Secondary autoimmune cytopenias in chronic lymphocytic leukemia

Seminars in Oncology 43 (2016) 300–310

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Secondary autoimmune cytopenias in chronic lymphocytic leukemia Kerry A. Rogers, Jennifer A. Woyachn Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH

a r t i c l e i n f o

abstract

Keywords: Chronic lymphocytic leukemia Autoimmune cytopenias Autoimmune hemolytic anemia Immune thrombocytopenia

Secondary autoimmune cytopenias in chronic lymphocytic leukemia are distinct clinical entities that require specific management. These autoimmune disorders have a complex pathogenesis that involves both the leukemic cells and the immune environment in which they exist. The mechanism is not the same in all cases, and to varying degrees involves the chronic lymphocytic leukemia (CLL) cells in antibody production, antigen presentation, and stimulation of T cells and bystander polyclonal B cells. Diagnosis of autoimmune cytopenias can be challenging as it is difficult to differentiate between autoimmunity and bone marrow failure due to disease progression. There is a need to distinguish these causes, as prognosis and treatment are not the same. Evidence regarding treatment of secondary autoimmune cytopenias is limited, but many effective options exist and treatment can be selected with severity of disease and patient factors in mind. With new agents to treat CLL coming into widespread clinical use, it will be important to understand how these will change the natural history and treatment of autoimmune cytopenias. & 2016 Elsevier Inc. All rights reserved.

1. Background and clinical experience The course of chronic lymphocytic leukemia (CLL) is frequently complicated by concomitant autoimmune cytopenias (AIC). The most common of these secondary AIC is autoimmune hemolytic anemia (AIHA), which is an antibody-mediated destruction of autologous red blood cells (RBCs). Second most common is immune thrombocytopenia (ITP), which shares some features with AIHA and has a similar mechanism targeting platelets. These two syndromes may occur in isolation, sequentially in the same patient, or present in combination as Evan’s syndrome. Pure red cell aplasia (PRCA) and autoimmune granulocytopenia (AIG) are comparatively rare and can occur alone or in combination with other AIC. PRCA can present with anemia as in AIHA, but involves a virtual absence of red cell precursors due to immune destruction of erythrocyte progenitor cells and no hemolysis. AIG has a similar mechanism to PRCA in which myeloid precursors are destroyed and patients develop infections. 1.1. Incidence While other autoimmune disorders have been reported in CLL, AIC are by far the most frequent immune complication [1,2]. n Corresponding author. Division of Hematology, 455A Wiseman Hall, 410 W 12th Ave, Columbus, OH 43210. Tel.: þ614 685 5667. E-mail address: [email protected] (J.A. Woyach).

http://dx.doi.org/10.1053/j.seminoncol.2016.02.011 0093-7754/& 2016 Elsevier Inc. All rights reserved.

Figures regarding the percentage of patients affected by secondary AIC vary. Exact numbers are difficult to ascertain as AIC can present at any time in the CLL disease course, including predating CLL diagnosis. Further complicating the issue, the rate in any given cohort will vary based on the cohort composition as AIC are associated with higher Rai stage, prior cytotoxic treatment, and more aggressive disease characteristics [3,4]. For example, heavily pretreated cohorts will have a higher incidence of AIC compared to populations enriched with asymptomatic, treatment-naïve patients. The retrospective nature of studies reporting incidence also limits their accuracy as not all patients underwent rigorous diagnostic testing for cytopenia diagnosis and some may have had cytopenias from alternate causes such as bone marrow infiltration with leukemia. Despite these challenges, a reasonable estimate is that AIC occur in 4%–10% of CLL patients with the highest reported rates coming from analysis of therapeutic clinical trials and lower estimates coming from large institutional studies [1,3–8]. This is a significant number of patients, as CLL is the most common adult leukemia with an incidence rate of 3.83 cases per 100,000 personyears. It is even more prevalent due to the long survival of CLL patients, making complicating AIC an important matter [9,10]. Relative frequency of the types of AIC is similar in nearly all reported cohorts with AIHA being the most common at 55%–70% of patients with AIC, ITP the second most common at 18%–47%, and PCRA and AIG being decidedly less common at 12% and 4%, respectively [1,3–8]. Patients prone to AIC may also develop more

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Table 1 Types of autoimmune cytopenias in CLL. Type

Clinical findings

Incidence* Mechanism

Common treatments

Autoimmune hemolytic anemia (AIHA) Immune thrombocytopenia (ITP) Pure red cell aplasia (PRCA) Autoimmune granulocytopenia (AIG)

Anemia, þ DAT, laboratory markers of hemolysis†

10%–15%

Antibody-mediated

Corticosteroids, IVIG, rituximab, rituximab w/ chemotherapy, cyclosporine

Unexplained low platelet count, increased megakaryocytes in bone marrow

2%–15%

Antibody-mediated

Corticosteroids, IVIG, rituximab, TPO receptor agonists, rituximab w/ chemotherapy

Antibody-mediated, T-cell–mediated Antibody-mediated, T-cell–mediated

Cyclosporine, alemtuzumab

n

†

Anemia, low reticulocyte count, absent red cell precursors o1% in bone marrow, no alternate cause Neutropenia, maturation arrest or absent neutrophil o1% precursors in bone marrow

Granulocyte colony-stimulating factor, cyclosporine

In CLL patients. Increased reticulocyte count, LDH, and direct bilirubin. Decreased haptoglobin.

