Primary Warm Autoimmune Hemolytic Anemia Research Articles

Second-Line Therapy Sequencing in Primary Warm Autoimmune Hemolytic Anemia: Splenectomy-Rituximab Versus Rituximab-Splenectomy

Introduction: Primary warm autoimmune hemolytic anemia (wAIHA) is a hematologic condition that affects approximately 1 in 8000 people and occurs when IgG antibodies directed against self-erythrocytes cause hemolysis and exceed the bone marrow's ability to compensate. First-line treatment with glucocorticoids yields sustained remission in less than 30% of individuals. For patients requiring second-line treatment, splenectomy was the preferred treatment for over 50 years, being recommended over rituximab by leading experts as recently as 2010. In the last decade this practice shifted due to concerns of tripartite risk of splenectomy with rates of infection, thrombosis, and perioperative mortality, juxtaposed against the increased experience with rituximab. These risks are outdated as they predate the modern-day vaccination, thromboprophylaxis, and laparoscopic splenectomy era, respectively. Summarily, the 2017 British guidelines dropped splenectomy from second- to third-line for patients with primary wAIHA, consistent with expert opinion in the United States in 2023. Over these same twenty years, the mortality and morbidity of splenectomy has greatly decreased, owing to updated preoperative vaccination standards, prevention of thrombosis, and widespread use of the laparoscopic (i.e., rather than open) technique. Despite these advancements, splenectomy is now used in fewer than 10% of patients with wAIHA. In the absence of a clinical trial randomizing patients to splenectomy versus rituximab, we conducted the first simulation of second-line therapeutic sequencing for adult patients with primary wAIHA with a focus on short-run and long-run effectiveness results. Methods: We constructed a Markov simulation of adult patients with primary wAIHA and requiring second-line therapy to compare the effectiveness of strategy #1: splenectomy followed by rituximab, if necessary, versus strategy #2: rituximab followed by splenectomy, if necessary. Transition probabilities for initial response and time-variant relapse risk with rituximab and with splenectomy were drawn from retrospective primary-wAIHA-specific literature. For rituximab, we assumed that 1) patients maintained in remission for three years following treatment with a rituximab cycle would remain in remission and 2) all patients who relapse more than a year after receiving rituximab, would be retreated with rituximab. Costs were sourced from the Centers for Medicare & Medicaid Services and the Centers for Disease Control and Prevention. Utility values for patients with wAIHA were specific to the reported patient disease status, whether in relapse, partial remission (defined as hemoglobin >10g/dL) or complete remission (defined as hemoglobin >12g/dL). These were sourced from the Global Burden of Disease Study and weighted for the degree of anemia corresponding with each status. Effectiveness was calculated in quality-adjusted life-years (QALYs). The primary outcomes were lifetime discounted costs, QALYs, and net monetary benefits across accepted willingness-to-pay thresholds over short- and long-runs at 5 years and lifetime, respectively. Probabilistic sensitivity analysis was employed to vary all parameters across their uncertainty intervals simultaneously over 10,000 Monte Carlo iterations. Results: In the short-run, strategies #1 and #2 cost $39,700 and $47,100 and accrued 5.21 and 5.26 QALYs (Table 1). In the long run, costs were $909,000 and $912,000 while accruing 22.0 and 22.1 QALYs. Net monetary benefit point estimates were similar across the accepted range of WTP thresholds in both the short- and long-runs. Conclusion: The balance of costs and quality-adjusted life expectancy appears to be comparatively tightly balanced in the sequencing of second-line therapies in primary wAIHA with splenectomy-rituximab versus rituximab-splenectomy. The absence of a clear rituximab-early advantage on a population level may be driven by advancements in vaccination, thromboprophylaxis, and laparoscopic surgical techniques for splenectomy over the last 20 years. Value-of-information analysis would help identify parameters whose uncertainty reduction with future study in the real world would yield a treatment strategy with a definitive advantage. In the meantime, patients whose values and preferences align with splenectomy should not be dissuaded from pursuing it due to perceived risk.

