Fostamatinib for warm antibody autoimmune hemolytic anemia: Phase 3, randomized, double‐blind, placebo‐controlled, global study (FORWARD)

Warm antibody autoimmune hemolytic anemia (wAIHA) is characterized by hemolysis and symptomatic anemia with no approved treatment options. Fostamatinib is an oral spleen tyrosine kinase inhibitor approved in the US and Europe for treatment of adults with chronic immune thrombocytopenia. In this phase 3 study, patients with an insufficient response to ≥1 prior wAIHA treatment were randomized to fostamatinib or placebo. The primary endpoint was the proportion of patients to achieve a durable hemoglobin (Hgb) response (Hgb ≥10 g/dL and increase from baseline of ≥2 g/dL on 3 consecutive visits) during the 24‐week treatment period. Ninety patients were randomized, 45 to each arm. Of the fostamatinib‐treated patients, 35.6% achieved a durable Hgb response versus 26.7% on placebo (p = .398). A post hoc analysis revealed a large placebo response in Eastern European patients. Significantly more patients on fostamatinib from North America, Australia and Western Europe exhibited a durable Hgb response compared to placebo (36% vs. 10.7%, p = .030). After censoring for Hgb values impacted by steroid rescue received during screening and excluding 2 placebo patients found to likely not have wAIHA, a reanalysis demonstrated a difference in durable Hgb response between fostamatinib and placebo (15/45 [33.3%] vs. 6/43 [14.0%], p = .0395). At least 1 AE was reported in 42 (93.3%) and 40 (88.9%) patients receiving fostamatinib and placebo, respectively. The most common AEs in the fostamatinib group were diarrhea (26.7%), hypertension (24.4%), and fatigue (15.6%). In this study, fostamatinib demonstrated a clinically meaningful benefit for patients in Western regions, and no new safety signals were identified.

7][8][9] Common symptoms of hemolysis and anemia (fatigue, dyspnea, and palpitations) may be severe, depending on the degree of hemolysis and anemia, and can impair patients' quality of life. 8Additionally, up to 33% of adults with wAIHA experience a venous thromboembolism, with an increased risk for thrombosis within 90 days of disease onset. 10The known challenges of managing severe cases of wAIHA, which are associated with an estimated mortality rate of 4%-8% 1,4 and up to 30% in critically ill patients, 11 underscore the importance of developing effective, well-tolerated therapies.4][15][16] IgG bound to RBCs may also activate the complement pathway, contributing to phagocytosis of C3b-opsonized erythrocytes by hepatic Kupfer cells. 1,8,12SYK inhibition offers a novel mechanism of action to treat wAIHA through reduced B-cell receptor mediated autoantibody production, inhibition of RBC phagocytosis via reduced activation of FcR, and inhibition of complement receptors.
Fostamatinib disodium hexahydrate is an orally administered SYK inhibitor that received FDA approval for the treatment of adults with chronic immune thrombocytopenia (ITP) in 2018.The pleiotropic effects of fostamatinib provide a novel targeted treatment option that abrogates the underlying mechanisms of disease.The major metabolite of fostamatinib (R406) was observed to be protective against the development of anemia in mouse models of AIHA. 17Fostamatinib was shown to be effective for achieving a hemoglobin (Hgb) response in a phase 2 open-label, multicenter study (NCT02612558) in patients with wAIHA. 18In the phase 2 study, 46% of patients treated with fostamatinib 150 mg BID achieved the primary endpoint by Week 24 (Hgb >10 g/dL with an increase of ≥2 g/dL from baseline).Adverse events (AEs) were manageable and consistent with the fostamatinib safety profile for ITP as well as a larger safety database of >4800 patients who received fostamatinib in multiple disease states. 18This report describes results from the FORWARD trial, a phase 3 randomized, double-blind, placebo-controlled study of fostamatinib in patients with wAIHA who have failed at least one prior treatment.

| Study design
This phase 3 multi-center, randomized, double-blind, placebocontrolled, parallel group study evaluated the safety and efficacy of fostamatinib in patients with wAIHA (Clinicaltrials.gov,NCT03764618; EudraCT 2018-004774-97; for list of study sites and investigators, see Supplementary Table 1).Patients were randomized 1:1 to receive fostamatinib or matching placebo twice daily (BID) for 24 weeks.Randomization was stratified at screening to balance patients by severity of anemia (baseline Hgb <9 vs. ≥9 g/dL) and dose of concomitant corticosteroid use at baseline (≥20 vs. <20 mg prednisone equivalent daily).

