by D. K. Cecconello, C. Rechenmacher, Prof. L. E. Daudt and M. B. Michalowski

Acute lymphoblastic leukemia is the cancer most commonly found in children and asparaginase (ASNase) is used in the treatment. Our laboratory has been working to establish a regular analysis of ASNase activity in Brazil. Monitoring ASNase activity can be an important tool to improve treatments in developing countries in order to avoid inferior prognosis.

Background

Acute lymphoblastic leukemia (ALL) is the cancer most commonly seen in children. Due to current chemotherapy regimens, long-term results have improved reaching event-free survival of 80% and overall survival rates close to 90%. One of the main drugs used in the treatment is asparaginase (ASNase). ASNase is a bacteria-derived enzyme that has an anti-leukemic function by catalysing the hydrolysis of the amino acid asparagine into aspartic acid and ammonia and is considered an essential component of therapies [1,2]. The rationale behind the use of ASNase as a therapeutic for ALL is that, unlike normal cells, ALL cells are unable to synthesize their own asparagine and so depend on circulating asparagine. The depletion of circulating asparagine by ASNase deprives the ALL cells of asparagine, leading to cell death.

Different formulations of ASNase

Three main ASNase formulations have been developed, one is derived from E. coli, another from Erwinia chrysanthemi (reassigned and now known as Dickeya dadantii), and a third, pegylated ASNase (PEG-ASNase), which is a conjugation of E. coli with polyethylene glycol that was created to reduce the immunogenic potential [3]. PEG-ASNase has a half-life of about 1 week, whereas native E. coli ASNase and Er. chrysanthemi ASNase have a half-life of 1.3 and 0.65 days, respectively [4]. ASNase generates adverse effects, including clinical hypersensitivity reactions, which occur as a result of the production of anti-ASNase antibodies. These antibodies may also cause rapid enzyme inactivation without clinical signs, referred to as silent inactivation. This situation may generate sub-therapeutic ASNase concentrations leading to a greater chance of relapse. For this reason, monitoring its activity levels was one of the tools that contributed to improving treatment outcomes [5,6,7]. This is the most sensitive and reproducible method with a relationship with asparagine depletion and clinical consequences, currently used regularly in high-income countries.

Analysis of ASNase activity

The analysis is based on a technique that uses aspartic acid B-hydroxamate (AHA) as the substrate for the quantification of ASNase derived from E. coli, Er. chrysanthemi and PEG-ASNase in human plasma and determines the effective activity of ASNase when this is ≥0.1 IU/mL at the time defined according to the formulation used (E. coli ASNase and Er. chrysanthemi ASNase 24 and 38 hours; PEG-ASNase 7 and 14 days after infusion) [7,8].

Acute lymphocytic leukemia cells in blood smear, ×1000 (Shutterstock.com)

Influence of ASNase formulations on levels of ASNase activity

Since 2018, our laboratory has been working to establish a regular analysis of ASNase activity in children being treated in Brazil. In 2015, the Brazilian Sistema Único de Saúde (SUS, the Brazilian Health System) provided ASNase for all oncological centres in the country. Aginasa® (Medac) was distributed until early 2017, and replaced afterward by Leuginase® (Beijing SL Pharmaceutical). This change happened when we were establishing a monitoring programme in our institution, implementing the laboratory test to measure ASNase activity to improve supportive care in patients with ALL [9].

As a result of this, in 2017 we analysed the activity of samples from children that were exposed to two different formulations of E. coli ASNase. At that time, we observed that most patients treated with Leuginase did not achieve adequate levels of enzymatic activity. In that analysis, 81% of patients who received Aginasa demonstrated an asparaginase activity greater than or equal to 0.1 IU/mL, whereas only 3% of patients who received Leuginase reached such levels. Our results were validated by international and national centres. These findings outlined a worrying scenario, as patients could be at risk of an inferior outcome. This study was able to implement an ASNase activity test to monitor the treatment of patients with ALL, allowing for possible adjustments and help policy makers to establish appropriate strategies to provide efficient treatment for patients [9].

At that time, the SUS began to provide a PEG-ASNase formulation for all children. After that, we conducted a multicentre clinical study with historical control in order to assess the impact of these changes in treatment on the prognosis of children with ALL at 10 different pediatric oncology centres in our country. With this analysis (which included over 400 children), we were able to demonstrate that children who received Leuginase had statistically lower event-free survival (EFS) and overall survival (OS) than children treated with Aginasa. Over the course of 3 years, the group treated with Aginasa had an OS of 91.8% and EFS of 84.8%, whereas the Leuginase-treated group had a 3-year OS of 83.8% (P=0.003) and EFS of 76.1% (P=0.008). The results obtained in our study demonstrated that the use of an ASNase formulation which, in previous analyses, had been shown not to have an adequate level of activity, had an impact on 3-year EFS of children treated for ALL in our country [10].

After the implementation and standardization of the technique in our laboratory, we started to receive samples from patients with suspected allergy/inactivation from other centres in Brazil. Thus, it is possible to define which patients who had had allergic reactions can undergo a reintroduction protocol with premedication or must change their formulation to Er. chrysanthemi ASNase or, even, discontinue therapy. It was possible to demonstrate how a simple technique can be efficiently incorporated into the treatment of ALL, improving the care of patients. We were also able to confirm the feasibility of desensitization protocols in patients who had clinical allergy but no drug inactivation [11].

Further considerations

Now that we understand the importance of this methodology, it is essential to create new centres capable of carrying it out. In 2020, we started a laboratory training programme, with the aim of teaching the ASNase activity technique to different laboratories. Our goal was to spread the technique in order to implement a reliable method of monitoring the treatment of patients with ALL in low–middle-income countries while at the same time bringing autonomy to the sites. More than 23 professionals were trained in six different centres in Brazil and one centre in Colombia. Many studies have been published showing the importance of such monitoring [12,13]. Some of them reinforced the importance, especially in developing countries. Sankaran et al. compared nine generic formulations of ASNase available in India with the reference medication used. They concluded that some generic formulations had inferior activity, which raises serious concerns about their quality and impact on final treatment results [14]. A study by Sidhu et al. showed that none of the seven biogeneric formulations of native ASNase marketed in India met the minimum manufacturing quality standards required for these products. In this context, regulatory oversight and periodic monitoring of all formulations are essential to ensure the quality of care provided to all patients, especially in countries that seek lower-cost drugs in the international market [15].

Summary

In summary, monitoring ASNase activity on a regular basis can be an important tool to improve treatments in developing countries in order to avoid inferior prognosis. The ability to individualize the ASNase therapy in patients, adjusting the dose, or switching patients with silent inactivation to an alternative ASNase preparation, may help improve the outcomes in those patients. Within this context, the commitment to the formation of networks that allow the training of new centres and access to the technique is essential
for ensuring high-quality care of patients with ALL.

The authors

Daiane Keller Cecconello1,2,3, Ciliana Rechenmacher1,2,3 PhD, Liane Esteves Daudt1,2,3 MD PhD and Mariana Bohns Michalowski*1,2,3 MD PhD
1 Post Graduate Program in Child and Adolescent Health, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
2 Translational Pediatrics Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
3 Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

* Corresponding author
E-mail: mmichalowski@hcpa.edu.br

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