than one, and it is not uncommon for patients to have AIHA and ITP occurring either together as Evan’s syndrome, or sequentially separated by years [1,3,8]. There are also many CLL patients who have a positive direct anti-globulin test (DAT) but no clinical evidence of hemolysis. This finding may predict risk of later developing AIHA but this does not occur in all cases [7,11]. Types and characteristics of AIC are found in Table 1. 1.2. Impact on clinical outcomes The effect of AIC on outcomes for CLL patients is a subject of debate. While patients with cytopenias due to autoimmunity clearly do better than those whose cytopenias are caused by bone marrow failure, it appears that the presence of AIC confers some negative impact with shortened survival and time to treatment [5,7]. In addition, patients with AIC may experience morbidity from anemia, bleeding, transfusion complications, and infection related to immunosuppressive treatment. CLL staging is useful for prognosis but is not straightforward in AIC patients. The widely used Rai and Binet clinical staging systems use presence of anemia and thrombocytopenia to increase stage and predict a poorer prognosis with decreased overall survival [12,13]. These staging systems do not distinguish between causes of anemia or thrombocytopenia and patients with autoimmune cytopenias receive the same stage as those with bone marrow infiltration and no evidence of autoimmune disease. Prognosis and treatment decisions should not be the same in these groups and staging was revisited in the most recent report from the International Workshop on Chronic Lymphocytic Leukemia. Cytopenias due to autoimmune causes are now not considered when assigning clinical stage to patients [3,8,14–16]. Retrospective series have examined the differences between patients with Binet stage C disease due to autoimmunity (stage C “immune”) and bone marrow failure due to leukemia infiltration (stage C “infiltrative”). In these series the overall survival for patients presenting with stage C “immune” was improved compared to those with stage C “infiltrative” or there was a trend towards improved survival [3,15,16]. However, when these patients were down-staged to Binet A after AIC directed therapy their survival was consistently worse than stage A patients without AIC and more closely approximated Binet stage B patients, suggesting that the presence of AIC does indicate a more aggressive phenotype [3,15,16]. Worse outcomes for AIC patients may be related to the association with poor prognostic features in the underlying CLL and it has been proposed that AIC are a potential marker for disease aggressiveness [15]. Both AIHA and ITP have been associated with advanced age, advanced stage, shorter lymphocyte doubling time, poor-risk cytogenetics (deletion 17p and deletion 11q), high Zap-70 expression, and unmutated IGVH status [2,17]. These are all markers

for worse survival from CLL and the association of AIC with these adverse disease features may account for some of the decreased survival seen in this group of patients [6,8,17–20]. The AIC themselves can decrease survival as patients with anemia and thrombocytopenia may experience end-organ ischemia or bleeding, which can be especially morbid in the older CLL population. In a series of secondary ITP patients, those with severe bleeding symptoms or refractory ITP had reduced overall survival compared to those who did not [8]. Infectious complications are also a major concern as risk for infection is already high in CLL patients. Morbidity due to infection has been documented in AIC patients treated with corticosteroids [6]. There are not enough patients with PRCA or AIG to determine associations with disease features or impact on survival. These patients, like those with AIHA and ITP, are at risk for morbidity from low cell counts and immunosuppressive treatments. This would logically impact their outcomes.

2. AIHA 2.1. Mechanism and pathogenesis of AIHA The mechanism of AIHA in CLL patients is not completely understood, but key clinical findings, in vitro experiments, and associations with CLL disease features give insight into pathogenesis. The CLL cells themselves have been implicated in several facets of autoimmunity including antibody production, antigen presentation, and inducing changes in T cells that favor the development of AIHA. The involvement of CLL cells in different aspects of AIHA pathogenesis is shown in Fig. 1. These mechanisms contribute more or less to the development of AIHA in different patients, accounting for variability in presentation and response to treatment. In autoimmune hemolytic anemia antibodies are produced targeting RBCs, which are then destroyed resulting in hemolysis. Most commonly the RBC targeting antibody is IgG, which coats RBCs. The IgG-coated cells are then cleared through the reticuloendothelial system in the liver and spleen. Some IgM antibodies lyse RBCs intravascularly resulting in massive acute hemolysis. As CLL cells are malignant B cells capable of producing antibody, the most concise explanation for AIHA is the malignant clone simply produces the anti-RBC antibodies directly. CLL cells do produce low amounts of polyreactive IgM and in select cases it has been demonstrated that the antibodies adherent to RBCs in circulation are of the same isotype found on the surface of the CLL cells [21–24]. However, this is a minority of cases and pathogenic antibody is usually a high-affinity, polyclonal IgG that is produced by stimulation and expansion of bystander B cells [21,22,25].

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Fig. 1. CLL cells are involved in many aspects of AIHA development. 1. They may act as antigen-presenting cells and present erythrocyte antigens to T cells. This subsequently stimulates polyclonal expansion of normal bystander B cells to produce anti-erythrocyte IgG. 2. In some cases the CLL cells produce the anti-erythrocyte antibody themselves. This is usually an IgM. 3. CLL cells induce changes in T cells that participate in or favor the autoimmune response. 4. Lastly, the B-cell receptor on some CLL cells may be stimulated by erythrocyte antigens causing activation and initiation of the response.

