Enhancing Effect on Tumour Apoptosis With the Use of Pentoxifylline in Patients With Hodgkin Lymphoma

Hodgkin's Lymphoma (HL) is a neoplasm that affects the lymph nodes and the lymphatic system. In Mexico, HL is the seventh most incident cancer and the ninth with the highest mortality. It is characterized by the presence of Reed-Sternberg (HRS) cells derived from B cells of the germinal center. They harbor mutations that activate the NF-κB pathway, favoring cell survival and their reprogramming. Currently, the available therapeutic options are chemotherapy and radiotherapy, achieving cure rates of 75% in patients in advanced stages, in which 70% of these are found at the time of diagnosis. The investigators proposed the use of pentoxifylline (PTX) as a therapeutic option to enhance the antitumor effect generated by the treatment since it can increase the efficacy of apoptosis, in vitro and in vivo, induced by doxorubicin, cisplatin, and adriamycin in human leukemic and cervical cancer cells, through inhibition of NF-κB by preventing phosphorylation of serine 32 of the inhibitor κB; it also decreases the expression of Bcl-2 and Bcl-XL, induces the releasement of cytochrome c and caspases 3, 9, and cleavage of caspase 8. The investigators evaluated the effects of PTX during the steroid window phase at induction to remission in pediatric patients with LLA of a recent diagnosis, where it was shown that the combined treatment of prednisone (PRD) with PTX achieves greater percentages of apoptosis compared to individual treatment. In addition, the effect of PTX on the expression of genes associated with apoptosis was evaluated; where it was shown that it activates the intrinsic and extrinsic pathways of apoptosis. Fortilin is a protein whose serum levels increase 2.4 times more after treatment with chemotherapy or radiotherapy in patients with malignancies, so it is considered a specific and sensitive biomarker of early apoptosis in vivo. The present protocol will evaluate the enhancing effect of PTX on tumor apoptosis in combination with chemotherapeutical agents in pediatric and AYA patients with HL. Apoptosis will be measured in vivo by quantifying serum levels of fortilin and cytochrome c in participants before and after treatment by ELISA; as well as an evaluation of the clinical response based on the results of the PET-Scan, overall and event-free survival according to the Kaplan-Meier curves, and the adverse effects associated with the use of PTX according to the common terminology criteria for adverse events and causality algorithms.

Hodgkin Lymphoma (HL) is a B-cell-derived neoplasm that involves the lymph nodes and lymphatic system. In Mexico, HL is seventh cancer with the highest incident rates and the ninth with the highest mortality.

The incidence of this pathology has a bimodal distribution, with the first peak of appearance among adolescents and young adults within an age range that goes from 15 to 35 years, followed by a second peak in adults 55 years of age and older.

This neoplasm is characterized by the presence of Reed-Sternberg cells (HRS), which are distinguished by being large with multinucleate or bilobed nuclei and being derived from B lymphocytes of the germinal center. They have a partial loss of the B phenotype and classical lineage markers. They harbor mutations that activate the NF-κB pathway, which regulates antiapoptotic factors, the expression of proinflammatory cytokines, and the reprogramming of B cells.

Patients with HL frequently present asymptomatic, painless, and slowly progressive lymphadenopathy. There may also be systemic symptoms such as B symptoms which are defined by the presence of deep night sweats, unexplained weight loss of \>10% of total body weight within the previous 6 months at diagnosis, and persistent or recurrent fever ≥38˚C.

Excisional biopsy of potentially involved lymph nodes is the gold standard for establishing the diagnosis of HL. A histopathological study is performed, in which the presence of diagnostic HRS cells must be found in an adequate microenvironment and the expression of CD30 and CD15.

Treatment is implemented based on the stage of the disease, with limited stages being an initial phase with chemotherapy followed by a consolidation phase with or without targeted radiotherapy. In limited favorable stages, it is recommended to apply 2 to 3 cycles of chemotherapy or 4 cycles for those with limited unfavorable stages. The scheme with the best cure rates is ABVD (adriamycin or doxorubicin, bleomycin, vinblastine, and dacarbazine). For advanced disease, the application of 6 cycles of combined chemotherapy using the BEACOPP scheme (bleomycin, etoposide, doxorubicin or adriamycin, cyclophosphamide, vincristine, procarbazine, and prednisone).

Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor belonging to the methylxanthine family. It has been reported that PTX can increase the efficacy of certain antitumor drugs through the inhibition of NF-κB. This can prevent the phosphorylation of serine 32 of the inhibitor of NF-κB (I-κB), thus preventing the activity of NF-κB and being able to activate certain proapoptotic genes. In addition, PTX is capable of sensitizing multidrug-resistant cells by down-regulating P-glycoprotein.

