key: cord-0866161-2inxsoqy
authors: Fattizzo, Bruno; Cantoni, Silvia; Giannotta, Juri Alessandro; Bandiera, Laura; Zavaglia, Rachele; Bortolotti, Marta; Barcellini, Wilma
title: Efficacy and safety of cyclosporine A treatment in autoimmune cytopenias: the experience of two Italian reference centers
date: 2022-05-14
journal: Ther Adv Hematol
DOI: 10.1177/20406207221097780
sha: 2b67766f59273a4b9a7c5b86988b1ba1be57919c
doc_id: 866161
cord_uid: 2inxsoqy

BACKGROUND: Immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) show good responses to frontline steroids. About two-third of cases relapse and require second-line treatment, including rituximab, mainly effective in AIHA, and thrombopoietin-receptor agonists (TPO-RAs) in ITP, while the use of splenectomy progressively decreased due to concerns for infectious/thrombotic complications. For those failing second line, immunosuppressants may be considered. OBJECTIVES: The aim of this study was to evaluate the efficacy of cyclosporine treatment in patients with ITP and AIHA. DESIGN: In this retrospective study, we evaluated the efficacy and safety of cyclosporine A (CyA) in ITP (N = 29) and AIHA (N = 10) patients followed at two reference centers in Milan, Italy. METHODS: Responses were classified as partial [Hb > 10 or at least 2 g/dl increase from baseline, platelets (PLT) > 30 × 10(9)/l with at least doubling from baseline] and complete (Hb > 12 g/dl or PLT > 100 × 10(9)/l) and evaluated at 3, 6, and 12 months. Treatment emergent adverse events were also registered. RESULTS: The median time from diagnosis to CyA was 35 months (3–293), and patients had required a median of 4 (1–8) previous therapy lines. Median duration of CyA was 28 (2–140) months and responses were achieved in 86% of ITP and 50% of AIHA subjects. Responders could reduce or discontinue concomitant treatment and resolved PLT fluctuations on TPO-RA. CyA was generally well tolerated, and only two serious infectious complications in elderly patients on concomitant steroids suggesting caution in this patient population. CONCLUSION: CyA may be advisable in ITP, which is not well controlled under TPO-RA, and in AIHA failing rituximab, particularly if ineligible in clinical trial.

Autoimmune cytopenias (AICs), namely immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA), are a heterogeneous group of diseases characterized by the presence of autoantibodies directed against platelets (PLT) and erythrocytes. 1, 2 Although frontline steroids are the mainstay of treatment, most patients relapse and require further lines of therapy which Efficacy and safety of cyclosporine A treatment in autoimmune cytopenias: the experience of two Italian reference centers slightly differ according to the type of cytopenia. [3] [4] [5] Rituximab is mainly effective in AIHA, although only a fraction of cases would experience longterm relapse-free survival. Splenectomy is still a valid option although progressively abandoned given the availability of alternative therapeutic options, and increasing awareness of thrombotic and infectious risks. Regarding ITP, thrombopoietin-receptor agonists (TPO-RA) are effective in more than 70% of patients but may result in great PLT fluctuations, increased bone marrow reticulin fibrosis, and increased thrombotic risk. 2 Therefore, both efficacy and safety issues of standard second-line treatment options still leave a fraction of patients without effective treatment. 1, 2, 6 For refractory ITP and AIHA cases, novel therapeutic options are being explored in clinical trials, although not all patients are eligible. Classic immunosuppressants have definitively moved to third-line options, although they are still largely used in clinical practice as steroid-sparing agents. 1, 2, 6 Some of them received recent attention, such as mycophenolate mofetil, that was used frontline in adult ITP patients with interesting results. 7 Cyclosporine A (CyA), which has been widely used in post-transplant settings and in aplastic anemia (AA) since about for more than 30 years, is an oral drug with known but easily manageable toxicities. 8 However, few data exist on CyA efficacy in refractory AIC used either alone or in combination with other treatments. In this study, we aimed at evaluating the efficacy and safety of CyA in a cohort of patients with ITP and AIHA, followed at two reference hematologic centers in Milan, Italy. In addition, a review of the literature on CyA use in AIC is provided.

