L-asparaginase in the treatment of patients with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a hematologic malignancy that predominantly occurs in children between 2 and 10 years of age. L-asparaginase is an integral component of treatment for patients with ALL and since its introduction into pediatric treatment protocols in the 1960s, survival rates in children have progressively risen to nearly 90%. Outcomes for adolescent and young adult (AYA) patients, aged 15-39 years and diagnosed with ALL, have historically been less favorable. However, recent reports suggest substantially increased survival in AYA patients treated on pediatric-inspired protocols that include a greater cumulative dose of asparaginase. All  currently available asparaginases share the same mechanism of action - the deamination and depletion of serum asparagine levels - yet each displays a markedly different pharmacokinetic profile. Pegylated asparaginase derived from the bacterium Escherichia coli is used as first-line therapy; however, up to 30% of patients develop a treatment-limiting hypersensitivity reaction. Patients who experience a hypersensitivity reaction to an E. coli-derived asparaginase can continue treatment with Erwinia chrysanthemi asparaginase. Erwinia asparaginase is immunologically distinct from E. coli-derived asparaginases and exhibits no cross-reactivity. Studies have shown that with adequate dosing, therapeutic levels of Erwinia asparaginase activity can be achieved, and patients switched to Erwinia asparaginase due to hypersensitivity can obtain outcomes similar to patients who do not experience a hypersensitivity reaction. Therapeutic drug monitoring may be required to ensure that therapeutic levels of asparaginase activity are maintained.

INTRODUCTION

Acute lymphoblastic leukemia (ALL) is a type of cancer of lymphoid progenitor cells. The two main subtypes of ALL, as categorized by immunophenotype, are B-cell ALL and T-cell ALL.[1] The most common type of cancer in children, ALL represents approximately 25% of cancer diagnoses among children aged younger than 15 years.[2] Incidence is highest among children aged 2-3 years and declines with age and is higher in males versus females and in Whites versus Blacks/African.[2] Advances in therapy have led to a substantial improvement in the 5-year survival rate for patients aged <20 years, from 54% between 1975 and 1977 to 90% between 2004 and 2010.[2] Contemporary therapy for ALL consists of four phases of treatment; specifically remission induction, consolidation/central nervous system-directed therapy, reinduction (delayed intensification), and maintenance/continuation [Table 1].[134] Total treatment duration is about 2-3.5 years, with intensive therapy occurring over the first 6-9 months.[134]

Table 1

Phases of therapy to treat patients with acute lymphoblastic leukemia[134]

HISTORY OF ASPARAGINASE TO TREAT PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA

The use of asparaginase to treat patients with ALL can be traced to the discovery by Kidd[5] in 1953 that guinea pig serum regressed Gardner 6C3HED lymphosarcoma xenografts implanted subcutaneously in mice. In a series of studies, Broome[678] subsequently demonstrated that asparaginase is responsible for the anti-lymphoma effect of guinea pig serum. The anti-leukemic effect of asparaginase is due to the fact that lymphoblastic leukemia cells are unable to synthesize adequate amounts of L-asparagine (Asn) and, therefore, depend on extracellular sources. Asparaginase catalyzes the conversion of Asn to aspartic acid and ammonia, thereby depleting serum Asn and starving leukemic cells of the Asn necessary for DNA, RNA, and protein synthesis, leading ultimately to cell death.[91011]

ASPARAGINASE AS PART OF FIRST-LINE THERAPY

Based on studies in the 1960s using bacteria to identify alternate sources of asparaginase,[121314] clinically available asparaginase is derived from two sources, namely Escherichia coli and Erwinia chrysanthemi. Native enzyme and an enzyme derivatized by the addition of monomethoxypolyethylene glycol (pegylated) are derived from E. coli. Until December 2012, native E. coli asparaginase was available in the USA as Elspar® (Lundbeck, Deerfield, IL, USA) when it was withdrawn by the manufacturer;[15] pegylated E. coli asparaginase is available as Oncaspar® (Baxalta Incorporated, Deerfield, IL; formerly Sigma-Tau Pharmaceuticals, Inc., Gaithersburg, MD); and Erwinia asparaginase is available as Erwinaze® (Jazz Pharmaceuticals, Palo Alto, CA). Both pegylated E. coli asparaginase and Erwinia asparaginase are approved for intramuscular (IM) and intravenous (IV) administration.[1617]