The efficacy of PTX has been reported, both in vitro and in vivo, in increasing apoptosis induced by some chemotherapeutic drugs such as doxorubicin, cisplatin, and adriamycin, both in humans leukemia cells and in cervical cancer cells and an increase in survival in murine models of lymphoma. It has also been shown that PTX decreases the expression of Bcl-2 and Bcl-XL. They also induce the release of cytochrome C and caspases 3, 9 and cleavage of caspase 8, resulting in increased apoptosis in the human leukemia cell line U937.

PTX also manages to significantly inhibit cellular senescence, a state that is induced by chemotherapy, which, although characterized by the non-replication of cells, continues to be alive, facilitating tumor growth.

Fortilin is a 172-amino acid protein that can be found in the cytosol, nucleus, extracellular space, mitochondria, and peripheral blood. In 2010, I. Sirois et al. identified the presence of fortilin as the most abundant protein in nanovesicles secreted by apoptotic cells. In addition, it was possible to demonstrate one of the extracellular functions of fortilin in the context of apoptosis, since these nanovesicles secreted by apoptotic cells were able to induce an antiapoptotic phenotype regardless of the cell type in question.

In a study conducted by Sinthujaroen et al., the potential use of serum fortilin as a peripheral biomarker associated with apoptosis was evaluated. Fortilin was shown to be present in the peripheral blood of healthy participants and patients with solid malignant tumors. In those patients without malignancies, the mean values were 75.57 ± 45.79 ng/mL with no significant difference between sex or age. Likewise, serum levels of this were measured in cancer patients before and after the administration of chemotherapy drugs or radiotherapy, and a 2.4-fold increase in their serum levels was observed after treatment.

Serum fortilin was considered a unique biomarker of apoptosis in vivo, thus, it is correlated that high serum levels are associated with greater tumour apoptosis.

Within the background that gave rise to this study, a pilot study was carried out where the effects of the use of PTX during the steroid window on the induction of remission in pediatric patients with a recent diagnosis of acute lymphocytic leukemia (ALL) were evaluated. For this study, patients were classified into 3 groups: Group 1 treated with prednisone (PRD) alone, Group 2 treated with a combination of PRD/PTX, and Group 3 with healthy control.

Significant differences were observed in the rate of apoptosis after treatment between the groups treated only with PRD and those treated together with PTX, with a higher percentage observed in group 2 treated with PTX (PTX 16.5±6.04% vs. PRD 9.2± 3.1; p=\<0.001). In addition, it was shown that the combined treatment of PRD with PTX gives higher percentages of apoptosis in leukemic cells compared to individual treatment with PRD, showing that PTX can increase glucocorticoid-induced cell death in pediatric patients with ALL.

In this same group of patients, the effect of PTX on the expression of genes associated with apoptosis was evaluated. A significant difference between the number of genes that are regulated, both up and down, when PTX is added compared to treatment with PRD alone was evidenced.

Among the genes that were modified in their expression by the PTX/PRD treatment, were those associated with FOXO3A, TNF receptors, DISC-associated genes such as FADD, and caspase-8 and -10 genes. The genes found to be downregulated were associated with BCL-2, the NF-κB pathway, and CDKN2A. However, there was also downregulation of proapoptotic genes (JUN, LTA, AKT, TRAF3, and PMAIP1) and upregulation of some antiapoptotic (BRIC3 and CFLAR).

Cancer is one of the leading causes of morbidity and mortality in children and adolescents around the world. In Mexico, is considered one of the biggest public health problems in pediatric patients since it represents the first cause of death in this age group.

HL comprises 6% of all childhood cancers. Although cure rates of up to 90-95% are currently reported in early stages, only 30% of patients are found in these stages at the time of diagnosis, leaving a large percentage of patients in advanced stages at diagnosis. For this latter group, currently available treatments have only achieved cure rates of approximately 75% of cases.

In addition, the therapeutic options are based on chemotherapeutic drugs and radiotherapy, which have repercussions on patients both in short and long term. Among the main associated adverse effects are cardiomyopathies, coronary heart disease, pulmonary toxicity, and the development of secondary neoplasms, both hematological and solid tumors; these constitute one of the main causes of mortality in long-term survivors.

Based on the antitumor effects of PTX and part of the pathophysiology of HL, the administration of PTX in combination with the drugs used in the standard chemotherapy scheme for the treatment of HL is proposed as a pharmacological strategy to increase the rate of apoptosis in lymphoma cells during the treatment of pediatric and adolescent/young adult patients with newly diagnosed HL.

This way, it is expected that the increase in cell death will have a positive impact on the clinical response and both global and event-free survival of patients, in addition, to giving rise to future research where this drug is used in conjunction with current treatments for different hematological neoplasms such as non-Hodgkin lymphoma.