Genome-wide DNA methylation profiles analysis in primary warm autoimmune hemolytic anemia patients

ABSTRACTBackgroundAutoimmune hemolytic anemia (AIHA) is caused by auto-antibodies, secreted by overactivated B cells, directed against self-red blood cells, resulting in hemolysis. It found that aberrant DNA methylation in B cells can induce the production of autoantibodies. Therefore, we attempted to explore if similar aberrant DNA methylation occur in AIHA patients.MethodsA 49-year-old female wAIHA patient and a 47-year-old female healthy control (HC) were enrolled. Peripheral blood (PB) B cells DNA was extracted. After constructing genomic libraries, bisulfite genomic sequencing (BSP) and DNA methylation profiles were analyzed. BSP was verified using PB B cells from 10 patients with hemolysis, 10 patients with hemolytic remission, and 10 healthy controls (HCs) by Methylation-specific PCR.ResultsTotal DNA methylation of whole-genome C bases (4.8%) and CG type bases (76.8%) in wAIHA patient were lower than those in the HC (5.3 and 82.5%, respectively) (p = 0.022 and p < 0.001). DNA methylation of C bases and CG type bases in whole-genome regulatory elements, such as coding sequence, up2Kb and down2Kb in the patient were also lower than those in the HC (p = 0.041, p = 0.038, and p = 0.029). 30,180 DNA-methylated regions (DMRs) on all 23 chromosomes were identified. DMR-related genes were mainly involved in the Rap1, phospholipase D, HIF-1, calcium, vascular endothelial growth factor (VEGF) and Ras signaling pathways.ConclusionThe DNA methylation spectrum of B cells in AIHA patients is different from that of HC, and the proportion of hypo-methylation regions is higher than that of HC. DMR-related genes are mainly related to some signaling pathways.

A Retrospective Study of the Combination of Rituximab, Cyclophosphamide and Dexamethasone for the Treatment of Relapsed/Refractory Warm Antibody Autoimmune Hemolytic Anemia

The combination of rituximab, cyclophosphamide, and dexamethasone (RCD) is highly effective in the treatment of warm autoimmune hemolytic anemia (WAIHA) associated with chronic lymphocytic leukemia (CLL). We treated a cohort of patients with relapsed/refractory WAIHA, without CLL, with RCD. The primary objective was to evaluate the overall response (OR) of RCD therapy. Complete response (CR) was defined as a hemoglobin (Hgb) ≥12 g/dL. Partial response (PR) was defined as Hgb 10–11.9 g/dL or ≥2 g/dL increase in Hgb. Sustained response was defined as Hgb ≥10 g/dL with no treatment changes. A total of 16 patients with relapsed/refractory WAIHA received RCD (7 primary WAIHA, 9 secondary WAIHA) for a median of 4 cycles (range: 2–6). The median pretreatment Hgb was 10.0 g/dL (range: 4.3–12.2). The median best Hgb achieved was 12.5 g/dL (range: 10.6–15.1) with a median of 2 cycles until best Hgb response. The OR was 94% (11 CR, 4 PR). Two immunocompromised patients were admitted for infections during RCD treatment. There were no deaths during the treatment or follow-up period. Following a response to RCD, 4 patients received noncorticosteroid immune modulation therapy and 4 patients continued on corticosteroid therapy. Seven patients received no additional treatment.

Probability of remaining in unsustained complete remission after steroid therapy withdrawal in patients with primary warm-antibody reactive autoimmune hemolytic anemia.

Primary warm autoimmune hemolytic anemia (WAIHA) is a rare autoimmune disorder frequently responding to corticosteroid first-line treatment and effective second-line treatment options such as splenectomy or anti-CD20 antibody therapy. Disease management is frequently hampered by a lack of evidence. We have investigated the probability of sustained treatment-free remission after steroid induction to facilitate clinical decision making regarding timing and necessity of second-line treatments. Response data from 31 patients with primary WAIHA initially treated with steroids were retrospectively analyzed. All patients responded by achieving a hemoglobin of at least 10mg/dl. After steroid tapering and final withdrawal, 9 of 30 patients remained in unsustained complete remission (CR). The probability of remaining in CR after steroid treatment only was 38.2 % (2 SD 20.6 %) at 15 months. The median remission duration was 100 + months with a range of 12 + to 163 + months. Of note, none of the remaining patients still on steroids achieved CR beyond 15 + months. These data indicate that a considerable proportion of patients do not need further treatment and that relapses will not occur after 15 months in CR.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.