| Patients
Eligible patients were ≥18 years old at screening and had primary or secondary wAIHA documented by a positive DAT specific for anti-IgG or anti-IgA.Inclusion criteria consisted of patients that failed or did not tolerate at least one prior wAIHA treatment, had a haptoglobin <lower limit of normal (LLN), or total bilirubin >upper limit of normal (ULN), or lactate dehydrogenase (LDH) >ULN.A Hgb level of less than 10 g/dL was required.Patients with a Hgb >9 and <10 g/dL at screening were required to have documented clinical symptoms related to anemia.Patients also had to have a Karnofsky performance status (KPS) ≥70 and could not be on more than two of the allowed concurrent therapies at a stable dose including azathioprine, steroids, erythropoiesis-stimulating agents (ESAs), mycophenolate mofetil (MMF), dapsone, or danazol.
Exclusion criteria included other types of AIHA (cold antibody AIHA, mixed type, cold agglutinin syndrome, or paroxysmal cold hemoglobinuria), AIHA secondary to autoimmune disease or lymphoid malignancy if the underlying disease was not stable or well-controlled, uncontrolled, or poorly controlled hypertension (SBP ≥135 mmHg or DBP ≥85 mmHg), or ≥1 of the following laboratory abnormalities including neutrophil count <1000/μL, platelet count <30 000 μL (unless due to Evans syndrome), transaminase levels >1.5x ULN, or previous treatment with fostamatinib for any indication.

| Treatment
Patients were allowed to continue up to two approved concomitant medications at a stable dose throughout their participation in the study.Doses and regimens were not to be changed during the screening period and the 24-week treatment period except in the case of a steroid dose taper or dose increase as rescue therapy.Other medications had to be discontinued within a pre-specified interval prior to Day 1 of fostamatinib.
The initial dose was fostamatinib 100 mg BID (or matching placebo), and if tolerated, the dose was increased to 150 mg BID at Week 4. Doses could be reduced to fostamatinib 100 or 150 mg once daily if a dose-limiting AE occurred.Rescue medication, including initiation of or an increase in steroid dose, RBC transfusion, or IVIg was permitted if a decrease in Hgb >1.5 g/dL from baseline occurred or patient experienced new or worsening symptoms of anemia.Importantly, if rescue was given during the treatment period, Hgb data were censored in the data analysis from all following visits during rescue and 4 weeks after rescue medication.The post hoc analysis extended this pre-specified censoring rule to cover rescue therapy received during the screening period.Steroid tapers per protocol were initiated in patients who reached Week 12 and achieved a Hgb response at ≥2 consecutive, scheduled visits.Patients who completed the 24-week blinded study period could enroll in the long-term, open-label extension study (NCT04138927).

| Endpoints
Assessments were conducted every 2 weeks during clinic visits.All randomized patients (intent-to-treat population) were evaluated for efficacy.The primary efficacy endpoint was the achievement of a durable Hgb response defined as achieving a Hgb ≥10 g/dL with an increase from baseline ≥2 g/dL on 3 consecutive available visits during the 24-week treatment period.Hgb values after rescue therapy were to be censored during and 4 weeks after rescue therapy.Secondary efficacy endpoints included: Hgb response on ≥1 visit, change in Hgb from baseline of ≥2 g/dL, use of permitted rescue therapy after Week 4, mean change in Hgb from baseline to end of treatment where end of treatment is defined as the mean of the Hgb values from the last 6 available visits between Weeks 14 and 24, and change from baseline to Week 24 in Functional Assessment of Chronic Illness Therapy -Fatigue (FACIT-F) scale.Safety was assessed in all patients who received at least one dose and included the incidence and severity of treatment-emergent AEs (TEAEs), including TEAEs of interest.

