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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.coli L-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 vitro sensitivity exceeded cell lines MOLT-4 (ALL) and HL-60 (AML). | Cell line | Sensitivity to L-ASPA (IC50 in IU/mL) | | ----------------------------------------------- | --------------------------------------------- | | HuT-78 (Peripheral T-cell lymphoma,PTCL) | 0.11 ± 0.02 | | Toledo (Diffuse large B-cell lymphoma, DLBCL) | 0.19 ± 0.03 | | SU-DHL-8(Diffuse large B-cell lymphoma, DLBCL) | 0.10 ± 0.04 | | SU-DHL-10(Diffuse large B-cell lymphoma, DLBCL) | 0.10 ± 0.01 | | REC-1 (Mantle cell lymphoma, MCL) | 0.15 ± 0.03 | | KHYG-1 (NK/T-cell lymphoma) | 0.16 ± 0.06 | | MOLT-4 (acute lymphoid leukemia, ALL) | 0.19 ± 0.07 | | HL-60 (acute myeloid leukemia, AML) | 0.23 ± 0.02 | Table 1 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). | ASNS 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) | 62,7 | 70,0 | 0,0 | 57,1 | 33,3 | 43,8 | | Low positive (1) | 21,8 | 25,0 | 66,6 | 35,7 | 66,6 | 56,3 | | Positive (2) | 7,3 | 5,0 | 33,3 | 7,1 | 0,0 | 0,0 | | Highly positive (3) | 8,2 | 0,0 | 0,0 | 0,0 | 0,0 | 0,0 | Table 2 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 vitro and 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.

Introduction The rational of use of L-asparaginase (L-Aspa) is based on asparagine synthetase (ASNS) deficiency in leukemic cells. Its efficacy is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. It is a mainstay in the treatment of acute lymphoid leukemia (ALL), where it is established that resistance to treatment is in part related to detectable expression of ASNS (Aslanian et al., 2001; Su et al., 2008). In acute myeloid leukemia (AML), promising results have been obtained in clinical trials (Capizzi et al., 1988), with an improvement of complete remission rates from 18% to 54% in refractory patients 60 year old. More recently, it has been reported that leukemic cells from AML patients with M1, M4 and M5 FAB subtypes were more sensitive to L-Aspa (Okada et al., 2003).The aim of this study was to investigate the sensitivity to L-Aspa and the ASNS expression in an AML cell line (HL-60) and in primary leukemic cells from newly diagnosed AML patients. Materials and methods AML (HL-60) and ALL (MOLT-4) cell lines were grown according to ATCC recommendations. Primary leukemic cells from the bone marrow of 24 AML patients (median age: 69 years; range: 2-83) were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was determined by western-blot on isolated leukemic cells and expressed as a ratio ASNS/cyclophilin A. When a sufficient amount of leukemic cells was available, sensitivity to L-Aspa (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of L-Aspa (0.001 to 10 IU/mL) with the cells and by measuring the cell viability with a counting kit (CCK-8 viability assay) at day 4. Results Determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro (0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low but higher than in MOLT-4 which is a well known ASNS deficient cell line. IC50 determination was possible on 17/24 patients. Seven displayed a high sensitivity to L-Aspa (IC50 < 0.01 IU/mL) whereas 5 displayed a moderate sensitivity (IC50 < 0.5 IU/mL). Remaining 5 patients were resistant to L-Aspa. The cells from the healthy subject were resistant (IC50 > 10 IU/mL). To date, ASNS expression has been evaluated on 16 patients with ratio ranges from 0 to 2.27: six were negative, 4 low positive (< 0.2), and 6 positive (> 0.5, amongst them 4 were > 1). No correlation was observed between ASNS expression and FAB grade. However, patients with blasts sensitive to L-Aspa had mainly M1 or M5 AML. Conclusion AML cell line HL-60 and 71% of primary cells from AML patients were found sensitive to L-Aspa. The sensitivity of cells from M1 and M5 AML patients is consistent with the findings of Okada et al. (2003). Globally, these results suggest that L-Aspa is effective for killing AML cells. Based on the epidemiology of AML subtypes (Selter et al., 2011) and our results, L-Aspa therapy may be beneficial for 50-70% of patients with AML. However, L-Aspa has only been used scarcely in the treatment of AML, mainly because of the commonly observed adverse effects that impair its use. A new formulation of L-Aspa encapsulated in homologous red blood cells was reported with a better safety profile, allowing its use even in elderly patients (Hunault et al., ASH 2012). A clinical study is currently recruiting patients unfit for intensive chemotherapy in order to evaluate its efficacy in combination with low-dose cytarabine (NCT01810705) in AML. In this study, L-Aspa sensitivity and ASNS expression in primary tumor cells at diagnosis will be explored to investigate the relationship with clinical response. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Gallix:ERYTECH Pharma: Employment. Bertrand:ERYTECH Pharma: Principal Investigator Other. Thomas:ERYTECH Pharma: Principal Investigator Other; Orphan Europe: Consultancy. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.