Normal B cells are theorized to produce this autoantibody after interacting with autoreactive T cells that have been presented with erythrocyte antigens by CLL cells. CLL cells are demonstrated in vitro to be efficient at presenting erythrocyte antigens to T cells and stimulating a response. In a series of in vitro experiments performed by Hall et al, CLL cells were able to stimulate the expansion of T cells in the presence of RhD antigen [26]. The RhD antigen was able to stimulate a greater response in the presence of CLL cells than with other antigen presenting cells. This effect was not seen for purified protein derivative (PPD), indicating an effect specific to RhD antigen. B-cell receptor (BCR) stimulation by autoantigens from blood cells may play a role in AIHA development as well. Sequence homology of the immune globulin heavy-chain variable region (IGVH) with the germline sequence is prognostic in CLL. Patients with a somatic mutation rate of o 2% are referred to as IGVH unmutated and have more aggressive disease with shorter survival [19]. This unmutated status is associated with the development of AIHA and ITP possible due to greater activity of the BCR in this patient population with a higher frequency of signaling from autoantigens. Further, a “sterotyped” heavy-chain complementary-determining (HCDR3) region in the BCR is found more commonly in AIC patients, with a particular subset (#3) increasing risk for AIHA development [17]. These subsets respond to a similar and restricted set of antigens. This may mean that subset 3 responds more efficiently to erythrocyte antigens leading to development of the immune response against RBCs. Lastly, there is clear evidence of alterations in T cells in CLL patients. This leads to immunocompromise and increased risk for infection, but also plays a role in AIC development. The exact mechanism of T-cell involvement is not well defined and is confused by reports of conflicting associations. For example, T regulatory cells (Tregs) which play a role in the suppression of autoimmune responses are reported to be both increased in CLL patients with AIC; however, T regs are decreased after fludarabine treatment, which frequently precipitates AIHA [27,28]. Further study of this relationship will be necessary and the picture is certainly more complex than our present understanding. A recent

report suggests that Th17 cells and not Tregs are implicated in ITP [29]. Further studies will be required to definitively elucidate the role of T cells in AIC development. 2.2. Association with purine analogues AIHA is famously associated with fludarabine treatment. It was frequently reported as a complication of treatment with singleagent fludarabine and has been documented with other purine analogues [30–33]. Patients who have received multiple prior chemotherapy treatments are at higher risk for this complication than treatment naïve patients and there is speculation that AIHA is related to disease factors more than fludarabine. However, the association is consistently reported and occurs at higher rates with fludarabine than when similar populations are treated with alternate regimens [30,34]. Treatment with fludarabine can trigger relapse of AIHA; single-agent fludarabine has been considered a contraindication in AIHA patients [35,36]. Development of AIHA during or after fludarabine treatment is speculated to be due to the decrease in regulatory T cells that have been shown to actively suppress autoimmunity [37]. This decrease was present in the 18 months following fludarabine treatment in one sample set and was not present after treatment with other forms of chemotherapy [27]. The whole picture may be more complicated as an increase in Tregs is seen in later stages of disease where AIHA is more common [27,28]. Understanding in this area is incomplete and the increased risk of AIHA may be due to an imbalance in T-cell subset populations. Clinically, using combination regimens containing cyclophosphamide and/or rituximab with fludarabine can mitigate the adverse effects on AIHA. In fact, patients treated with fludarabine and cyclophosphamide (FC) have a much lower incidence of AIHA than patients treated with fludarabine alone [7,38]. Further, in the prospective, randomized, phase III trial of FC compared to fludarabine, cyclophosphamide, and rituximab (FCR) both groups had a very low rate of AIHA at 1% and o1%, respectively [34]. These combination treatment regimens are both more effective and more commonly used than single-agent fludarabine and precipitating

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AIHA should no longer be a concern with modern chemoimmunotherapy. A history of AIHA should not be considered a contraindication to using FCR provided the hemolysis is controlled when starting treatment. Best clinical judgment should be used when deciding whether to discontinue fludarabine based combinations when patients develop AIHA during their use. 2.3. Diagnosis of AIHA The diagnosis of AIHA is less difficult than other AIC as evidence of overt hemolysis and a positive DAT are usually present. Patients tend to present with an acute drop in hemoglobin and strong suspicion for AIHA should be maintained for any CLL patient presenting with anemia without overt cause. An elevated reticulocyte count and features of hemolysis should be present such as elevated bilirubin, lactate dehydrogenase (LDH), and low haptoglobin. Detection of antibody on the surface of RBCs with the DAT agglutination assay is helpful in facilitating diagnosis, although DAT negative cases do occur [39,40]. A negative DAT should not preclude the diagnosis of AIHA in a patient with other findings consistent with hemolytic anemia. More sensitive tests such as mitogen stimulated DAT, flow cytometry, or enzyme-linked assays can detect autoantibody in DAT-negative cases but are not widely available [41–43]. Determination of the type of pathogenic antibody is important as it has implications for management. While in the majority of cases the pathologic antibody is a polyclonal IgG, IgM, and IgA are seen. IgM can cause both warm antibody hemolytic anemia and cold agglutinin disease [44]. Knowing which immune globulin isotypes are detected by the clinical lab is important. Most clinical labs only detect IgG, but a positive DAT for complement and not IgG points to a low-affinity or low-titer IgG or a warm IgM [44]. Cold agglutinin testing can be requested in cases where hemolysis is related to cold exposure or DAT is negative and there is clinically apparent hemolysis. Delayed transfusion reactions due to alloantibodies should to be considered in the differential diagnosis in patients with hemolysis, a positive DAT, and a history of recent blood transfusion. Anemia due to bone marrow failure should always be thought of, but is unlikely in patients with an elevated reticulocyte count. However, advanced or progressive disease may blunt reticulocyte response and obscure the diagnosis of AIHA in some cases. Laboratory markers of hemolysis can be unreliable as LDH can be elevated owing to the underlying malignancy and liver disease can complicate interpretation of bilirubin. Given the limitations of this testing it is important to consider all these factors when diagnosing the anemia. Other causes of anemia should be excluded including acute blood loss, anemia of inflammation, recent chemotherapy administration, and nutritional deficiencies such as copper, zinc, B12, and folic acid. Peripheral blood smear is helpful when classic findings of a warm autoantibody hemolysis, such as microspherocytes, reticulocytosis, and nucleated red blood cells, are present. Review of the peripheral blood smear will also exclude a thrombotic microangiopathy. Bone marrow aspiration to examine for bone marrow failure or myelodysplastic syndromes (MDS) can be helpful in more complex cases but is not mandatory when classic findings are present. 2.4. Treatments for AIHA Patients generally require treatment at the time of AIHA diagnosis. Immediate blood transfusions may be required for symptomatic anemia. However, transfusion is not an effective long-term strategy and may be difficult in patients with a warm autoantibody, making control of the hemolysis imperative.