| Statistical analyses
The sample size of 90 patients was estimated to provide ≥84% power for the primary efficacy endpoint, using a 2-sided, Fisher's Exact Test with alpha level of 0.05.All randomized patients were evaluated in the intent-to treat (ITT) population and all patients that received at least one dose of either fostamatinib or placebo were included in the safety analysis.A per-protocol (PP) analysis including any patient in the ITT population without major protocol deviations that could affect the primary efficacy analysis was also performed.
All efficacy and safety endpoints were summarized descriptively.The primary efficacy endpoint was analyzed using Cochran-Mantel-Haenszel (CMH) test stratified by concomitant steroid use and screening Hgb levels.CMH was also used to analyze binary secondary efficacy endpoints.The difference in proportion between the two treatment groups was estimated, and 95% Miettinen-Nurminen score confidence intervals computed.Analysis of covariance was used to analyze change in Hgb levels from baseline to end of treatment.Least squares mean with the 95% CI for the mean was constructed for each treatment group and for the differences between treatment groups.
A post hoc analysis was performed to evaluate differences in outcomes by region.Additionally, a re-analysis was conducted to exclude patients with no evidence of hemolysis (likely misdiagnosed as wAIHA) and to apply identical censoring rules for Hgb values influenced by rescue not only during the treatment period, but also during the screening period.

| Baseline characteristics and fostamatinib treatment
Demographics and baseline characteristics were well-balanced between the two treatment arms (Table 1).Most patients had primary wAIHA (89%), and median age was 58 and 64 years in the fostamatinib and placebo groups, respectively.The majority (76%) of patients in each group were treated with >1 prior wAIHA therapy; all but one patient (randomized to placebo) had been previously treated with corticosteroids, and 53.3% of patients were previously managed with rituximab.Some patients (21.1% and 13.3%) were previously treated with azathioprine and/or MMF, respectively, while 13.3% of the overall population had undergone a splenectomy.Most patients in both arms were on a concomitant prednisone equivalent of <20 mg/day.Median baseline Hgb was 8.4 (range 5-10.7) and 8.9 (range 6.1-10.3)g/dL for the fostamatinib and placebo groups, respectively (Table 1).
Patients (74/90, 82%) increased their dose from the starting dose of 100 mg BID to 150 mg BID after 4 weeks on study with the remaining patients increasing after Week 4. The mean total dose on study was 256.2 and 267.8 mg/day in the fostamatinib and placebo groups, respectively, during Weeks 6 to 24.

| Primary outcome
Based on the original pre-specified analysis, the primary endpoint of durable Hgb response was attained in 16 (35.6%)fostamatinib-treated patients and 12 (26.7%)patients on placebo (p = .398).A post hoc regional evaluation revealed that most placebo responders were from Eastern Europe, and durable Hgb response rates in that region were not statistically significant between fostamatinib and placebo groups.
By contrast, durable Hgb response rates for patients in the US, Canada, Australia, and Western Europe were significantly different between the fostamatinib and placebo groups (36% vs 10.7%, p = .030)(Table 2).It was noted that some sites had administered rescue therapy during the screening period against protocol guidance and the statistical analysis plan had not originally pre-specified to apply censoring rules during screening.Hence, a post hoc reanalysis of the overall population was conducted that censored Hgb values secondary to rescue therapy received during the treatment and screening period (Supplementary Table 2A-C and Supplementary Figure 3).The reanalysis resulted in T A B L E 1 Baseline characteristics.2B).In addition, two patients with elevated haptoglobin levels (168 and 188 mg/dL) and no evidence of hemolysis were excluded from the analysis because they were assessed likely to not have an accurate diagnosis of wAIHA.(Supplementary Table 2A).

| Secondary endpoints
Hgb response at ≥1 visit was achieved by 46.7% of patients who received fostamatinib compared to 35.6% of patients who received placebo.In the post hoc analysis of patients from US, Canada, Australia, and Western Europe, a Hgb response at ≥1 visit was achieved by 52% of fostamatinib-treated patients compared with 17.9% of placebo patients.A change in Hgb ≥2 g/dL from baseline was achieved by 48.9% vs. 35.6% of patients in the overall fostamatinib and placebo groups, respectively; however, in the US, Canada, Australia, and Western Europe, more patients on fostamatinib achieved this endpoint compared to placebo (56% vs. 17.9%,respectively) (Figure 1A).After Week 4, 40% of patients in both arms of the pre-specified groups required no rescue therapy, but more patients in the fostamatinib group were free of rescue therapy in the post hoc analysis (48% vs. 28.6%placebo) (Figure 1A).The change in mean Hgb from baseline to end-of study (mean of last 3 visits) was 1.8 and 1.85 g/dL in the overall fostamatinib and placebo groups, respectively.In the post hoc analysis, the change in mean Hgb was 2.25 and 1.27 g/dL in the fostamatinib and placebo groups, respectively, with the median Hgb values increasing >2 g/dL by Week 12 through Week 24 (Figure 1B).
The FACIT-F instrument was designed to assess fatigue/tiredness and its impact on daily activities and functioning in several chronic diseases including anemia.FACIT-F assessments were administered at baseline, Week 12, and Week 24/Early Termination.Mean changes in FACIT-F scores from baseline were improved in the fostamatinib group compared with placebo in both the pre-specified analysis (2.9 vs. 1.9) and post hoc analysis (7.0 vs. 0.7).

