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L-asparaginase (L-ASPA) is included in current chemotherapy regimen for the treatment of acute lymphoblastic leukemia (ALL). The sensitivity of ALL to L-ASPA has been linked to the incapacity of leukemic cells to produce their own asparagine, since they are deficient in a specific enzyme, asparagine synthetase (ASNS). Thus, ASNS-negative leukemic cells rely on plasmatic asparagine and can be starved to death by L-ASPA treatment. Several studies evidenced the potential of asparagine depletion to treat lymphomas. Indeed, L-ASPA is routinely administered to treat canine lymphomas and its adjunction in chemotherapy regimens significantly improves the outcome of patients with NK/T cell lymphoma. Some studies suggest its benefit for the treatment of B and T-cell non-Hodgkin lymphomas (NHL). In this study, we assessed the in vitro sensitivity to L-ASPA of 11 lymphoma cell lines, including diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL) and other NHL cell lines. We analyzed ASNS expression in tissue microarrays and biopsies from 226 cases of lymphomas, including 158 DLBCL, 15 PTCL, as well as other NHL and Hodgkin lymphomas. Sensitivity to L-ASPA (expressed as an IC50) was assessed by measuring the cell viability in the presence of various concentrations of L-ASPA. ASNS expression was assessed with a validated immunohistochemistry method attributing a score based on 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. All lymphoma cell lines were proved to be sensitive to L-ASPA. Their in vitro sensitivity usually exceeded well-known sensitive cell lines (MOLT-4 and HL-60) representative of acute leukemia. ASNS expression was null/low in 171/226 (76%) total cases of lymphomas. 118/158 (75%) DLBCL cases expressed no/low ASNS, whereas ASNS expression was low/null in 2/15 (13%) PTCL cases. Globally, B-cell NHL displayed a lower expression of ASNS than T-cell NHL, Hodgkin lymphoma and non tumoral lymph nodes. The in vitro sensitivity to L-ASPA of all lymphoma cell lines and the low/null expression of ASNS in the majority of the lymphoma tissues tested suggest that L-ASPA may be effective for the treatment of various lymphomas. As suggested by ASNS expression, L-ASPA treatment may be particularly effective in DLBCL and others B-cell NHL, whereas its efficacy in T-cell NHL is probably more limited. 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…) render its use particularly hazardous in older or frail patients. The development of a new formulation of L-ASPA with better safety profile must be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Anne-Marie Chevrier, Alexandra Traverse-Glehen, Yann Godfrin. Low asparagine synthetase expression and in vitro sensitivity highlights L-asparaginase potential for the treatment of aggressive lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2015-5369

Due to asparagine synthetase (ASNS) deficiency, tumors cells auxotrophic for L-asparagine lack capability to synthesize this amino acid and depend to circulating L-asparagine for survival. Based on this rational, L-asparaginase (L-Aspa), an enzyme depleting L-asparagine, is an essential drug in the treatment of acute lymphoblastic leukemia (ALL), as these hematological cancer cells express low levels of ASNS. Recent evidence suggests that some solid tumors are also deficient in ASNS (Lorenzi, 2008; Dufour, 2012; Zhang, 2013), providing rationale for testing L-Aspa treatment in other contexts. In this study, we investigated the levels of ASNS expression in tumor tissues from patients with urological cancers (bladder, prostate and kidney) and we investigated the sensitivity to L-Aspa of bladder and prostate cell lines. Material & Method: ASNS expression was evaluated by a validated immunohistochemistry (IHC) method on tissue biopsie microarrays (USBiomax, Rockville, MD) including 384 cases of bladder, 94 cases of prostate and 75 cases of kidney tumors. A score was attributed to each tumor based on ASNS IHC labeling intensity from 0 (no labeling) to 3 (strong labeling). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. Sensitivity to L-Aspa (expressed as an IC50) was assessed in vitro by measuring the cell viability of bladder cancer cell lines (RT-4 and UM-UC-3) and prostate cancer cell line PC-3 in the presence of various concentrations of L-Aspa. ASNS expression in cell lines was evaluated by western-blot. Results: ASNS expression was low/null in 77.3% of bladder tumors, 86.2% of prostate tumors and 72% of kidney tumors. In bladder tumors, no correlation was found between the grade of the tumor and its expression of ASNS. Bladder and prostate cancer cell lines were all sensitive to L-Aspa (IC50 = 0.31, 0.12 and 0.22 IU/mL for RT-4, UM-UC-3 and PC-3 cell lines, respectively) at a level similar to the sensitivity observed with the L-Aspa-sensitive MOLT-4 ALL cell line (IC50=0.19 IU/mL). All cell lines displayed low basal expression of ASNS. Conclusion: The sensitivity of bladder and prostate cancer cell lines to L-Aspa and the low/no expression of ASNS in the majority of urological tumor biopsies suggest that an extensive population of patients with urological tumors may respond to asparagine depletion. However, L-Aspa is accompanied with well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, liver failure) and this drug should be used very carefully especially in older or frail patients. A new safer formulation is requested to make easier the clinical development of L-Aspa in urological cancer. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Fabien Gay, Gautam Borthakur, Yann Godfrin. Potential of asparagine depletion for the treatment of bladder cancer and other urological tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2014-5436

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.