these studies were conducted in vitro and reported a higher percentage of cells undergoing apoptosis than those observed in our patients (25). It is crucial to consider that clinical trials take place in a less controlled microenvironment and involve various factors, such as the mononuclear phagocytic system, which continuously eliminates apoptotic bodies (26), as well as the daily production of hematopoietic cells (27).

One discovery regarding senescence is that we observed in our study a consistently low level of senescent cells throughout the induction phase. This finding can be attributed to the nature of leukemia, a type of cancer characterized by rapid cell proliferation, and it is found in premalignant lesions or slow-replicating neoplasms where higher rates of senescence have been reported (28).

There is evidence suggesting that therapy-induced senescent cells may acquire a pro-tumor effect by escaping senescent arrest and re-entering a proliferative state and, also because the senescent cells release a secretome that may contain factors with pro-tumorigenic properties (29, 30). Those phenomena raise concerns about the long-term efficacy of treatment. A significant observation of this study is that PTX did not induce senescence: we observed consistently low levels of senescent cells throughout the induction therapy in our study. This finding can be attributed to the nature of leukemia, a type of cancer characterized by rapid cell proliferation, and it is found in premalignant lesions or slow-replicating neoplasms where higher rates of senescence have been reported (28).

In the context of ALL, MRD represents the most significant prognostic indicator. Typically, the analysis of treatment response using MRD is conducted at the end of induction therapy, as outlined in different chemotherapy protocols (7, 9, 10, 24). However, in our study, we opted to assess bone marrow MRD at an earlier stage, specifically after the first week of steroid treatment and following the initial dose of intravenous chemotherapy, corresponding to days 8 and 15. Only a few studies have explored the correlation between achieving very early remission at these specific time points and its impact on treatment outcomes, as described in the following studies (4, 10, 31).

The Children’s Oncology Group, as evidenced in a study conducted by Borowitz et al., revealed that patients with a peripheral-blood MRD level exceeding 1% on day 8 exhibited 5-year Event-Free survival (EFS) of 79%, whereas those with lower MRD levels achieved a 5-year EFS of 90%, even if they eventually tested negative for MRD by day 29 (4).

Similar to our findings, a study conducted by Loosveld et al. also demonstrated a more favorable prognosis in patients who tested peripheral-blood MRD-negative on day 15 (post diagnosis). This assessment was performed after the steroid prophase and following 1 week of chemotherapy. The study reported a significantly improved 4-year Disease-Free Survival (DFS) rate of 91.6% compared to 67.6% in patients who remained MRD-positive (p = 0.0013) (31).

Nevertheless, unlike our study, previous studies employed peripheral blood and not bone marrow to analyze treatment response. We employed bone marrow because we sought to increase sensitivity (32). As in our clinical trial, Basso et al. utilized bone marrow to perform MRD; these authors found that flow-cytometry bone-marrow MRD at day 15 was the most important prognostic factor, and they classified patients according to three risk groups based on the level of MRD at day 15: standard (<0.1%); intermediate (0.1 to <10%), and high (>10%); their 5-year cumulative relapse incidences were 7.5%, 17.5%, and 47.2%, respectively (11).

Our findings align with the previously reported results by our group, indicating that better survival outcomes were observed when patients achieved an MRD level of less than 0.01% on days 8 and 15 (post-diagnosis). Notably, this effect was particularly pronounced in patients who received PTX, as they exhibited significantly lower MRD levels on both days 8 and 15 compared to the group that did not receive the drug (see Table 3 ).

The cohort in our study exhibited a 3-year OS of 71.5%, similar to the 5-year OS reported in Mexico, ranging from 43.7%-74.7% according to risk, and with an average 5-year OS of 61.8% (33). Moreover, it is also similar to the survival rates reported in Latin-American countries, where 5-year net survival was still less than 70%, even after adjusting for very high background mortality in childhood: with in Brazil, Chile, Colombia, and Peru, and less than 60% in Mexico and Ecuador (23).

However, our results are considerably lower than the OS rate reported by St. Jude Children’s Research Hospital, which reported a 5-year OS of 93.5% using the same treatment protocol (34). It is essential to consider that Mexico belongs to middle-income countries, and our circumstances here are significantly different. Other countries in the same economic category as Mexico, which also implemented the same chemotherapy regimen, have reported low survival rates. For instance, a study by Chona de Armas et al. reported a 2-year OS of 57% and an EFS of 18.8% (35).

The overall conditions of our patients and their families, the nutritional status of the patients, and the limited availability of healthcare services in various areas contribute to increased morbidity and mortality from chemotherapy toxicity (36). Adjusting the treatment approach for children demonstrating a very early response could aid in reducing treatment-related toxicities. Indeed, tailoring treatment based on the patient’s response forms the foundation of the most innovative protocols (37).

In this clinical trial, most patients were classified in the high-risk group compared to other international publications (38, 40). We think that this difference is due to a conditioning factor in the selection of patients for the study; it was established as an inclusion criterion that patients have had a minimal weight of 20 kg and swallowing capacity since there was no presentation of suspension of the PTX, and the tablets could not be split or crushed due to pharmacological considerations. The former led to the inclusion of older patients, which increased the proportion of high-risk patients.

During the clinical trial, no patients experienced any adverse effects related to PTX. This finding leads us to consider PTX as a safe drug for administration in pediatric patients, which is consistent with results reported in other pediatric studies (39, 40).

However, it is essential to acknowledge that further research and larger-scale studies are necessary to validate these findings and to ensure the long-term safety of PTX administration in pediatric patients.

Chemotherapy exerts its primary action by causing irreparable cell damage through various pathways, predominantly inducing apoptosis as a mechanism of cell death. However, novel approaches in both in-vitro and in-vivo settings aim to enhance the effectiveness of chemotherapy, induce cell death, or halt proliferation using non-cytotoxic drugs to reduce the tumor burden before the emergence of resistance.

Increasing apoptosis while minimizing adverse effects holds significant potential for improving the survival outcomes of children with cancer. In this study, patients receiving a combination of PTX and chemotherapy achieved an MRD of <0.01% at an earlier stage. Early remission is known to be associated with better survival and serves as an independent prognostic factor. The favorable therapeutic response observed may be attributed to the substantial increase of apoptotic cells induced by PTX.

Based on the findings of the current clinical trial and in line with previous studies conducted in pediatric populations, PTX appears to be a safe therapeutic option for pediatric patients. The absence of reported adverse effects during this trial offers valuable insights and encourages further exploration of the potential benefits and safety of PTX in pediatric populations.