We retrospectively investigated consecutive adult patients diagnosed with ITP or AIHA and followed at two Italian reference centers for AIC who received CyA treatment between June 2010 and February 2021. Diagnosis of AIHA and ITP was established according to current guidelines. 1, 2, 6 AIHA was classified according to direct anti-globulin test (DAT) results as warm (either IgGpositive or IgG-positive plus complement, C, at low titer), cold (C-positive), mixed (IgG plus complement positive autoagglutination at room temperature, and high titer cold agglutinins), or atypical (DAT-negative, IgA-positive). Anemia severity was categorized according to previous scores for AIHA 6 in moderate (Hb < 10 g/dl), severe (Hb < 8 g/dl), and very severe (Hb < 6 g/dl). ITP was categorized as severe (PLT < 30 × 10 9 /l), or moderate (PLT 30-100 × 10 9 /l). 2 The positivity of anti-PLT autoantibodies was registered when available. The study was conducted according to the Declaration of Helsinki and approved by the local Ethical Committee (Comitato Etico Milano Area 2, code CYTOPAN, date of approval 9 March 2021). All patients' information has been de-identified. At the time of treatment, patients had given informed consent to therapy.

Hematological data at the time of CyA start, and previous AIC history was retrospectively collected. Where available, bone marrow features were registered, including evaluation of cellularity, presence of fibrosis and its degree, lymphoid infiltrate and its phenotype, and cytogenetic study. The treatments administered were collected, including steroids, intravenous immunoglobulins (IVIG), rituximab, splenectomy, cytotoxic immunosuppression, thrombopoietin-receptor analogues (TPO-RA) eltrombopag and romiplostim, and recombinant erythropoietin (rEPO).

CyA treatment: efficacy and safety All patients received a CyA starting dose of 3-5 mg/kg/day in two divided doses, and cyclosporine blood levels were initially monitored to avoid overdosing. The drug was subsequently tapered to a maintenance dose of 1.5-3 mg/kg/day according to hematological response. Responses were assessed at 3, 6, and 12 months and divided into partial (PR, for Hb > 10 or at least 2 g/dl increase from baseline, PLT > 30 × 10 9 /l with at least doubling from baseline) and complete (CR, for Hb > 12 g/dl and normalization of hemolytic markers; PLT > 100 × 10 9 /l). All relapses were registered, and adverse events, including infectious, thrombotic, and bleeding complications, graded according to the common terminology criteria CTCAE version 5. The occurrence of death and the relative causes were registered for all patients. 

A total of 39 patients, 29 ITP (74%) and 10 AIHA (26%), 17 men (44%) and 22 women (66%), with a median age of 51 (range 21-81) years were included in the analysis (Table 1) . Anti-PLT autoantibodies tested positive in 52% of ITP cases. AIHA were all warm forms, with DAT positivity for IgG in three patients, IgG + C in six, and IgA in one subject only. Bone marrow evaluation was performed in 33 cases (85%) and showed reduced cellularity in 11 (28%). ITP patients predominantly showed normal/increased megakaryocytes (75%), a low prevalence of dysplastic features, and had a polyclonal lymphoid infiltrate in 28% of cases, mainly of T cell phenotype (62%). Bone marrow fibrosis was detected in 53% of patients, of whom one grade 2 and one grade 3, both in patients treated with TPO-RA. Among AIHA patients, 40% of subjects displayed dyserythropoiesis, and two patients had a mixed (B/T cell phenotype) polyclonal lymphoid infiltrate. Overall, three patients (two ITP and one AIHA) showed a non-myelodysplastic syndrome defining cytogenetic abnormality.

Median time from diagnosis to CyA start was 35 months (range 3-293), and patients had required a median of 4 (1-8) previous therapy lines, including steroids (100%), IVIG (49%), splenectomy (41%), rituximab (13%), TPO-RA (65% of ITP cases, seven subjects had received both eltrombopag and romiplostim in sequence prior to CyA), danazol (13%), and cytotoxic immunosuppressors (10%; two azathioprine, one mycophenolate mofetil, one vincristine). Only two patients received CyA as second-line treatment: an ITP elderly patient who was not eligible to either splenectomy or TPO-RA therapy due to age and recent ischemic cardiopathy, and a young steroidrefractory wAIHA patient who refused splenectomy in the pre-rituximab era (2001). 

AIC, autoimmune cytopenia; AIHA, autoimmune hemolytic anemia; DAT, antiglobulin test; ITP, immune thrombocytopenia; IVIG, intravenous immunoglobulins; PLT, platelets. a DAT positivities were as follows: 3 IgG + ; 6 IgG + C; 1 IgA. b Bone marrow reticulin fibrosis WHO grade 1, before treatment with eltrombopag or romiplostim. (3), or TPO-RA (16) . The main reason to start CyA in ITP was no response to previous treatments (72%), followed by the development of bone marrow fibrosis on TPO-RA (20%), contraindication for splenectomy (14%; two due to age and two due to recent ischemic cardiopathy on double anti-PLT agents), platelets fluctuations (10%), and wish to become pregnant (7%). In AIHA, CyA was mainly started due to non-response to previous therapy (100%), followed by contraindication for splenectomy in six patients (60%; four due to age and two due to recurrent infections). Finally, in three ITP and two AIHA patients, the choice of CyA was also supported by the presence of a polyclonal T cell infiltrate at bone marrow evaluation.