| Safety
The incidence of AEs was similar between fostamatinib (93.3%) and placebo (88.9%) groups (Table 3).The most commonly occurring AE in patients treated with fostamatinib was diarrhea (26.7%).Hypertension and fatigue were also common, occurring in 24.4% and 17.8% of fostamatinib-treated patients, respectively.Serious AEs occurred in T A B L E 3 Incidence of adverse events.
33.3% and 37.8% of patients receiving fostamatinib and placebo, respectively.Five deaths, 2 (4.4%) in the fostamatinib group and 3 (6.7%) in the placebo group, occurred during the study.None were considered by the investigator to be related to treatment.In the fostamatinib group, one patient died due to multi-organ failure and one due to cardiac failure.In the placebo group, one patient died due to COVID-19 infection, one due to bacterial endocarditis, and one due to sudden death.In the regional analysis, similar patterns of AEs were observed with slightly fewer AEs being reported in Eastern Europe.

| DISCUSSION
This phase 3 randomized, double-blind study investigated the efficacy and safety of the SYK inhibitor fostamatinib in patients with wAIHA who had an insufficient response to at least one prior treatment.In the pre-specified primary analysis, there was no statistically significant difference in durable Hgb response (Hgb ≥10 g/dL with an increase from baseline of ≥2 g/dL on 3 consecutive visits) in the fostamatinib group compared with the placebo group.There was an unexpectedly large number of placebo responders in patients from Eastern Europe.A post hoc regional analysis without any modification to the data analysis methods demonstrated a statistically significant difference in the Hgb response of patients treated with fostamatinib versus placebo in North America, Australia, and Western Europe, but not Eastern Europe.While baseline characteristics of patients across groups appeared to be generally similar, the placebo patients in Eastern Europe had a notably shorter duration of AIHA and fewer prior treatments.
The two main explanations for the differences in response rates were: (a) some sites had administered rescue medication during the screening period before randomization without censoring Hgb values impacted by the rescue therapy (the prespecified censoring rules applied only to rescue therapy given during the treatment period), and (b) there were 2 patients from the same site in Eastern Europe who were probably misdiagnosed as having wAIHA, as there was no evidence of hemolysis.Hence, an exploratory reanalysis of the data was performed with two simple modifications, (1) to apply identical censoring rules used in the treatment period also to the screening period and (2) to exclude patients with no evidence of hemolysis (and likely an incorrect diagnosis of AIHA).With the new censoring rules applied in the reanalysis, 5 patients no longer qualified for stable response to treatment including 1 patient in the fostamatinib group and 4 patients in the placebo group (Supplementary Table 2B and Supplementary Figure 3).Additionally, the two patients with no evidence of hemolysis who experienced spontaneous recovery to normal Hgb while on placebo were excluded from the re-analysis.These two adjustments resulted in a significant difference (p = .0395)favoring fostamatinib in the overall population and a highly significant difference in Western Countries (32.0% vs. 0%; p = .0021).
It should be noted that the study protocol had directed to keep the background AIHA medication stable during the screening period with an option to rescue if the investigator deemed such rescues clinically necessary.This was accounted for in the treatment period with pre-specified censoring rules agreed upon with regulatory agencies, but not during the screening period.As a consequence, some placebo patients became responders shortly after randomization due to rescue medications started in the screening period.
Despite these two adjustments in the analysis, there were still six remaining placebo responders, all from Eastern European countries.Results from various studies have been published. 2,26It is important to recognize that most trials reported data based on heterogeneous outcome measures, which make the trials difficult to compare.
While short-term response rates to corticosteroids can range from 75%-80%, 9 studies measuring response as one Hgb value >10 g/dL with a ≥2 g/dL change from baseline resulted in response rates of 38%-43% in newly diagnosed patients. 4,32ng-term use of steroids is not recommended due to adverse effects.Despite risk of infection and infusion complications, rituximab is often used as a subsequent-line therapy.Three studies evaluated rituximab using a similar endpoint achieved at one visit, and the response rates were 16%-45% in relapsed patients [33][34][35] and 75% in newly diagnosed patients treated with the combination of rituximab and corticosteroids. 36,37In the FORWARD study, 47% of previously treated patients achieved response to fostamatinib at one visit (or more).0][21][22][23][24][25] No new safety signals were detected.
From a mechanistic perspective, there is a considerable body of evidence for SYK inhibition as a rationale for treating wAIHA.SYK inhibition with fostamatinib has the potential to target multiple mechanisms of wAIHA pathophysiology.The critical position of SYK in the FcɣR signaling pathway mediates both phagocytosis of antibodycoated RBCs 3,13,14,16 and production of cytokines, which are known to have a role in cytotoxic responses against RBCs. 27,28SYK has been shown to be a key transducer of the B-cell receptor signaling required for activation and development of B cells, indicating its role in production of autoantibodies. 29Additionally, there is evidence for a role for SYK signaling in complement-mediated phagocytic processes. 30The potential for immunosuppression with SYK inhibition was evaluated in a series of host immunotoxicology studies, which showed that fostamatinib (and its active metabolite) did not inhibit innate or acquired immunity in Listeria, Streptococcal, or Influenza host resistance mouse models; the study also showed a lack of effect on humoral immune response to KLH challenge in rats. 31garding trial design for wAIHA, FORWARD has provided Patients with wAIHA continue to need treatments that provide durable efficacy without long-term safety concerns.The results presented here provide encouraging data that fostamatinib may induce durable Hgb responses that continue to build over time with a safety profile consistent with that of long-term studies in ITP and rheumatoid arthritis.Altogether, fostamatinib may represent a viable treatment option for patients with wAIHA.
important insights on how to optimize clinical data generation.The use of rescue medication and the analysis of data from such patients appears to be critically important.In addition, a strict assessment of accuracy of diagnosis, duration of disease, and definition of previously failed medications is essential.With post hoc exploratory adjustments of such important factors, we observed results in Western Countries that suggest clinically meaningful efficacy in AIHA patients that are generally consistent with a previous phase 2 study.While there are limitations to conducting post hoc analyses, these results are insightful and future studies may confirm the efficacy of fostamatinib for wAIHA.