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Selection of an appropriate treatment strategy hinges on whether or not treatment for the underlying CLL is required. For patients without treatment indications for their CLL, the first-line treatment should be immunosuppression. Only after a reasonable course of immunosuppression with more than one sequential agent has failed should consideration be given to treating the underlying CLL in order to control hemolysis [14]. If the underlying CLL also requires treatment, either the AIHA can be controlled with prednisone while CLL treatment is planned, or a regimen with some efficacy for the CLL should be chosen, such as rituximab or rituximab with chemotherapy. Supportive care is important and patients should receive appropriate infection prophylaxis and folic acid to support erythrocyte production. Laboratory markers of hemolysis should normalize and transfusion should no longer be required with effective treatment. This usually occurs in the timeframe of 1–3 weeks. There are no large prospective studies on the treatment of secondary AIHA in CLL patients and evidence for current treatments comes mostly from retrospective cohort studies. Corticosteroids are the most widely used initial treatment, although only limited data are available regarding their efficacy [14,45]. Prednisone at a dose of 1 mg/kg for 4–12 weeks is commonly used. Prednisone dose should be reduced after 2–4 weeks and subsequently tapered off over the next months if hemolysis is controlled. Pulse-dose dexamethasone is an excellent treatment for primary AIHA but not commonly used in secondary AIHA and is not well studied in CLL patients. Intravenous immunoglobulin (IVIG) is an effective initial treatment for AIHA in CLL patients and can be used alone or in conjunction with prednisone for patients in whom rapid control of hemolysis is required [46,47]. Patients needing to avoid corticosteroids due to diabetes or uncontrolled infection may want to trial IVIG as an initial therapy and forgo prednisone unless needed. If the initial treatment with steroids or IVIG is not effective, and AIHA relapses during treatment or when it is tapered, second line treatment options need to be considered. If initially effective, highdose corticosteroid treatment can be repeated while further treatment is being planned or taking effect. There are a variety of regimens that have been used in refractory patients. Table 2 contains a review of published studies reporting on treatments for AIHA or ITP associated with CLL. It is notable that nearly all reported patients had received corticosteroid treatment prior to the therapy being studied but limited evidence exists on the effectiveness of corticosteroids themselves. The anti-CD20 monoclonal antibody rituximab is quite effect for the treatment of secondary AIHA and is generally well tolerated [48,49]. Rituximab, dosed at 375 mg/m2 intravenously weekly for 4 weeks, should be considered in any patient who is refractory to corticosteroids alone or has an early relapse. Response rates are 70%–100% and it has the benefit of avoiding cytotoxic chemotherapy and providing some treatment for the underlying CLL. Rituximab works by targeting B cells, reducing production of the pathogenic antibody. It will have some effect on the CLL as well, although as a single agent at the AIHA treatment dose of 375 mg/ m2 weekly it is not as effective as other treatments for CLL [50,51]. While the anti-CD20 monoclonal antibodies ofatumumab and obinutuzumab are approved by the US Food and Drug Administration for CLL, there is limited experience with their use for AIHA. There is one case report of a CLL patient with AIHA refractory to rituximab after multiple administrations who was successfully treated with ofatumumab without AIHA recurrence [52]. This may warrant further study as a therapeutic option. Alemtuzumab, an anti-CD52 monoclonal antibody, has similarly been successfully used to treated refractory CLL at both the standard dosing of 30 mg three times a week and at a lower dose of 10 mg [53,54]. Alemtuzumab targets both B cells and T cells

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Table 2 AIC treatment studies. Author Year

Agent and dosing

Total patients AIC type

Responses*

Duration of response (mo)

Hegde 2002 [69]

Single-agent rituximab (weekly x4) – Rituximab 375 mg/m2

N ¼ 3 – ITP ¼ 3

CR ITP ¼ 2 PR ITP ¼ 1

6, 6, 17

Zaja 2003 [49]

Single-agent rituximab (weekly x4) – Rituximab 375 mg/m2

N ¼ 6 – AIHA ¼ 4 – CAD ¼ 1 – ITP ¼ 1

CR AIHA ¼ 1 CR CAD ¼ 1 CR ITP ¼ 1

NR

Narat 2005 [117]

Single-agent rituximab (weekly x4) – Rituximab 375 mg/m2

N ¼ 3 – AIHA ¼ 3

CR AIHA ¼ 1 PR AIHA ¼ 2

NR

D’Arena 2006 [48]

Single-agent rituximab (weekly x4) – Rituximab 375 mg/m2

N ¼ 14 – AIHA ¼ 14

CR AIHA ¼ 3 PR AIHA ¼ 7

NR

D’Arena 2010 [68]

Single-agent rituximab (weekly x4) – Rituximab 375 mg/m2

N ¼ 21 – ITP ¼ 19 – Evan’s syndrome ¼ 2

CR ¼ 12 PR ¼ 6

21

Gupta 2002 [57]

RCD (every 4 weeks x1-4 cycles) N ¼ 8 – Rituximab 375 mg/m2 D1 – AIHA ¼ 8 – Cyclophosphamide 750 mg/m2 IV D2 – Dexamethasone 12mg IV D1 þ2, 2 mg PO D3-7

R ¼ 8

13

Kaufman 2009 [58]

RCD (Every 3-4 weeks, x1-9 cycles) – Rituximab 375 mg/m2 D1 – Cyclophosphamide 750-1,000 mg/m2 D2 – Dexamethasone 12 mg D1-7