Until the discovery of asparaginase, standard chemotherapy involved the use of vincristine and prednisone to induce first remission in patients with ALL. The earliest case reports with asparaginase in humans were reported in the mid-1960s by Dolowy et al.[18] and Hill et al.[19] using asparaginase derived from guinea pig serum and highly purified asparaginase, respectively. Trials with larger populations were commenced in the early 1970s following the development of large-scale production methods.[202122] Comparison of treatment protocols with/without asparaginase revealed that clinical outcome was improved with those incorporating asparaginase [Table 2].[232425262728] In the Children's Cancer Group (CCG) 101/143 study,[23] the addition of native E. coli asparaginase to the treatment schedule during induction resulted in an increased percentage of patients (93%) who achieved complete remission (CR) at the end of induction compared with 86% of patients who achieved CR in the CCG 903 study[24] where only vincristine and prednisone were used. In the Dana-Farber Cancer Institute (DFCI) 77-01 trial, patients randomized to receive E. coli asparaginase during intensification had a significantly greater probability of disease-free survival (DFS) compared with those receiving treatment that did not include asparaginase (P = 0.04).[25] At a median follow-up of 9.4 years, event-free survival (EFS) was 71% versus 31% for patients treated with/without asparaginase, respectively (P = 0.03).[26] Similarly, the 4-year continuous CR rate of patients with T-cell ALL in the Pediatric Oncology Group (POG) 8704 was greater for those who also received native E. coli asparaginase during the maintenance phase.[27] In a study conducted by the Italian, Dutch, and Hungarian Pediatric Oncology Cooperative Groups, patients randomized to receive an additional 20 weeks of asparaginase (mostly Erwinia asparaginase) during continuation had a significantly greater probability of DFS at 10 years versus those who did not receive asparaginase (88% vs. 79%; P = 0.03).[28]

Table 2

Protocols of trials showing improved efficacy with addition of L-asparaginase

Study results also suggest the effect of asparaginase on outcome is frequency- or intensity-dependent [Table 3].[2930] In the POG 9310 study, children with B-precursor ALL in first marrow relapse with/without concomitant extramedullary relapse had a significantly higher CR rate and an approximate eightfold lower risk of induction failure when investigators increased the frequency of pegylated asparaginase administration, 1-week versus 2-week intervals.[29] Nearly all patients (95%) in this study were previously exposed to native E. coli asparaginase during induction therapy and many patients likely developed antiasparaginase antibodies. Asparaginase clearance can increase in patients with antiasparaginase antibodies, and these patients would benefit from a more frequent dosing schedule. The DFCI ALL consortium protocol 91-01 prolonged E. coli asparaginase therapy from 20 to 30 weeks during intensification; 5-year EFS was significantly increased in children with newly diagnosed ALL compared retrospectively with previous DFCI protocols.[30] In this study, patients who tolerated ≤25 weeks of asparaginase therapy had worse EFS than those who received ≥26 weeks of the enzyme (73% vs. 90%, respectively; P < 0.01). Older children were less tolerant of more intensive therapy. The authors report that 5-year EFS was not significantly different in patients treated with pegylated asparaginase (78%; n = 106) compared with native E. coli asparaginase (84%; n = 92; P = 0.29); however, the study was not sufficiently powered to compare survival between asparaginase preparations.[30]

Table 3

Protocols of trials showing improved efficacy with increased dose intensity of L-asparaginase

OPTIMAL DOSING OF ASPARAGINASE

The goal of asparaginase therapy is to deplete serum Asn. The relationship between the serum asparaginase activity and Asn concentration in humans has been the focus of many studies even though measurement of Asn in the presence of asparaginase has been controversial due to rapid ex vivo hydrolysis. The optimal degree and length of asparaginase depletion required for leukemic cell death are not known; however, results from several studies suggest an asparaginase activity level of 0.1 IU/mL as the target necessary to ensure adequate Asn depletion.[3132333435] Thus, doses and schedules, for example, Berlin-Frankfurt-Münster (BFM) and Dutch Childhood Oncology Group protocols, that ensure a nadir serum asparaginase activity (NSAA) ≥0.1 IU/mL, have become standard.[36] Accordingly, a target NSAA ≥0.1 IU/mL was the primary endpoint in studies that led to the Food and Drug Administration approval of Erwinia asparaginase given IV or IM.[3738] Several investigators have reported Asn depletion or positive outcomes in patients with asparaginase activity as low as 0.05 IU/mL, challenging the strict ≥0.1 IU/mL criterion.[36394041] Conversely, Avramis and Panosyan have proposed that asparaginase activity levels of >0.4-0.7 IU/mL are required for optimal Asn depletion.[42]

