L-Asparaginase Ex Vivo Activity Distorts Plasmatic Asparagine Depletion Assessment
Authors: Berlier Willy , Godfrin Yann 
Published: October 2019

Abstract
L-asparaginase (L-ASP) is a well-established and important agent having demonstrated significant clinical efficacy in the treatment of acute lymphoblastic leukemia (ALL) Many cancerous cells are deficient in asparagine synthetase (ASNS), requiring endogenous plasma asparagine (ASN) for proliferation. Without this essential amino acid, the malignant cells undergo apoptosis.
Plasma levels of ASN have been considered a relevant surrogate biological parameter to assess L-ASP efficacy. The aim is to maintain ASN levels below a threshold varying from 0.1 to 3µM. However, the measurement of ASN in presence of L-ASP is problematic, considering that 1 International Unit (IU) of L-ASP is able to cleave 1µmol of ASN per minute at 37°C. As the normal value of plasma ASN is around 50µM in humans, it requires about 30 seconds for 100 IU/L of L-ASP in the blood stream to fully deplete L-ASN, also considering there is no de novo ASN production., Plasma ASN is provided by normal cells expressing ASNS and by food intake. Once blood is drawn, no endogenous source of ASN is present and in the presence of active L-ASP a rapid ASN ex vivodepletion is expected to occur in the sampling tube.
L-ASP activity can be reduced by low temperature (iced water) and/or by lowering the pH through addition of sulfosalicylic acid (Pieters et al, Blood, 2008). It is recommended to cool down the sample for 15 minutes on iced water immediately after the patient’s blood is drawn, and to centrifuge at 1000g for 15 minutes at +4°C to recover the plasma. A solution of 10% sulfosalicylic acid is added to the plasma 1:4 (v/v) before centrifugation at 3000g for 5 minutes to extract the proteins, including L-ASP. Here we report our investigation on the impact of the duration between blood draw and immersion in “iced water” and the impact of the sulfosalicylic acid concentration when “free” E. coliL-ASP (L-ASP) or L-ASP encapsulated in erythrocytes (L-ASP/RBC) is present.
L-ASP or L-ASP/RBC was added to human blood, in concentrations comparable to patients receiving L-ASP treatment (0-200 IU/L). The sampling process used in clinical practice was strictly applied to the sample, except that different times (5 sec to 30 min) at room temperature (RT) between the addition of L-ASP (considered as the moment of the blood draw) and the step of cooling were tested. Two quantities (100µL and 500µL) of sulfosalicylic acid were also tested. ASN was quantified in the samples after the completion of the whole process to assess whether variations in these parameters could impact the measurement.
When 20 IU/L of L-ASP was present in the sample, ASN was depleted by 75% after a 5-sec delay at RT, and below the limit of quantification (BLLQ=2µM) after 10 min. When reducing sulfosalicylic acid to a suboptimal quantity (100µL), complete depletion (BLLQ) of ASN was obtained after 5 sec at RT with 20 IU/L of L-ASP. These results confirmed the necessity of the acidic deproteinization of the samples but also revealed a significant ex vivo activity of L-ASP leading to overestimation of ASN depletion in plasma. Considering L-ASP/RBC, 25% ASN depletion was observed at 200IU/L after 3 min. at RT, and ex vivocomplete depletion was reached with 2000IU/L and a 30-minute delay at RT.
In conclusion, considering current methods, due to inability to control ex vivo L-ASP metabolism, it is practically impossible to have a measurement of plasmatic ASN which reflects the in vivo reality. Indeed, in the presence of low concentrations of free L-ASP, a very rapid depletion of ASN is observed ex vivo. In the time needed to cool down and centrifuge samples further “artificial” depletion occurs rapidly and the patient can be erroneously considered ASN depleted. However, the comparison of the phamacodynamics between “free” asparaginases may be still considered valuable because the error is probably equal in both sides. On the contrary, “artificial” depletion is much less when the L-ASP is encapsulated in RBCs (thanks to the delay of asparagine to pass through the RBC membrane), leading to a potential bias to compare L-ASP or L-ASP/RBC. Finally, L-ASP activity is believed to be a more relevant marker than the measurement of ASN depletion. This is consistent with the approach taken by regulatory agencies in recent years, favoring the duration of L-ASP activity over 100IU/L as endpoint over depletion endpoints.

Disclosures
Berlier: ERYTECH: Employment, Equity Ownership. Godfrin:ERYTECH Pharma: Employment, Equity Ownership.