N ¼ 21 – AIHA ¼ 18 – ITP ¼ 1 – Evan’s syndrome ¼ 2

R ¼ 20

22

Rossignol 2011 [59] Michallet 2011 [60]

RCD Schedule 1 (every 4 weeks, N ¼ 27) – Rituximab 375 mg/m2 D1 – Cyclophosphamide 750 mg/m2 D1 – Dexamethasone 12 mg PO D1-7

N ¼ 48 – AIHA ¼ 26 – ITP ¼ 9 – Evan’s syndrome ¼ 8 – PRCA ¼ 5

CR AIHA ¼ 21 CR ITP ¼ 8 CR Evans ¼ 6 PRCA ¼ 5

24

N ¼ 20 – AIHA ¼ 11 – ITP ¼ 3 – Evan’s syndrome ¼ 4 – AIHA & PRCA ¼ 1 – AIHA, ITP, & PRCA ¼ 1

CR ¼ 14 PR ¼ 5

NR

CR AIHA ¼ 8 R AIHA ¼ 21 (CLL response: 77%)

NR

Schedule 2 (every 2 weeks, N ¼ 21) – Rituximab 375 mg/m2 D1 – Cyclophosphamide 100 mg/m2 IV D1 – Dexamethasone 40 mg PO D1 Bowen 2010 [61]

R-CVP x2-6 cycles – Dosing not reported

Quinquenel 2014 [63]

BR x6 cycles N ¼ 26 – AIHA ¼ 26 – Bendamustine 70, 90, or 100 mg/m2 D1-2 – Rituximab 375 mg/m2 D1 (increased to 500 mg/ m2 in 75% of patients)

Laurenti 2007 [53]

Alemtuzumab x30 doses (3 times per week) – 10 mg SC

N ¼ 3 – AIHA ¼ 3

R AIHA ¼ 3

10

Karlsson 2007 [54]

Alemtuzumab x12 weeks (3 times per week) – Up to 30 mg SC or IV

N ¼ 5 – AIHA ¼ 5

R AIHA ¼ 5

NR

Cortes 2001 [55]

Cyclosporin A – 300 mg PO daily continuous

†

R Thrombocytopenia ¼ 19 R Anemia ¼ 11

10

Gudbrandsdottir 2012 [73]

Romiplostim or Eltrombopag – Dosing not reported

N ¼ 3 – ITP ¼ 3

R ITP ¼ 1

9

N ¼ 31 – Thrombocytopenia ¼ 29 – Anemia ¼ 16

Table includes English language publications reporting on treatment for AIHA or ITP in CLL patients. All studies report on at least three patients and their responses to therapy. PO ¼ oral; IV ¼ intravenous; SC ¼ subcutaneous; CAD ¼ cold agglutinin disease; NR ¼ not reported. n

†

Response as defined by study authors. CR = Complete response. PR = partial response. R = response. Response reported by AIC type when known. Patients were defined and assessed separately for anemia and thrombocytopenia. Several patients had both anemia and thrombocytopenia and response of each cytopenia in the same patient was reported separately.

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reducing the immune response and CLL burden. While effective in all eight of the reported cases of use for AIHA, infectious risks limit the clinical utility of this treatment. Less toxic options are available and alemtuzumab should be considered mainly for patients who are refractory to other treatments. Cyclosporine has been evaluated in refractory cases in a prospective study of 31 patients with either autoimmune or fludarabine-associated cytopenias, as well as case reports [55,56]. In the prospective study more than half the patients had at least a good response with 63% responding. Main toxicities were increases in creatinine and hypertension [55]. Cyclosporine works by inhibiting T cells. Treatment with cyclosporine avoids cytotoxic chemotherapy and has convenient oral administration. Given its efficacy, cyclosporine is a good option for refractory patients and should certainly be considered in cases of chronic AIHA and for patients who are unable to discontinue prednisone treatment. Combination treatment with anti-CD20 monoclonal antibodies, steroids, and cytotoxic chemotherapy is an option for highly refractory patients. These combinations target the CLL as well as the immune mechanism of the AIHA. Rituximab, cyclophosphamide, and dexamethasone (RCD) was developed specifically to do just that and has relatively significant reported experience in patients with CLL-associated AIHA. It has 81%–100% response rates in patients who have received prior AIHA therapy [57–60]. The similar regimen of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) has also been used in a population of AIHA patients who required treatment for progressive CLL with high response rates [61]. However, R-CVP is no longer recommended by the NCCN Guidelines for the treatment of CLL as more effective options exist [62]. More recently, treatment with bendamustine and rituximab (BR) has been reported to be effective in patients with active hemolysis [63]. It is an excellent option for patients who require CLL treatment in addition to therapy for AIHA [64]. The relatively large series of 26 patients offers at least as much evidence as for other treatments. In this series 81% of patients had a response to BR with 31% of those achieving a complete remission. Response rate of the CLL was 77% making this an excellent choice for patients who require both AIHA and CLL directed treatment. CLL response rates to BR in patients without AIHA are high and it is well tolerated, adding to its appeal [64]. If none of the above treatments are effective or appropriate due to patient factors, treatments used for primary AIHA should be tried. Mycophenolate mofetil and azathioprine are reasonable options or can be useful as steroid sparing agents. Given the many available alternative treatments splenectomy is a last resort. Splenectomy exposes patients to the short terms risk of surgery and longer term risk of infection in already susceptible patients, but can be effective in select cases [65,66]. AIHA can relapse years after initial diagnosis [6]. When this is the case treatment considerations are similar as when deciding on an initial treatment. Frequently corticosteroids or rituximab can be repeated if previously effective.