Asselin et al.[43] first recognized the clinical implications on the dosing schedule imparted by differences in the pharmacokinetics of the various preparations. Specifically, they demonstrated that the half-lives (t΍) of serum asparaginase activity following IM administration were 1.28, 5.73, and 0.65 days for native E. coli, pegylated E. coli, and Erwinia asparaginases, respectively; t΍ of serum asparaginase activity correlated with the t΍ of the protein level. The t΍ for native E. coli (0.27-0.76 days) and Erwinia (0.31 days) asparaginases was lower with IV administration[16174445] but was independent of dose or dosing history, age, or disease-risk profile.

Results from studies comparing the effects of native E. coli and Erwinia asparaginases administered on the same schedule and dose suggested that Erwinia asparaginase is less effective. In the European Organisation for Research and Treatment of Cancer-Children's Leukemia Group (EORTC-CLG) 58881 trial, 700 patients aged <18 years were randomized to E. coli asparaginase or Erwinia asparaginase 10,000 IU/m2 IV twice weekly for 4 and 2 weeks during induction and reinduction, respectively.[46] Log-rank tests revealed that the 6-year rates of EFS and overall survival for Erwinia asparaginase versus E. coli asparaginase were 60% versus 73% (P = 0.0004) and 75% versus 84% (P = 0.002), respectively. Similarly, in the DFCI 95-01 trial, 5-year EFS was inferior with Erwinia asparaginase.[47] In this study, patients were given a single dose of E. coli asparaginase or Erwinia asparaginase 25,000 IU/m2 IM during induction and 25,000 IU/m2 IM at weekly intervals for 20 weeks during intensification. The apparent difference in efficacy between asparaginases noted in the EORTC-CLG 58881 and DFCI 95-01 trials is consistent with the differences in pharmacokinetics,[43] suggesting that the difference in efficacy is related to suboptimal dosing rather than a less effective compound.

In light of the differences in the pharmacokinetic properties of the asparaginase preparations, results from several studies suggest that dose adjustments to yield therapeutic levels of asparaginase activity are necessary if conditions dictate the need to switch between E. coli-derived and Erwinia asparaginase preparations. In a trial following the ALL/non-Hodgkin lymphoma BFM 95 protocol,[48] Erwinia asparaginase 20,000 IU/m2 given for 9 doses during reinduction resulted in a trough enzyme activity level comparable with that measured in a previous study using native E. coli (Crasnitin) asparaginase given for 4 doses of 10,000 IU/m2.[39] Albertsen et al.[49] reported that trough asparaginase activity was 1.75 IU/mL versus 0.272 IU/mL following administration of Erwinia asparaginase 30,000 IU/m2 IM daily for 10 days during induction versus E. coli asparaginase (medac) 1000 IU/m2 IM, respectively. In the Children's Oncology Group (COG) AALL07P2 study,[38] substitution of Erwinia asparaginase at a dose of 25,000 IU/m2 IM given 3 times weekly for 2 weeks for each dose of pegylated E. coli asparaginase yielded an overall median NSAA of 0.645 IU/mL at 48 h after dosing for all treatment cycles, with NSAA ≥0.1 IU/mL in 96% of all 48 h samples; the overall median NSAA at 72 h after dosing was 0.248 IU/mL during all treatment courses, with NSAA ≥0.1 IU/mL in 85% of all 72 h samples. A key finding of this study was that 80% of the evaluable patients completed all remaining courses of planned asparaginase therapy.[38] A study to evaluate the pharmacokinetics of IV administration of Erwinia asparaginase at a dose of 25,000 IU/m2 found that 83% and 43% of patients had an NSAA ≥ 0.1 IU/mL at 48 h and 72 h, respectively.[37]