Median duration of CyA therapy was 28 (2-140) months, and response to treatment was achieved in 25 ITP (86%) and 5 AIHA (50%) patients. Median time from CyA start to response was 68 (31-131) days. Considering the various time points, in ITP overall response raised from 72% at month 3 to 91% of evaluable subjects at month 12, and in AIHA from 40% at month 3 to 83% at month 12 (Table 2) . Specifically, in ITP responders median PLT increase from baseline was 32 × 10 9 /l at month 3, 121 × 10 9 /l at month 6, and 43 × 10 9 /l at month 12. In AIHA, median Hb improved by 0.5 g/dl at month 3, by 1 g/dl at month 6, and by 2.6 g/dl at month 12. As regards additional endpoints, concomitant medications were reduced or discontinued in 81% of ITP and 50% of AIHA patients, including 10 subjects who stopped or tapered TPO-RA (five each). Moreover, 3/3 ITP patients resolved PLT fluctuations on CyA treatment. By statistical analysis, we did not identify any baseline clinical or laboratory factors significantly associated with response to CyA, including age, gender, disease duration, bone marrow features, or DAT positivity.

Overall, 16 patients (9 ITP and 7 AIHA) stopped therapy, mainly due to non-response (33% ITP and 72% AIHA) or relapse of the autoimmune cytopenia (33% of ITP and 28% of AIHA). Interestingly, three ITP patients who had achieved a complete response (CR) on treatment were able to taper and discontinue CyA, maintaining a long-term remission off-treatment. These subjects were one male and two females, with a median age of 66 (52-77) years, who had received a median of 4 lines (4-5) of therapies before CyA, including splenectomy.

Adverse events were mainly grade 1-2, occurring in 28% of patients, and included asthenia, dyspnea, myalgia, nausea, vomiting, diarrhea and abdominal pain, hypertrichosis, epistaxis, petechiae, and an Escherichia coli cystitis. Three patients had a slight increase in creatinine values (median 1.5 g/dl) that was resolved by increasing water intake and by reducing CyA dose. Two patients on concomitant long-term steroids developed a G ⩾ 3 event, including one aspergillus lung infection (in a previously splenectomized AIHA patient) and one fatal pneumocystis jirovecii pneumonia. Notably, none of the patients was receiving anti-pneumocystis prophylaxis. Finally, lymphocyte counts did not show any significant changes during CyA treatment, and some subjects previously treated with rituximab showed persistent lymphopenia.

Review of the literature A total of 23 reports have been published involving the use of CyA in AIC for a total of 441 patients (Table 3 ). They were mainly pediatric studies (60%) and included 10 case reports/ser ies, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 8 clinical trials, [19] [20] [21] [22] [23] [24] [25] [26] and 5 retrospective studies [27] [28] [29] [30] [31] of CyA used as single agent or in combination.

ITP reports included patients with relapsed or refractory disease, and the overall response to CyA was about 65% (180/278 patients, case reports excluded), without differences among pediatric and adult settings. [20] [21] [22] [23] [24] [25] [27] [28] [29] Regarding combined regimens, the association of CyA and recombinant thrombopoietin (rTPO) versus rTPO single agent yields similar response rates (over 80%), but fewer relapses (29% versus 88% at 3 months). 21 In a clinical trial of 20 adult ITP patients, the association of CyA plus steroids and rituximab achieved a relapse-free survival of 92% and 76% at 12 and 24 months, respectively. 22 These results, although in a small series, seem better than those observed in larger studies with journals.sagepub.com/home/tah 5 

rituximab monotherapy, where long-term remission was 20-30%. [32] [33] [34] In the setting of secondary ITP, a recent study including 83 adult patients with ITP associated with connective tissue disease showed that CyA was inferior to rituximab in terms of response rates (82% versus 54% at 6 months). 28 AIHA studies mainly consisted of case reports [12] [13] [14] [15] [16] [17] [18] with only one retrospective cohort study including relapsed or refractory subjects. 30 Overall response rate was about 70% (51/73, case reports excluded). In a clinical trial enrolling adult patient with AIHA and Evans syndrome (the association of ITP and AIHA), the combination of CyA plus danazol and steroids was more effective than steroids alone with a 89% versus 58% of patients achieving a response and a relapse rate of 3% versus 70%. 26 Finally, in a recent French retrospective study involving 34 pediatric AIC patients, response rates were higher in AIHA and Evans syndrome (about 50%) compared to ITP subjects who did not respond. 31