3. ITP 3.1. Mechanism and pathogenesis of ITP The mechanism of ITP in CLL patients is similar to that of AIHA as an antibody-mediated process. ITP is less well understood with key experiments that built an understanding of AIHA not being done in ITP. The pathogenic antibody is not as easy to detect on platelets and less is known about isotypes. Further, in vitro work implicating CLL cells in antigen presentation has not been repeated in ITP.

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What is known is that ITP is associated with many of the same disease characteristics as AIHA, such as unmutated IGVH status. It is also associated with a stereotyped BCR HCDR3, but with different subsets than AIHA (#1 and #7) [67]. Similar changes in T cells are seen as in AIHA, but the issue is complicated by the fact that AIHA and ITP patients are often considered together when examining for disease associations. 3.2. Diagnosis of ITP Determining that immune destruction is the cause of thrombocytopenia in patients with CLL can be difficult. Unlike AIHA, there are no ready markers for platelet destruction. Commercially available anti-platelet antibody testing is generally unhelpful and has not been expressly studied in CLL patients. Clues that ITP is the cause of thrombocytopenia are a rapid fall in platelet count by at least 50% without administration of myelosuppressive chemotherapy or alternative explanation and lack of response to platelet transfusion in patients not known to be alloimmunized or previously refractory. Causes such as splenomegaly, medications, or thrombotic microangiopathy should be considered. Review of peripheral blood smear is helpful in excluding thromboctic microangiopathy and pseudothrombocytopenia. It is worth noting that some patients may have chronic low-level thrombocytopenia from ITP; dramatic decreases in platelet count are not always seen. In these cases immediate treatment may not be required. Bone marrow aspiration and biopsy is helpful in diagnosing ITP as the finding of increased megakaryocytes is classic. This points to immune peripheral destruction as a cause of thrombocytopenia and also excludes bone marrow failure due to leukemic infiltration. Bone marrow pathology and cytogenetics are similarly helpful in excluding a MDS in patients at risk for therapy related MDS. A bone marrow biopsy should be performed any time treatment is being contemplated on the basis of thrombocytopenia [14,62]. 3.3. Treatment of ITP The goal of ITP treatment is to prevent or treat bleeding complications. As in primary ITP, patients with stable mild thrombocytopenia may be observed and this pattern can persist for months or years without requiring intervention. Decision to start treatment should be based on the patient’s risk of bleeding and trend in platelet count. Treatment is generally considered when the platelet count falls to 20–30 K/μL or less and for bleeding symptoms. Platelet goals should be individualized to patient risk. Patients on systemic anticoagulants or antiplatelet agents and those at high risk for injury may need to initiate treatment to maintain a higher platelet level. Complete normalization of platelet count is not necessary and most patients do not have bleeding if platelet count is maintained 4 30 K/μL. The treatment paradigm is similar to AIHA where immunosuppression is used first-line provided patients do not have treatment indications for their CLL. A therapeutic trial of IVIG can also clarify the diagnosis as rapid recovery of platelet would be expected only in ITP. As in AIHA, first-line treatment is generally prednisone at a dose of 1 mg/kg for weeks with a slow taper [14]. IVIG is also an appropriate acute treatment for patients who need rapid increase in platelet count as before surgery or for bleeding, although this is usually temporary. ITP may not relapse after initial treatment in all cases, but when it does, several second line treatments are available. As with AIHA, late relapsing patients can often be retreated with corticosteroids or rituximab if effective for the first episode. Studies on the treatment of ITP in CLL patients are found in Table 2. As with AIHA, rituximab is an excellent option for refractory or relapsed patients and has similar response rates

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[68,69]. RCD has also been studied and effective, but as in AIHA should be reserved for patients who are refractory to other treatments. Cyclosporine is also useful [55]. A new and exciting option for ITP patients are the thrombopoietin receptor agonists romiplostim and eltrombopag. These agents bind to and stimulate the thrombopoietin receptor causing platelet production to increase [70]. Widely used for primary ITP, retrospective case series show responses in CLL-associated ITP [71–73]. While very few cases are reported at present, there are ongoing prospective studies evaluating the oral agent eltrombopag in this setting which have the potential to increase the usage of this class of agents in CLL patients. Since these thrombopoietic receptor agonists are not immunosuppressants, they have a low infectious risk which is attractive in CLL [74,75]. Splenectomy remains an option of last resort due to better available options and risk for infection. However, splenectomy can be an effective treatment for those patients who are refractory to medications [76]. Rh(D) immune globulin is approved for primary ITP and has a similar mechanism to IVIG of blocking clearance through the reticuloendothelial system [77]. Use of Rh(D) immune globulin is not advisable in CLL patients as it can induce anemia related to hemolysis, which can be problematic in patients with limited bone marrow reserve. Additionally, hemolysis related to Rh (D) immune globulin administration could confound any cooccurrence of AIHA. Lastly, Rh(D) immune globulin is less welltolerated in older adults and is best utilized in the pediatric population.

approaches used for AIHA and ITP may not result in similar clinical responses. 4.2. Diagnosis of PRCA The diagnosis of PRCA should be suspected in any CLL patient with normochromic anemia and decreased or absent reticulocytes. Reticulocytopenia may be relative to the degree to anemia and the absolute reticulocyte count may be normal. Anemia is typically severe and many patients require transfusions at diagnosis. PRCA can be distinguished from AIHA by low reticulocyte count and absent serum markers of hemolysis. Once suspected, the diagnosis should be confirmed by bone marrow biopsy and alternate causes must be carefully excluded. There should be no evidence of viral infection such as parvovirus B-19, human immunodeficiency virus, and Epstein-Barr virus as they can cause defects in erythropoiesis [82]. Nutritional deficiencies and MDS are considerations and should be excluded through laboratory testing and bone marrow pathology. Bone marrow biopsy is mandatory for the diagnosis of PRCA. Parvovirus infection is a known cause of severe underproduction anemia in CLL patients and bone marrow should be evaluated for evidence of parvovirus B19 infection with polymerase chain reaction or immunohistochemistry. The classic finding in PRCA is virtual absence of erythroid precursors. Characteristic defects of erythroid maturation may be seen as well. TCR gene rearrangement studies should be done on peripheral blood as T large granular lymphocytic (LGL) clones are found in combination with PRCA and have been seen in PRCA secondary to CLL [83].