HYPERSENSITIVITY TO ESCHERICHIA COLI-DERIVED ASPARAGINASE AND MAINTENANCE OF EFFICACY AFTER SUBSTITUTION WITH ERWINIA ASPARAGINASE

Hypersensitivity caused by the introduction of a foreign protein such as asparaginase is a common toxicity.[50] Of note, up to 30% of individuals develop a treatment-limiting allergic reaction to E. coli-derived asparaginase, necessitating a switch to Erwinia asparaginase.[305152] Erwinia asparaginase is immunologically distinct from E. coli-derived asparaginases and, therefore, lacks immunologic cross-reactivity.[5354] There are two patterns of hypersensitivity responses including antibody production concomitant with an overt clinical reaction and antibody production in the absence of an overt clinical reaction, referred to as “silent inactivation” or “subclinical hypersensitivity.” The presence of antiasparaginase antibodies has been documented in numerous studies, ranging in incidence from 26% to 71% of patients.[51535556575859606162636465] Results from several studies suggest that pegylated asparaginase is less immunogenic than the native E. coli enzyme[33666768] and patients who develop antibodies are more likely to suffer an allergic reaction.[51565758626469]

The development of a hypersensitivity reaction and/or the production of anti-asparaginase antibodies can have a significant impact on t΍, the serum levels of asparaginase protein and activity, and consequently, clinical outcome.[293343535759606162647071] Several studies have demonstrated that switching patients who develop hypersensitivity to E. coli-derived asparaginase to Erwinia asparaginase, at a dose level adequate to maintain Asn depletion, yields clinical outcomes equivalent to patients who never experienced a hypersensitivity reaction.[515972] In the DFCI ALL consortium protocol 00-01, children with newly diagnosed ALL who developed hypersensitivity to native E. coli asparaginase were switched to treatment with twice-weekly IM Erwinia asparaginase at a dose of 25,000 IU/m2.[72] Measurements of asparaginase activity showed that 89% of Erwinia patients had at least one trough asparaginase activity level > 0.1. Importantly, the investigators showed that patients who switched to Erwinia asparaginase due to hypersensitivity to native E. coli asparaginase showed similar EFS at 5.4 years compared with patients who never developed hypersensitivity (86.5% vs. 81.3%, respectively; P = 0.55).[72] Similarly, recently reported data from CCG-1961 show that 5-year EFS was similar in patients who were able to tolerate pegylated E. coli asparaginase throughout postinduction compared with patients who displayed clinical hypersensitivity and were switched to Erwinia asparaginase (80.8% vs 81.6%, respectively; P = 0.66).[73] In the St. Jude Children's Research Hospital front-line protocol XIII-HR, Woo et al.[51] noted no difference in the 4-year EFS rate between patients who developed hypersensitivity and were switched to Erwinia asparaginase compared with patients who did not develop hypersensitivity and continued treatment with E. coli asparaginase (82% vs. 78%, respectively; P = 0.68).

In a prospective drug-monitoring study by Tong et al., patients who developed silent inactivation or allergy to pegylated E. coli asparaginase were given Erwinia asparaginase 20,000 IU/m2 3 times weekly for up to 30 weeks during intensification.[74] Approximately 96% of the patients had at least one NSAA level ≥0.1 IU/mL during the first 2 weeks, and all patients had at least one NSAA level ≥0.1 IU/mL; thereafter, 47% of the patients had all 48 h samples ≥0.1 IU/mL from week 6 to 30.

Erwinia asparaginase is indicated for those patients who have developed hypersensitivity to native or pegylated E. coli asparaginase.[17] A treatment algorithm created by Bleyer et al.[75] proposes that if a reaction is suspected to have occurred after infusion of pegylated E. coli asparaginase, serum should be collected after 4-7 days if the full dose was given, or earlier for an incomplete dose. Based on the finding of Rizzari et al.,[40] the algorithm recommends switching to Erwinia asparaginase if the serum NSAA is <0.05 IU/mL. Of note, NSAA put forth in this algorithm is below the generally accepted threshold of 0.1 IU/mL as an index of asparaginase efficacy in pediatric patients,[36] and others have suggested a target level of 0.05 IU/mL to be inadequate. Specifically, results from two studies of adolescent and young adult (AYA) and adult patients with newly diagnosed ALL suggest that minimal serum asparaginase activity levels of 0.2 IU/mL[76] and 0.4 IU/mL[77] are associated with significant Asn depletion. Moreover, in the study by Angiolillo et al.,[77] Asn began to rebound after asparaginase activity fell below 0.4 IU/mL.

