First Report of Eltrombopag Induced Severe Autoimmune Hemolytic Anemia in the Management of Waldenström Macroglobulinemia-associated Autoimmune Thrombocytopenia

Background: Autoimmune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) can be observed in Waldenström Macroglobulinemia (WM). The cause of AIHA should be carefully distinguished during the disease management. Case Presentation: A 63-year-old female WM patient complicated with thrombocytopenia, who was admitted to our department with a complaint of abdominal pain. After �rst half of bortezomib/dexamethasone/rituximab (BRD) chemotherapy, her platelet level recovered, but subsequently decreased to extremely low level (around 1-2×10 9 /L), and the patient suffered from platelet transfusion refractoriness. During the management of refractory thrombocytopenia, the patient developed severe hemolytic anemia, and further tests con�rmed warm AIHA. FcγRIIα polymorphism test showed that the patient had FcγRIIα-131RH, which implied that the AIHA may not be WM-related. Given the effects of ibrutinib in controlling WM, ibrutinib single treatment was started, which quickly corrected the thrombocytopenia within �ve days, but not hemolysis. With a relatively safe platelet level, eltrombopag was stopped, and the hemolysis alleviated within three days after eltrombopag withdrawal. Conclusion: This is the �rst report on eltrombopag-induced AIHA, and we should always keep in mind of the drug induced hemolysis even in disorders with autoimmune background.