In this study, we show that CyA is effective in a large proportion of pretreated and refractory AIC patients with an effect lasting more than 2 years. Responses required at least 3 months to be observed, and some patients showed benefit as late as at 12 months. This may be due to the immunomodulatory effect of CyA, including promotion of regulatory T cells differentiation with restoration of a tolerogenic milieu, which takes time to establish. 31 The rationale of using a T cell targeting agent in autoantibody-mediated conditions, such as ITP and AIHA, resides in the dysregulation of cellular immunity in the pathogenesis of these diseases. This includes direct cellular damage, presentation of self-antigens, and cytokine production leading to amplification of the dysregulated immune response and disease chronicization. Consistently, a T cell polyclonal infiltrate may be observed in the bone marrow of a proportion of ITP and AIHA patients, 35, 36 and T cell enrichment has been recently reported by RNA single-cell sequencing in ITP and AIHA. 37, 38 These observations are also in keeping with the efficacy of other anti-T cell agents, that is, mycophenolate mofetil in the treatment of ITP. 7 In our case series, best responses were observed in ITP (up to 86%), fairly greater than those reported in the literature, although the great heterogeneity of published studies (clinical trials, retrospective studies, and combination regimens) impedes a direct comparison.

Considering current guidelines, 2 it is difficult to establish the position of CyA in ITP. Rituximab, TPO-RA, and splenectomy are considered as equivalent second-line options to be adopted in accordance with patient' age, comorbidities, and quality of life. 2 Splenectomy has been The two AIHA patients achieving a PR only had persistently altered LDH and unconjugated bilirubin levels, while those achieving CR, by definition, reached full normalization of hemolytic markers. c Causes of death in ITP included fatal pneumocystis jirovecii pneumonia, COVID pneumonia, end-stage liver disease, and elderly marasmus in two; in AIHA, sepsis, heart failure, thromboembolic event, and elderly marasmus. All events, but the first, occurred in patients of CyA treatment. progressively abandoned or refused due to thrombotic and infectious complications, and rituximab induces up to 70-80% responses that are however durable in about 20-30% of cases only. [32] [33] [34] 39 Nowadays, rituximab is discouraged, given the B cell depleting effect impacting on response to vaccination, particularly anti-SARS-CoV-2. 40 Thus TPO-RA are becoming the preferred second-line choice, and the spleen tyrosine kinase inhibitor fostamatinib has been recently licensed, although not worldwide available. 2 Our results and the review of the literature suggest that CyA can be an option for ITP patients whose disease is not well controlled by TPO-RA, who develop side effects (mostly bone marrow reticulin increase) and those who are not eligible for clinical trials. In addition, our data show that the addition of CyA to TPO-RA not only increased PLT counts in refractory patients but also stabilized fluctuations and allowed TPO-RA tapering or discontinuation in a fraction of subjects. This may be pointed at as a 'TPO-RA sparing' effect, which may be particularly relevant in the case of bone marrow fibrosis and in the settings of increased thrombotic risk.

Finally, only about one-third of patients relapsed after CyA, confirming relapse-free survival data from the literature. Of note, three ITP patients obtained a treatment-free remission after CyA therapy, an increasingly recognized outcome for this patient population.

Approximately 50% of AIHA patients achieved a response on CyA, although with the caveat of an old series mainly including patients who did not receive rituximab. The efficacy of the latter in warm AIHA is about 70-80%, clearly better than that reported for ITP and represents the recommended second-line treatment, although not indicated or available worldwide. 1, 3, 5, 6 However, all CyA responders were able to reduce or discontinue steroids, in keeping with well-known steroid-sparing effect of CyA. 30 Translated nowadays, CyA may be an option in AIHA patients who fail rituximab, are not candidate or refused splenectomy, and result ineligible in clinical trials.

Regarding safety, CyA was generally well tolerated with mainly grade 1-2 adverse events.

Although infections under CyA are less frequently reported than under other immunosuppressants (i.e. cyclophosphamide, mycophenolate mofetil), 41 we observed two serious infectious complications (5%, one fatality in an elderly) both in patients concomitantly receiving steroids. Thus, particular attention is warranted for elderly, heavily pretreated patients, including anti-pneumocystis prophylaxis in patients receiving >25 mg/day prednisone or equivalent for >4 weeks. Finally, CyA plasma concentrations may be monitored to optimize treatment and reduce renal or liver toxicity.