4. PRCA 4.3. Treatment of PRCA 4.1. PCRA features and pathogenesis Pure red cell aplasia is distinguished from the more common AIHA by severe reticulocytopenia and lack of hemolysis. It is rare, occurring in o 1% of CLL patients and care must be taken to differentiate it from alternative causes of normocytic anemia, such as marrow infiltration with leukemic lymphocytes and nutritional deficiencies. The driving factors in the development of PRCA are different from AIHA or ITP with evidence of both an antibody mediated mechanism and direct suppression of bone marrow erythroid progenitor cells through T-cell–mediated events. Indication of IgG-mediated suppression of erythroid precursor cells comes from the description of a patient with lymphoma and PRCA where immunoglobulin purified from the patient’s serum inhibited normal bone marrow progenitor cell colony formation in vitro, whereas the non-immune globulin fraction of patient’s serum and that of healthy donors did not [78]. This supplies indirect evidence that an auto reactive immunoglobulin may be inhibiting growth of erythroid progenitor cells. The pathogenic antibody is suspected to target erythroid progenitor cells, although antibodies targeting erythropoietin directly have been demonstrated in primary PRCA [79]. More intriguing is the role of T cells in the mechanism of PRCA. Bone marrow T cells from CLL patients with PRCA have been shown to suppress erythroid colony growth from autologous bone marrow, when serum from these same patients did not [80]. The role of T cells is further supported by studies where CLL patient groups with earlier Rai stage, later Rai stage, or hypoproliferative anemia where studied. Compared to the other groups, CLL patients with hypoproliferative anemia had the largest amount of T-gamma cells in the bone marrow. T-cell depletion of the marrow in this group significantly augmented erythroid colony growth [81]. Given the involvement of T cells in this process it follows that T-cell–directed therapies may be more effective and the same

Treatment of PRCA is different from that of AIHA or ITP. Although some responses have been seen with corticosteroids and rituximab, cyclosporine is more effective and should be used preferentially over corticosteroids in patients able to tolerate it [84,85]. Most patients require long-term therapy to maintain a response and toxicity of extended treatment should be considered when selecting an agent. Infection is a major concern with cyclosporine as well as hypertension and renal insufficiency; these factors should be considered when contemplating its use. The goal of PRCA treatment is to minimize or eliminate the need for blood transfusion so as to avoid iron overload, antibody formation, and other risks associated with chronic packed RBC transfusions. Reticulocyte count should increase in 2–3 weeks and is an indication of response. Transfusion requirements may fall off over months in successfully treated patients. For those who do not respond to cyclosporine or who lose their response, alemtuzumab has been successful in a handful of cases and can be tried [86]. The RCD regimen has been reported as successful in refractory cases and is reasonable to consider if other treatments are not sufficient [59,61]. Given the limited evidence, second-line treatment can be selected based on patient factors and toxicity profile.

5. AIG 5.1. AIG features and pathogenesis Like PRCA, autoimmune neutropenia or granulocytopenia (AIG) occurs very rarely in CLL and complicates o1% of cases [87]. Highlighting the rarity of this condition, a study from a Mayo Clinic by Zent et al followed 1,750 CLL patients with respect to complicating autoimmune cytopenias and only three patients were identified with AIG. All presented with serious infections [5].

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Study of secondary AIG is difficult due to its rarity and made even more difficult as CLL patients frequently have confounding factors to which neutropenia could be attributed, such as receipt of cytotoxic chemotherapy or anti-CD20 monoclonal antibodies. Further muddling the picture, it has been speculated that AIG may not exist strictly as an isolated complication of CLL, but rather in association with T LGL clones. LGL leukemia is strongly associated with neutropenia and T LGL clones are found in the blood of CLL patients with otherwise unexplained neutropenia [88–91]. The mechanism by which LGL leukemia causes granulocytopenia is not well elucidated and may occur by more than one mechanism. In some cases it may be antibody-mediated, or mature neutrophils may be triggered to undergo apoptosis through a FAS-dependent pathway [90,92]. Rituximab usage can cause a late-onset neutropenia (LON) in patients treated for lymphoid malignancies. This should not be confused with AIG. Rituximab LON is well described as occurring approximately 6 months after last rituximab administration in patients treated with rituximab either as a single agent or in combination with chemotherapy [93–95]. Interestingly, activated T LGL clones are also found in the blood of patients with rituximab LON further solidifying their association with neutropenia [96]. Imbalance of lymphocyte populations during recovery from lymphocyte depletion is speculated to be the mechanism. Fortunately, LON secondary to rituximab only infrequently causes infectious complications and generally resolves without long-term treatment [93,95].

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at a risk for infectious complications when the absolute neutrophil count (ANC) falls below 500 /μL and action should be taken. Most experts recommend G-CSF, which can cause the ANC to increase within several days; however, the ANC often will fall once G-CSF is discontinued. In cases of rituximab LON a short course of G-CSF may be sufficient to get patients through the period of risk, but in AIG remission may not be seen. Many patients require chronic therapy for AIG or do not respond to treatment [5]. Current treatment recommendations for AIG come from case reports. There are two cases of prolonged successful treatment of AIG in CLL patients with combination rituximab and cyclosporine with continued cyclosporine administration [104,105]. One of these patients responded briefly to G-CSF, azathioprine, and IVIG prior to achievement of remission with cyclosporine. This was after suffering infectious complications [104]. As cyclosporine treats LGL leukemia and PRCA, it should be considered if AIG does not spontaneously remit or recurs after G-CSF is stopped. If cyclosporine is not effective, other agents used to treat primary AIG should be considered, including sirolimus and alemtuzumab [106,107]. Splenectomy has been undertaken for treatment of AIG, but was not highly successful and is no longer recommended with availability of more effective options [102,106,108]. Attention to supportive care and prevention of infection is important; prophylactic antibiotics should be considered in patients with an ANC of o500/μL.