Introduction
Waldenström Macroglobulinemia (WM) is a chronic lymphoproliferative disorder, with an indolent clinical course.Autoimmune thrombocytopenia (ITP) has been observed in approximately 3.8% of patients with WM 1 and 10-20% of patients may complicated with autoimmune hemolytic anemia (AIHA) 2 .The occurrences of autoimmune disorders are thought to be related to a dysfunctional immune response associated with WM process.
The preferred management of ITP is corticosteroids if thrombocytopenia is the sole manifestation in WM (normal hemoglobin and without symptoms), and anti-lymphoma therapies such as chemotherapy should be initiated if the thrombocytopenia does not improve with previous treatment 3 .Herein, we report a WM patient who rapidly developed pancytopenia (especially with severe ITP) refractory to dexamethasone (DXM) containing chemotherapy (BRD: Bortezomib, DXM and Rituximab), which progressed to very severe thrombocytopenia.During the management of thrombocytopenia with eltrombopag, severe AIHA occurred.Disease remission was nally achieved with the application of ibrutinib, which is an effective Bruton's tyrosine kinase inhibitor, and the AIHA ceased after withdrawal of eltrombopag.To the best of our knowledge, this is the rst report of eltrombopag-induced AIHA, and ibrutinib is an ideal option for the autoimmune disorder in WM, and its effect in controlling WM-related ITP is inspiring.
Case Report 2020, and blood routine test showed pancytopenia (WBC 1.8×10 9 /L, Hb 82g/L and PLT 14×10 9 /L).The patient recalled that, three months ago, she suffered from gingival bleeding for four days, but did not take it seriously after healing.Besides moderate anemia and subcutaneous bleeding, she also had multiple enlarged lymph nodes in retroperitoneum, hepatic portal area and pelvic cavity, as well as splenomegaly, as observed by abdominal computerized tomography (CT).Further examinations showed an extremely high ratio of CD19 positive cells (17.63%) in the peripheral blood.Hence, we screened for B cell originated disorders.Histopathology result showed 4% small lymphocytes and occasional lymphoplasmacytic cells in bone marrow aspiration, and there was an active proliferation of megakaryocytes (approximately 126/slide) with dysmaturity (only 9% were plate-producing).The immunophenotype of this abnormal population was positive for CD19, CD20, CD22, CD79b, CD200, sIgM and κ light chain, weak expression of CD25 and negative for CD103, CD10, CD5, CD11c, CD23, CD27, CD56, CD43, FMC-7, CD38, λ light chain, and CD138, which indicated an abnormal clonal mature B cell population.The next generation sequencing (NGS) indicated positive for MYD88 L265p mutation (c.794T > C).There was also a clonal IgM in serum (total IgM 7.65 g/L, immunoelectrophoresis showed positive for IgM and κ light chain).
These clinical data supported the diagnosis of WM.In addition, the detection of Coombs' (both direct anti-globulin test and indirect antiglobulin test), CD55/CD59, lactate dehydrogenase (LDH) showed negative.
Given the ITP-like manifestation in bone marrow morphology, a dexamethasone containing chemotherapy (BRD: Bortezomib 2 mg weekly×4, DXM 20 mg weekly×4 and Rituximab 375 mg/m 2 monthly) was initiated.The platelet counts increased to normal after the rst two rounds of DXM, but decreased to 12×10 9 /L after the third dose of DXM, and became refractory to platelet transfusion (Fig. 1).
The platelet count was maintained at 1-2×10 9 /L, and the patient did not respond to second round continued dose of DXM (20 mg qd for 4 days).The patient refused further chemotherapy, and accepted ITP-based treatment including methylprednisolone (12 mg qd), intravenous immunoglobulin (IVIG, 20 g qd×4), recombinant human thrombopoietin (TPO, 15,000U qd), and TPO receptor agonist (Eltrombopag 75 mg qd) subsequently, but remained unresponsive to all these treatment.Rituximab was continued during the course (500 mg qw×3) considering the primary WM disease (Fig. 2).
During the management of refractory thrombocytopenia, the patient suffered from severe hemolytic anemia (hemoglobin decreased from 104 g/L to 41 g/L), with increased serum indirect bilirubin (IBIL maximum was 119.7 µmol/L), LDH (maximum was 559U/L) and reticulocyte (maximum was 25.25%) (Fig. 3).The cold agglutinating titer is under 1:4, while the direct anti-globulin test (DAT) was positive for IgG and C3.And the Anti-nuclear antibody (ANA), Ham's, Rous test, as well as ADAMST13 activity and Flare test (PNH) showed normal.To further distinguish whether the hemolysis originated from the primary disease (WM) or was drug-induced, we tested the polymorphism of FcγR.The results showed that the patient was FcγR α 131H/R and FcγR α 158F/V (Fig. 4), which indicated that the hemolysis may not have developed from the progression of WM 4 .Eltrombopag was highly suspected as the cause of hemolysis, since it was initiated 10 days before the onset of hemolysis, but no published evidence was available for its withdrawal regardless of its potential bene t in platelet recovery.Given the patient's refusal of chemotherapy, we nally started ibrutinib with her informed consent after explaining the high risk of hemorrhage, accompanied by continuous eltrombopag daily and IVIG twice a week.Five days later, the platelet count increased signi cantly to 15×10 9 /L, but hemolysis did not improved within 15 days, and even got worse.The dosage of ibrutinib was increased from 140 mg to 420 mg per day, and the platelet count continuously increased to around 25×10 9 /L.With decreased risk of hemorrhage, eltrombopag was withdrawn, and the hemolysis stopped immediately (indicators improved every day).
IBIL, LDH and absolute value of reticulocyte decreased to almost normal within 10 days, and the hemoglobinn recovered in 50 days (Fig. 5).The patient continued the ibrutinib maintenance therapy for three months, and achieved very good partial remission (VGPR), with normal IgM, hemogram and stable disease.