The investigators hypothesize that pentoxifylline, when used in conjunction with chemotherapeutic agents, manages to enhance the effect that the latter has in inducing tumor cell apoptosis in vitro and in patients with Hodgkin lymphoma, which leads to an improvement in survival, and their clinical response.

The aim of the study is to evaluate the potentiating effect on cell apoptosis generated by the combined use of pentoxifylline with chemotherapeutic agents in patients with Hodgkin lymphoma during pharmacological treatment.

The universe of study will be pediatric, adolescent, and young adults patients with a recent diagnosis of Hodgkin lymphoma without prior treatment, from Pediatric Oncology and Hematology Service of the Hospital Civil de Guadalajara Dr. Juan I. Menchaca, and the Hospital Civil de Guadalajara Fray Antonio Alcalde in the Adult Hematology Service.

The sample size was evaluated based on the sample size formula for a given proportion, considering the proportion obtained in the protocol "Very early remission and increased apoptosis with the use of pentoxifylline in children with lymphoblastic leukemia acute" (Salceda Rivera et al., 2020).

To achieve statistically significant results a sample size of 30 patients was obtained, considering a proportion of 99%. Patients will be classified into two study groups: Group A: Patients with conventional treatment based on the OEPA/COPDAC, ABVD, or BEACOPP chemotherapy scheme plus placebo, during the first two cycles of chemotherapy. Group B: patients with conventional treatment based on the OEPA/COPDAC, ABVD, or BEACOPP chemotherapy scheme plus pentoxifylline, during the first two cycles of chemotherapy.

The dose of pentoxifylline is 20 mg/kg/day (maximum dose of 1200 mg), which will be indicated orally, without chewing or crushing, preferably accompanied by food. It will be administered one hour before the rest of the drugs that are part of the respective chemotherapy regimen.

Three peripheral venous blood samples will be obtained from the patients before the start of treatment (day 0), at the end of the first cycle of chemotherapy (day 30), and at the end of the second cycle (day 60). Plasma levels of indirect biomarkers of apoptosis (fortilin and cytochrome c) will be determined by ELISA; the results will be represented as the mean ± standard deviation in pg/mL.

The clinical response will be evaluated comparatively based on the physical examination and the data provided by the clinical file. Likewise, a positron emission tomography (PET) or Computed Axial Tomography will be performed at the time of diagnosis (day 0) and the end of the second cycle of chemotherapy (day 60). Event-free survival will be assessed using the Kaplan-Meier log-rank test.

Finally, the adverse effects will be determined according to the Common Terminology Criteria for Adverse Events. In addition, it will be determined if the presence of an adverse effect is correlated to the use of pentoxifylline using Karch \& Lasagna, Naranjo, and FDA causality algorithms for adverse drug reactions.

The data will be represented as the mean ± the standard deviation of the values obtained. The data obtained will be analyzed by inferential statistics using the Mann-Whitney U test for parametric data. For qualitative variables, contingency tables will be used, to use squared Xi, in addition to multiple linear regression since multi variables will be used. The kappa (k) statistical test will be used to measure the strength of agreement between the causality algorithms. A statistically significant difference between the data will be considered when the p-value is less than 0.05.

Eligibility Criteria:
Inclusion Criteria:

* Pediatric and AYA (adolescents and young adults) patients (age up to 35 years of either sex) newly diagnosed with Hodgkin lymphoma regardless of clinical stage.
* Patients with the ability to swallow tablets.
* Patients who agree to enter the protocol by signing the informed consent personally or by the parent/guardian

Exclusion Criteria:

* Patients previously treated with chemotherapy, corticoids, and/or radiotherapy
* History of active acid peptic disease or gastrointestinal bleeding Intolerance to pentoxifylline and in general to xanthines
* Patients under treatment with anticoagulants, cimetidine, ciprofloxacin or theophylline
* Patients with severe bleeding, retinal hemorrhage or bleeding diathesis
* Serious cardiac arrhythmias (E.g. paroxysmal supraventricular tachycardia, congenital AV block, arrhythmias associated with congenital heart disease, digitalis poisoning, postoperative cardiac surgery, hypoxia, hypercapnia, electrolyte disturbances)
* Patients with hypotension
* Severe liver failure
* Moderate to severe renal insufficiency (with a glomerular filtration rate ≤ 30 mL/min)
* Patients admitted to the Intensive Care Unit at diagnosis
* Patients with treatment adherence of less than 80%
* Patients who wish to withdraw from the study or withdraw informed consent
* Patients who present grade III adverse events related to the drug under study
* Patients who become pregnant during the study