Emergence of Long-Lived Autoreactive Plasma Cells in the Spleen of Primary Warm Auto-Immune Hemolytic Anemia Patients Treated with Rituximab

Introduction: We recently proposed that B-cell depletion in immune thrombocytopenia (ITP) promotes the generation of long-lived plasma cells in the spleen, some of them being auto-reactive; but it remained possible that this observation was related to ITP itself rather than to B-cell depletion. Primary warm autoimmune hemolytic anemia (wAIHA) is a rare disease characterized by IgG auto-antibodies directed against antigens at the surface of red blood cells (RBCs) antigens, leading to their accelerated destruction. The use of B-cell depletion in wAIHA leads to 60 % to 70% of overall response at one-year and beyond. Nevertheless, 30-40 % of patients may resist to rituximab and then require a splenectomy. Nothing is known about antibody secreting cells (ASC) in the spleen of wAIHA patients, who have previously been treated or not with rituximab. In this study, we analyzed at the single cell level the splenic ASC from patients with chronic and active wAIHA, previously treated or not with rituximab (RTX), and we compared them with splenic ASC from ITP patients, and with splenic and bone marrow plasma cells from healthy donors (HD).Methods: We took advantage of the different therapeutic outcomes to analyze the splenic B-cell compartment of wAIHA patients, not previously treated with RTX (n=6), or after failure RTX treatment (n=3).Splenic tissues from organ donors and bone marrow from cardiovascular thoracotomy were used as controls. Blood samples from wAIHA (n=19), and (HD) (n=8) enrolled in this study were obtained after giving written informed consent in accordance with the Declaration of Helsinki.Results: We observed by flow cytometry and microscopy that the spleen from wAIHA patients who received less than 3 months of steroid therapy was the site of a B-cell response characterized by the presence of Bcl6+ germinal-center (GC) B-cells. Furthemore, splenic ASC secreted anti-red blood cell IgG in vitro. In line with this observation, we observed in the peripheral blood from patients with a newly diagnosed wAIHA (n=11), that short-lived IgG plasmablasts were increased compared with HD (n=8) (Mean 4.2 ± 0.84 % vs 0.99 ± 0.19% of CD19+ cells, p< 0.01). Moreover, for patients receiving long term steroid therapy (> 6 months) the plasmablast response was suppressed in the peripheral blood (Mean: 0.68 ± 0.2 % of CD19+ cells, n=8) and the splenic GC B-cell reaction was impaired (n=3). We conclude that short-lived IgG ASC result from an over-activity of GC reactions in wAIHA. We then analyzed the spleen of 3 patients who failed to respond to RTX, and observed a residual population of CD19+B-cells (median: 0.9% of lymphoid cells), including non-proliferative memory B-cells and plasma cells (PC). A fraction of these residual PC secreted anti-red blood cells IgG in vitro, thus accounting for the faillure of the B-cell depletion therapy. By using a single cell multiplex quantitative RT-PCR (Fluidigm dynamic arrays), we showed that such RTX-resistant plasma cells display a long-lived transcriptional program, which differs from PC from untreated wAIHA patients or HD, as well as from plasmablasts. Interestingly, the gene expression profile of wAIHA long-lived plasma cells segregated with long-lived PC previously observed in the spleen of ITP patients treated with rituximab. By a principal component analysis, we observed a gradient of maturation from plasmablasts to bone marrow plasma cells in which PC from RTX-treated spleens segregated close to bone marrow PC. We also observed that the cytokine BAFF was increased in the supernatants of spleen cell cultures from wAIHA patients treated with rituximab compared with controls (p< 0.05), suggesting, in keeping with our previous report in ITP, a role for BAFF in the differentiation of short-lived plasma cells into long-lived plasma cells.Conclusion: The presence of splenic long-lived autoreactive PC in wAIHA may explain why some patients cannot achieve a response after RTX. Our results show that, the B-cell depletion induced by rituximab itself, as opposed to the underlying auto-immune condition, promotes a suitable environment for the maturation of auto-immune long-lived plasma cells in the spleen. Targeting specifically some factors such as BAFF right after rituximab injection could be an interesting therapeutic option in the future. DisclosuresNo relevant conflicts of interest to declare.