THERAPEUTIC DRUG MONITORING

The wide interpatient variability with respect to trough asparaginase activity levels in serum, the development of subclinical hypersensitivity, and differences in the pharmacokinetic properties among the different asparaginase preparations have underscored the need for therapeutic drug monitoring (TDM). The importance of TDM has been highlighted in several studies. In a study following the ALL-BFM 2000 protocol, children were given eight and four doses of native E. coli asparaginase (medac or Crasnitin) 10,000 IU/m2 IV during induction and reinduction, respectively; patients were switched to Erwinia asparaginase if they developed an allergy or untoward reaction.[39] During induction, median trough asparaginase activity was higher and t΍ was longer with medac versus Crasnitin. During reinduction, the rank order of median trough asparaginase activity was medac (0.528 IU/mL), Crasnitin (0.049 IU/mL), and Erwinia asparaginase (<0.02 IU/mL). Boos et al., therefore, concluded that monitoring is necessary to ensure efficacy targets are reached following substitution of therapy due to an allergic reaction.[39] In their study to assess the pharmacokinetics of IV administration of Erwinia asparaginase, Vrooman et al. concluded that every 48 h dosing should be evaluated, given that NSAA ≥0.1 IU/mL was achieved in 83% of patients after 48 h versus 43% of patients after 72 h.[37] By monitoring patients given native E. coli asparaginase 5000 or 10,000 IU/m2, pegylated E. coli asparaginase 1000 IU/m2, or Erwinia asparaginase 10,000 IU/m2 according to the ALL-BFM 2000 protocol, Schrey et al. found that the wide range of serum asparaginase activity regardless of the asparaginase preparation highlighted the need for TDM.[78]

Results from a recent study to evaluate the relative tolerability and efficacy of fixed versus individualized dosing suggest that individual dosing may be an effective strategy to improve outcome.[79] Patients treated according to the DFCI ALL consortium protocol 00-01 were given 30 weekly doses of E. coli asparaginase either at a fixed dose of 25,000 IU/m2 IM or an individualized dose, based on monitoring of the NSAA, starting at 12,500 IU/m2, and adjusted to maintain NSAA between 0.10 and 0.14 IU/mL.[79] Clinical outcomes were superior with individualized dosing compared with fixed dosing: Fewer relapses (9% vs. 15%, respectively) and significantly greater overall 5-year EFS (90% vs. 82%, respectively; P = 0.04). Moreover, 5-year EFS was 95% in patients placed on individualized dosing, but who were switched to another preparation because of silent inactivation compared with 76% for those in the FD arm with NSAA <0.1 IU/mL and never switched.[79] These results suggest that individualized dosing may improve clinical outcome by monitoring asparaginase activity and prospectively identifying subclinical hypersensitivity.

SPECIAL POPULATION: OPTIMAL TREATMENT PARADIGM IN ADOLESCENT AND YOUNG ADULTS

Despite the significant advances made in the treatment of children with ALL, the outcome for AYAs, defined by the National Cancer Institute as patients aged 15-39 years,[2] has historically been considerably less favorable. A period analysis of trends in 5-year survival based on the US National Cancer Institute Surveillance, Epidemiology and End Results 9 registry has shown that relative survival in children aged younger than 15 years increased from 80% between 1990 and 1994 to 88% between 2000 and 2004.[80] The 5-year survival for adolescents aged 15-19 years also increased during that time, but from 41% to 61%, respectively; lesser but significant improvements were seen for older age groups as well.[81] Similarly, an analysis of 21,626 patients aged 0-22 years enrolled in the COG ALL clinical trials showed that the 5-year survival rate was 91% for children aged younger than 15 years and 75% for those aged 15-19 years in 2000-2005.[82]

The reasons for the disparity in outcome are multifactorial and not completely understood. Adult patients have a poorer tolerance to intensive chemotherapy involving asparaginase. Evidence of increased toxicity in adults was noted as early as 1970 by Oettgen et al.[55] As a result of using different treatment protocols, there is an abrupt drop in the 5-year survival/age relationship at the age at which pediatric versus adult therapy regimens are administered.[83]