6. Current areas of investigation 5.2. Diagnosis of AIG Diagnosis of AIG remains challenging and mainly focuses on excluding alternate causes of isolated neutropenia. Criteria for diagnosis recommended by D’Arena et al and include (1) persistent and “unexplained” neutropenia, (2) decreased or absent granulocyte precursors in bone marrow, (3) presence of anti-neutrophil antibodies, and (4) more than 4–8 weeks since the last chemotherapy infusion [31]. These are helpful guidelines provided confounding factors are carefully excluded. As rituximabcontaining chemoimmunotherapy is highly effective and commonly used to treat CLL, rituximab LON must be excluded by history [34,97–99]. This is important as rituximab LON can be treated with granulocyte colony-stimulating factor (G-CSF) and usually resolves spontaneously without requiring prolonged therapy [93]. MDS occurs in CLL patients as a complication of prior chemotherapy administration [100,101]. In patients who previously received purine analogues or alkylating agents, neutropenia related to MDS is a consideration. A bone marrow aspiration and biopsy in conjunction with cytogenetic studies can be highly useful in excluding MDS. Findings of a lack of myeloid dysplasia and decreased granulocyte precursors or late maturation arrest of granulocytes would be consistent with AIG. Bone marrow biopsy can also exclude neutropenia due to extensive bone marrow infiltration with CLL. Bone marrow biopsy should be considered mandatory in patients with abnormalities in peripheral blood counts aside from a low neutrophil count. Peripheral blood TCR gene rearrangement studies should be done in all cases due to the frequent association of T LGL clones and neutropenia specifically in CLL. Anti-neutrophil antibody testing can be undertaken but sensitivity and specificity are not defined in CLL [102,103]. Lack of widespread availability is another barrier to utility of this test. 5.3. Treatment of AIG Given the rarity and diversity of AIG it is not surprising that there is no high-quality evidence regarding treatment. Patients are

As new treatments are developed for CLL it will be important to understand their impact on autoimmune cytopenias. Inhibitors of BCR signaling are now in clinical use; little is known about their effect on secondary AIC. Ibrutinib is an inhibitor of Bruton tyrosine kinase (BTK), which is downstream of the BCR. In addition to demonstrating remarkable efficacy in relapsed and genetically high-risk CLL, it impacts the immune system [109–112]. Ibrutinib is now in widespread clinical use and understanding its influence on the development and treatment of secondary autoimmune cytopenias is paramount. Given the central role of BCR signaling in several aspects of the mechanism of AIHA, inhibition of BCR signaling is anticipated to have a therapeutic effect on AIC independent of controlling the underlying CLL. Ibrutinib theoretically may block stimulation by the BCR and decrease Rh antigen presentation by CLL cells. Ibrutinib also causes shifts in T-helper subsets towards a Th1 response, which is dissimilar from AIHA that is associated with a Th2 profile [112,113]. Moreover, as BTK is found in normal B cells, hypothetically, inhibition may block polyclonal expansion of bystander B cells and reduce autoantibody production. Supporting a positive effect of BTK inhibition on AIC, the reported incidence rate of AIHA or ITP in patients taking ibrutinib is low. In a cohort of 301 ibrutinib-treated patients only six cases of AIC developed. This is a low incidence in a cohort consisting of largely pretreated patients with IGVH unmutated disease. In fact 26% of these patients had a history of AIC prior to starting ibrutinib and only two relapsed while receiving the drug [114]. Further study will be necessary to determine rates of AIC during ibrutinib treatment over time and if inhibition of BTK may reduce the risk of AIC development. Towards this point, other inhibitors of BCR signaling used in the treatment of CLL have immune mediated side effects. Idelalisib is an inhibitor of phosphatidylinositol 3-kinase p110δ (PI3K), downstream from BTK in the BCR pathway. It has been associated with severe diarrhea and colitis with increased duration of use [115,116]. The colitis is similar to inflammatory bowel disease colitis and responds to steroid treatment. This may be related to

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the influence of PI3Kδ blockade on regulation of autoimmunity. Little is known about the effect of idelalisib on AIC. In addition to studying novel CLL treatments for their impact on AIC, current therapies for primary ITP and AIHA should be evaluated specifically in CLL patients. Secondary AIHA and ITP are common enough that prospective studies can be completed. This would provide information about toxicity and response rates that is currently lacking. Eltrombopag is currently undergoing prospective study and may offer a less toxic option with a lower risk of infection due to its non-immunosuppressive mechanism.

7. Conclusions Secondary AIC are frequent and important complications of CLL and have implications for the disease course and treatment. The diagnosis of secondary AIC should be considered for any CLL patient presenting with an otherwise unexplained new onset cytopenia. Current management of CLL-associated AIC is with immunosuppression and adds to treatment burden and infection risk. Evidence in this area is lacking and further study is necessary to define optimal treatment regimens. Management strategies for secondary AIC should evolve with advances in CLL treatment. New targeted drugs treating CLL have the potential to change secondary AIC as they are immunologically active agents and impact the course of the underlying malignancy. Defining optimal treatment of secondary AIC is an ongoing challenge and will be increasingly important as new advances in CLL treatments allow patients with CLL to live longer, increasing the number of years they are at risk.

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