100 Durable Hgb Response Hgb Response on ≥1 visit Achieved a Change in Hgb ≥2 g/dL Free of Rescue after Week 4 Percentage of Patients
Primary efficacy outcome: Durable Hgb rsponse.Two patients from Eastern Europe with the highest haptoglobin and no evidence of hemolysis were excluded due to a likely misdiagnosis of wAIHA.
a Western Europe: Austria, Germany, Spain, France, Italy, Belgium, UK, Netherlands, Norway.b Eastern Europe: Bulgaria, Czech Republic, Russia, Ukraine, Georgia, Belarus, Serbia.c Prednisone or equivalent.d One placebo patient was not on prior steroids.T A B L E 2 c treatment.Hgb, hemoglobin; ITT, intent to treat; PBO, placebo. 1 fostamatinib-treated patient and 4 placebo-treated patients no longer having valid Hgb values that met the definition of response (Supplementary Table 6% vs. placebo, 11.1%), but FACIT-F scores were generally improved in patients treated with fostamatinib.Overall, fostamatinib has a manageable and predictable safety profile, with few serious or severe AEs and no known cumulative toxicities.Most AEs were mild or moderate and could be managed as needed.The safety profile based on this phase 3 study is consistent with the known safety profile of fostamatinib, established in the fostamatinib safety database of >4000 patients across multiple disease programs, including wAIHA, immune thrombocytopenia, rheumatoid arthritis, and of concomitant anti-hypertensive medication.Similarly, diarrhea was generally managed with fostamatinib dose modifications and/or using anti-motility medication, with a very small percentage of patients discontinuing therapy for these or other AEs.The majority of patients were maintained on the highest recommended dose of fostamatinib.The incidence of fatigue was similar between both groups (fostamatinib, 15.