Retrospective comparisons have consistently shown that clinical outcome is improved in AYAs treated with pediatric versus adult treatment protocols [Table 4].[84858687] Prospective studies have shown that clinical outcome is improved in AYAs treated with pediatric or pediatric-inspired protocols [Table 5][888990919293] when compared with historical controls of patients treated on adult protocols.[9495] A feature common to the pediatric protocols was the higher cumulative dose of asparaginase, as well as that of glucocorticoid and vincristine, compared with the adult protocols. The asparaginase-free regimen comprising hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD) has been widely used to treat AYA patients.[96979899100] Although most retrospective studies have documented inferior survival rates in AYA patients treated with hyper-CVAD versus pediatric-inspired protocols,[101102] Rytting et al.[103] found that the 3-year OS was 71% and 74% for AYA patients treated with hyper-CVAD and an augmented BFM protocol, respectively.

Table 4

Trials involving adolescent and young adult patients with acute lymphoblastic leukemia treated with pediatric versus adult protocols

Table 5

Trials involving adolescent and young adult patients with acute lymphoblastic leukemia treated with pediatric or pediatric-inspired protocols

As described, pediatric protocols are distinguished by the use of intensive asparaginase, vincristine, and glucocorticoid therapies. A meta-analysis of total therapy studies XIIIA, XIIIB, XIV, and XV revealed that despite the more intensive treatment regimen, the incidence of asparaginase-related allergy was not higher in patients aged 15-18 years treated with any of these protocols versus those aged 1-14 years; conversely, the incidences of thromboembolic complications, pancreatitis, osteonecrosis, and hyperglycemia were greater in the older patients.[104] Central venous thrombosis (CVT) is a potentially life-threatening event that has been reported in a minority of patients receiving asparaginase and corticosteroids.[105106] Early monitoring and detection of CVT are critical to ensure positive outcomes with anticoagulation therapy.[105106]

In a compassionate-use trial with patients switched to Erwinia asparaginase after developing a hypersensitivity reaction to native E. coli or pegylated E. coli asparaginase, Plourde et al.[107] found that the safety profile in 147 patients aged ≥16-<40 years was consistent overall with that of the full trial population. In this trial, Erwinia asparaginase was given at a dose of 25,000 IU/m2 IM 3 times/week for 2 weeks for each dose of pegylated E. coli asparaginase remaining or 1:1 for each dose of native E. coli asparaginase remaining. In addition, in the USA intergroup study C10403, the largest prospective study to date to assess the feasibility of pediatric protocols in AYAs, toxicities were manageable in AYA patients treated using the COG AALL0232 regimen administered by adult hematologists/oncologists; 2-year OS and EFS rates were 78% and 66%, respectively.[108]

SUMMARY

Following the seminal discovery by Kidd in 1953,[5] asparaginase has been a mainstay of pediatric chemotherapy protocols to treat patients with ALL. Since the incorporation of asparaginase into treatment protocols, clinical outcomes have improved significantly, with an NSAA ≥0.1 IU/mL widely accepted as the therapeutic level necessary to achieve efficacy. Pegylated E. coli asparaginase remains first-line treatment, but the occurrence of an allergic reaction necessitates a switch to Erwinia asparaginase. Studies have shown that substitution of Erwinia asparaginase for E. coli-derived asparaginase following an allergic reaction and/or silent inactivation is an effective therapeutic option to complete the treatment protocol as planned. The use of pediatric-inspired protocols has been shown to improve outcome in the AYA population with an acceptable safety profile. TDM may also improve clinical outcome by prospectively identifying patients who develop subclinical hypersensitivity.

Financial support and sponsorship

This work was supported by Jazz Pharmaceuticals.

Conflicts of interest

Rachel A. Egler has nothing to disclose. Sanjay P. Ahuja has received honoraria from Bayer and Biogen Inc., served as a consultant for Bayer and Biogen Inc., and served on the speakers’ bureau for Biogen Inc., Novo Nordisk Inc., and Grifols. Yousif Matloub has served as a consultant for Novartis AG, has received honoraria from Novartis AG and Jazz Pharmaceuticals, and owns stock in Amgen Inc.