Discussion
Autoimmune phenomenon is well recognized in B cell-associated disorders, which is mostly mediated by autoimmune monoclonal gammopathy, including IgM or IgG 1,3,5,6 .The secondary immune thrombocytopenia in WM may be complement-mediated, and the severity of thrombocytopenia varies from mild to fatal 1 .The primary therapy for ITP in WM is corticosteroids, or corticosteroids containing chemotherapy.For the refractory and relapse cases, splenectomy, vinca alkaloids, azathioprine, and highdose intravenous gamma-globulin therapy may not work.According to the National Comprehensive Cancer Network (NCCN) guideline, the preferred regimens for WM include bendamustine/rituximab (BR), bortezomib/dexamethasone/rituximab (BRD), ibrutinib ± rituximab (I ± R), and rituximab/cyclophosphamide/dexamethasone (BCD), which can achieve > 80% e cacy 7 .
In this case, the patient achieved quick platelet response in the rst half of BRD therapy, but subsequently suffered from refractory platelet transfusion and severe thrombocytopenia.The refractory thrombocytopenia did not respond to high dose IVIG, recombinant human thrombopoietin (rhTPO), methylprednisolone and azathioprine, respectively.Given the high response rate of eltrombopag for secondary immune thrombocytopenia in chronic lymphoproliferative disorders 8 , TPO receptor agonist (TPO-RA) was tried, but the platelet count showed no improvement.
To the best of our knowledge, there is only one report about concomitant AIHA and ITP in WM, which was resolved by the combination treatment with rituximab and romiplostim 9 .AIHA includes both warm AIHA and cold agglutinin disease (CAD), and the most common cause of hemolysis in B cell disorders is primary disease related 10 , in which the malignant cells serve as antigen-presenting cells to T lymphocytes, and the generated autoreactive lymphocytes are involved.In WM, the majority of cases with AIHA are CAD, and DAT results always show positive for anti-C3 but negative for anti-IgG 10 .Inconsistent with the literature, our case was positive for both anti-IgG and anti-C3, and warm AIHA was con rmed.IgG fragment C (Fcg)-mediated erythrophagocytosis has been recognized as the major pathogenic mechanism responsible for AIHA [11][12][13] .To distinguish whether the warm AIHA in WM was disease-related or drug-induced, the polymorphism of FcγRIIα was detected by Sanger sequencing.As reported, all the IgG-mediated warm AIHA in WM mostly showed FcγRIIα-131 homozygous (131 HH or 131RR) 4 , and the heterozygous 131RH in our case indicated that AIHA may not be WM clone-related, and drug induced AIHA was suspected.However, no clear drug was found after searching published literatures and from our experience.According to the patient's preference, ibrutinib single treatment was initiated, which may be bene cial for the primary disease as well as secondary ITP and AIHA [14][15][16] .As expected, the thrombocytopenia was quickly resolved, and became transfusion independent, but the AIHA did not resolved and even worsen, which further indicated that the AIHA was not WM-related.With a relatively safe platelet level, we discontinued eltrombopag, which was a potential suspicious drug that was started 10 days before hemolysis.Notably, the hemolysis stopped within three days (indicator improved every day) after eltrombopag withdrawal.Based on the clinical manifestations and consequent changes, we deduced that the AIHA was induced by eltrombopag.
In summary, this is the rst report about eltrombopag-induced AIHA in the management of WMassociated ITP.Eltrombopag is widely used for primary immune thrombocytopenia (ITP), but eltrombopag-induced AIHA has not been reported.The mechanism of hemolysis in eltrombopag-induced WM is unclear, and may be associated with the speci c disease background.Further investigations are needed.Physicians should consider the possibility of eltrombopag-induced AIHA if hemolysis develops, and drug withdrawal should be immediately initiated.

Figures
Figure 1 Changes of hemogram during BRD (Bortezomib 2mg weekly×4, DXM 20mg weekly×4 and Rituximab 375mg/m2 monthly) chemotherapy.The platelet counts increased to normal after rst two rounds of BD, but decreased to 12×109/L after the third round, and became refractory to platelet transfusion.Additional, the hemoglobin also decreased 8 days after third round of BD.
Changes of platelet count after multiple managements on thrombocytopenia.The response of platelet was not su cient after second round DXM (20mg qd for 3days).With the consequence treatment including methylprednisolone, intravenous immunoglobulin (IVIG), recombinant human thrombopoietin (TPO) and TPO receptor agonist, no response was observed as well, and patient suffered from with platelet transfusion refractoriness.
Severe hemolytic anemia occurred during the management of thrombocytopenia.During the management of thrombocytopenia, the patient suffered from severe hemolytic anemia (hemoglobin decreased from104g/L to 41g/L), with high level serum indirect bilirubin (IBIL maximum to 119.7μmol/L), lactic dehydrogenase (LDH maximum to 559U/L) and increased reticulocyte (Ret maximum to 25.25%).

Figure 5 Changes
Figure 5