In conclusion, CyA was effective in a high proportion of heavily pretreated ITP and AIHA patients and allowed tapering or discontinuation of concomitant treatments in about two-third of cases. Responses were higher in subjects with ITP with an interesting TPO-RA sparing effect and reduction of PLT fluctuations.

Autoimmune hemolytic anemias

American Society of Hematology 2019 guidelines for immune thrombocytopenia

A phase III randomized trial comparing glucocorticoid monotherapy versus glucocorticoid and rituximab in patients with autoimmune journals.sagepub.com/home/tah haemolytic anaemia

Epidemiology of incident immune thrombocytopenia: a nationwide population-based study in France

A randomized and double-blind controlled trial evaluating the safety and efficacy of rituximab for warm auto-immune hemolytic anemia in adults (the RAIHA study)

Diagnosis and treatment of autoimmune hemolytic anemia in adults: recommendations from the First International Consensus Meeting

Mycophenolate mofetil for first-line treatment of immune thrombocytopenia

A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders

Low-dose cyclosporin A therapy in children with refractory immune thrombocytopenic purpura

Cyclosporin A in the treatment of refractory immune thrombocytopenia purpura in children

Refractory immune thrombocytopenia. Successful treatment with repeated cyclosporine A: two case reports

Efficacia del trattamento con ciclosporina A in paziente con anemia emolitica autoimmune resistente a terapia con steroidi

Glucocorticoid-resistant Evans' syndrome successfully controlled with low-dose cyclosporine

Management of autoimmune hemolytic anemia in children and adolescents: a single center experience

Alemtuzumab plus cyclosporine treatment of the autoimmune hemolytic anemia in an adult bowel transplant

Incidence and outcomes of refractory immune thrombocytopenic purpura in children: a retrospective study in a single institution

Treatment of refractory Evans syndrome with alternate-day cyclosporine and prednisone

Successful management of severe chronic autoimmune hemolytic anemia with low dose cyclosporine and prednisone in an infant

Long-term salvage therapy with cyclosporin A in refractory idiopathic thrombocytopenic purpura

Treatment with short-term, high-dose cyclosporin A in children with refractory chronic idiopathic thrombocytopenic purpura

Recombinant human thrombopoietin in combination with cyclosporin A as a novel therapy in corticosteroidresistant primary immune thrombocytopenia

A novel triple therapy for ITP using high-dose dexamethasone, low-dose rituximab, and cyclosporine (TT4)

More about the combination of rituximab, cyclosporine and dexamethasone in the treatment of chronic ITP. A useful option on an environment with limited resources

Sirolimus versus cyclosporine for the treatment of pediatric chronic immune thrombocytopenia: a randomized blinded trial

Long-term salvage treatment by cyclosporin in refractory

autoimmune haematological disorders

The effectiveness of cyclosporin A in the treatment of autoimmune hemolytic anemia and Evans syndrome

Cyclosporin A for persistent or chronic immune thrombocytopenia in children

Rituximab or cyclosporin in refractory immune thrombocytopenia secondary to connective tissue diseases: a real-world observational retrospective study

Immunological characteristics and effect of cyclosporin in patients with immune thrombocytopenia

Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients

Treatment with cyclosporin in auto-immune cytopenias in children: the experience from the French cohort OBS'CEREVANCE

Safety and efficacy of rituximab in adult immune thrombocytopenia: results from a prospective registry including 248 patients

Rituximab as second-line treatment for adult immune thrombocytopenia (the RITP trial): a multicentre, randomised, double-blind, placebo-controlled trial

Longterm safety and efficacy of rituximab in 248 adults with immune thrombocytopenia: results at 5 years from the French prospective registry ITPritux

Bone marrow characteristics predict outcome in a multicenter cohort of primary immune thrombocytopenia patients treated with thrombopoietin analogs

Prognostic impact of bone marrow fibrosis and dyserythropoiesis in autoimmune hemolytic anemia

Single-cell characterization of hematopoietic stem and progenitor cells in immune thrombocytopenia

Dissection of bone marrow microenvironment by single cell RNA sequencing in warm AIHA patients: a proof-of-concept analysis

Rituximab in immune thrombocytopenia: gender, age, and response as predictors of long-term response

Practical guidance for the management of adults with immune thrombocytopenia during the COVID-19 pandemic

Infectious complications in autoimmune hemolytic anemia

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: B.F. received consultancy from Apellis, Momenta, and Novartis and lecture fee or congress support from Alexion and Apellis. W.B. received consultancy from Agios, Alexion, Apellis, Biocryst, Bioverativ, Incyte, Momenta, and Novartis and lecture fee or congress support from Alexion, Incyte, Novartis, and Sanofi.

The authors received no financial support for the research, authorship, and/or publication of this article.