Asparagine Synthetase Expression and L-Asparaginase Sensitivity in Aggressive Lymphomas
Authors: Berlier Willy , Aguera Karine , Chevrier Anne-Marie , Gallix Fanny , Traverse-Glehen Alexandra , Salles Gilles , Godfrin Yann 
Published: October 2019

Abstract
L-asparaginase (L-ASPA) displays a strong clinical benefit in the treatment of acute lymphoblastic leukemia (ALL), where it is included in most of current chemotherapy regimen. L-ASPA depletes plasmatic asparagine (ASN), an amino acid essential for the proliferation of leukemic cells. Since these cells are deficient in asparagine synthetase (ASNS), they rely on external (plasmatic) source of ASN and can be starved to death by L-ASP treatment.
Several studies evidenced the potential of ASN depletion to treat lymphomas. Indeed, many animal and human lymphoma cell lines have been shown to be sensitive to L-ASPA in vitro. In veterinary medicine, L-ASPA is routinely administered to treat effectively both feline and canine lymphomas (Wypig et al., 2013). L-ASPA regained attention in the treatment of human lymphomas since its adjunction in current chemotherapy regimens significantly improved the outcome of patients with NK/T cell lymphoma (Zou et al., 2014). Some studies also evidenced its benefit in combined chemo or monotherapy for the treatment of B-cell and T-cell lymphomas (Sun et al., 2006; Takahashi et al., 2010).
In this study, we assessed the in vitro sensitivity to L-ASPA of 6 lymphoma cell lines and we analyzed ASNS expression in biopsies from 166 cases of lymphomas (130 B-cell lymphomas and 17 T-cell lymphomas). Sensitivity to L-ASPA (expressed as an IC50) was assessed in vitro by measuring the cell viability in the presence of various concentrations of E.coliL-ASPA. ASNS expression in biopsies (TMA, USBiomax, Rockville, MD) was assessed with a validated immunohistochemistry (IHC) method attributing a score to each tumor based on ASNS labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion.
As shown in the following table, all cell lines were proved to be sensitive to L-ASPA. Their in vitrosensitivity exceeded cell lines MOLT-4 (ALL) and HL-60 (AML). Table 1Cell lineSensitivity to L-ASPA (IC50 in IU/mL)HuT-78 (Peripheral T-cell lymphoma,PTCL)0.11 ± 0.02Toledo (Diffuse large B-cell lymphoma, DLBCL)0.19 ± 0.03SU-DHL-8(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.04SU-DHL-10(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.01REC-1 (Mantle cell lymphoma, MCL)0.15 ± 0.03KHYG-1 (NK/T-cell lymphoma)0.16 ± 0.06MOLT-4 (acute lymphoid leukemia, ALL)0.19 ± 0.07HL-60 (acute myeloid leukemia, AML)0.23 ± 0.02
As shown in the following table, ASNS expression was null/low in 85% in the entire population of patients with B-cell lymphomas. Considering DLBCL, 63% of patients displayed no ASNS expression at all. ASNS expression was also null/low in 88% of patients with T-cell lymphomas (n=17). Table 2ASNS expression (IHC score)Type of lymphoma(% of cases)DLBCL (n=110)Others BCL (n=20)PTCL (n=3)Others TCL (n=14)MCL(n=3)Hodgkin (n=16)Negative (0)62,770,00,057,133,343,8Low positive (1)21,825,066,635,766,656,3Positive (2)7,35,033,37,10,00,0Highly positive (3)8,20,00,00,00,00,0
Globally, these results suggest that L-ASPA is potentially effective for the treatment of several lymphomas. Indeed, B-cell as well as T-cell lymphoma cell lines are sensitive to L-ASP in vitroand the majority of lymphoma tissues express no/low ASNS. Based on our results on ASNS expression in lymphoma biopsies, L-ASPA therapy may be beneficial for up to 85% of patients with DLBCL. Up to 90% of patients with other B-cell lymphomas or T-cell lymphomas may be sensitive to L-ASPA treatment as well.
However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, and liver failure) render its use hazardous, particularly in older or frail patients. Therefore, the development of a new formulation of L-ASPA with safer profile has to be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas.

Disclosures
Berlier: ERYTECH: Employment, Equity Ownership. Aguera:ERYTECH: Employment. Chevrier:ERYTECH: Employment. Gallix:ERYTECH: Employment. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.