Rituximab Therapy for Chronic LymphocyticLeukemia-Associated Autoimmune Hemolytic Anemia

Giovanni D’Arena,1* Luca Laurenti,2 Silvana Capalbo,3 Alfonso Maria D’Arco,4

Rosaria De Filippi,1 Gianpaolo Marcacci,1 Nicola Di Renzo,5 Sergio Storti,6 Catello Califano,4

Maria Luigia Vigliotti,1 Michela Tarnani,2 Felicetto Ferrara,7 and Antonio Pinto1

1Hematology and Bone Marrow Transplantation Unit, National Cancer Institute, IRCCS Fondazione ‘‘G. Pascale,’’ Naples, Italy2Hematology Department, Catholic University of ‘‘Sacred Heart’’ Rome, Italy

3Hematology Department, University of Bari, Italy4Hematology Oncology Unit, ‘‘Umberto I’’ Hospital, Nocera Inferiore, Italy

5Hematology Oncology Unit, CROB Hospital, Rionero in Vulture, Italy6Hematology Oncology Unit, Catholic University, Campobasso, Italy

7Hematology and Bone Marrow Transplantation Unit, ‘‘A. Cardarelli’’ Hospital, Naples, Italy

Autoimmune hemolytic anemia (AIHA) is a well-known complication of chronic lymphocytic

leukemia (CLL). In recent years the anti-CD20 monoclonal antibody rituximab has been used

for the therapy of steroid-refractory AIHA and autoimmune thrombocytopenia, either idio-

pathic or in association with CLL. We report the results of rituximab treatment for 14 pa-

tients suffering from CLL-associated AIHA. They developed a direct antiglobulin test positive

AIHA at a mean time of 47 months (range 0–135 months) from the diagnosis of CLL. In

3 cases AIHA was diagnosed at the same time as CLL. Only 1 patient had fludarabine-related

AIHA. All patients received steroids as first-line treatment. At a mean time of 46 days (range

1–210 days) from the diagnosis of AIHA all patients received rituximab at a dosage of

375 mg/m2/weekly for 4 weeks. All patients except 3 (2 died of cardiac failure or sepsis soon

after the third cycle and 1 HCV-positive patient experienced a rise in serum amino transfer-

ases) completed the scheduled four programmed cycles. First injection side effects of rituxi-

mab were minimal. All but 2 patients showed an increase in hemoglobin levels in response

to rituximab (mean value 3.6 g/dl; range 0.7–10 g/dl) and a reduction in the absolute lympho-

cyte count and lymph nodes and spleen volume. Nine patients required packed red cell

transfusions before starting rituximab; 5 no longer needed transfusions just after the sec-

ond cycle and another patient after the fourth cycle. Three patients (22%) were considered

to fully respond and 7 (50%) only responded partially. At a mean follow-up of 17 months,

8 patients were still alive, 6 of them transfusion-free. Our results prove that the anti-CD20

monoclonal antibody is an effective and well-tolerated alternative treatment for CLL-associ-

ated AIHA. Am. J. Hematol. 81:598–602, 2006. VVC 2006 Wiley-Liss, Inc.

Key words: chronic lymphocytic leukemia; autoimmune hemolytic anemia; rituximab

INTRODUCTION

Autoimmune hemolytic anemia (AIHA) may de-velop as a complication of, or may rarely herald, alymphoproliferative disorder [1–3]. AIHA is the bestknown autoimmune complication of chronic lym-phocytic leukemia (CLL), occurring in 10–20%cases [4,5]. Although the origin of the warm or coldautoantibodies against antigens on the red blood cellsurface in CLL-associated AIHA is controversial(malignant B-cell clone or residual normal B-lym-phocytes), steroids are the first-line treatment choice.Different immunosuppressive agents are also usedfor steroid-refractory disease. Some authors report

the prognosis of CLL-associated AIHA patients tobe poor, while others feel that this complication isunchanged for the overall survival of CLL patients[4,5]. In recent years the chimeric anti-CD20 mono-

*Correspondence to: Giovanni D’Arena, Hematology and BoneMarrow Transplantation Unit, IRCCS Fondazione ‘‘G. Pas-cale,’’ Via Mariano Semmola, 80131 Naples, Italy.E-mail: giovannidarena@libero.it

Received for publication 12 July 2005; Accepted 6 February 2006

Published online in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/ajh.20665

American Journal of Hematology 81:598–602 (2006)

VVC 2006 Wiley-Liss, Inc.

clonal antibody rituximab has been used in the ther-apy of AIHA and autoimmune thrombocytopenia,including cases with concomitant CLL [6–12]. Wereport the results of rituximab treatment in 14 pa-tients suffering from CLL-associated AIHA, mostlysteroid-refractory.

PATIENTS AND METHODS

Fourteen patients (8 males and 6 females) suffer-ing from immunologically typical CLL (CD19þ

CD5þCD23þsIglow) were treated for AIHA at ourcenters between March 2001 and September 2004.The mean age at CLL diagnosis was 64 years old

(range 48–87 years old) and 68 years old (range 48–87 years old) at AIHA diagnosis. A direct antiglo-bulin test (DAT) positive AIHA developed at amean time of 47 months (range 0–135 months) fromthe diagnosis of CLL. In 3 cases AIHA was diag-nosed at the same time as CLL. For only 1 patient(case 10, in Table I) AIHA was considered fludara-bine-related in terms of a limited time intervalwithin the use of this purine analog (1 monthbefore). In all patients, laboratory signs of hemolysiswere present (high serum levels of LDH and indirectbilirubin as well as reticulocytosis). Finally, in 2 pa-tients (cases 5 and 6) a sudden reduction of plateletlevels was observed (Fisher-Evans syndrome). Nopatient displayed monoclonal gammopathy on serumelectrophoresis or any autoantibodies that could sug-gest the presence of an autoimmune disorder otherthan AIHA. Three patients carried antibodies againsthepatitis C virus (cases 6, 11, 12) and 1 against hepa-titis B virus (case 7).All patients received steroids at therapeutic dos-

ages (methylprednisolone 1-1,5 mg/kg body wt daily)as first-line treatment.As second-line treatment of AIHA, some patients

were given intravenous Cytoxan (cases 6, 7 and 8)and in 1 case (case 8) 2 mg of vincristine was intra-venously infused. After a mean time of 46 days(range 1–210 days) from the diagnosis of AIHA allpatients received rituximab (Mabthera, Roche S.p.A.,Milan, Italy) at a dosage of 375 mg/m2/weekly for4 weeks. All patients remained on steroids when rit-uximab was started, and the dose was tapered after2 weeks in responding patients. Moreover, the doseof steroids was not increased in nonresponding sub-jects. Eleven of 14 patients were considered steroid-refractory because they did not experience a rise inhemoglobin level and/or a stable improvement oftheir AIHA after therapy. In 3 patients (cases 10, 11,and 12) the anti-CD20 monoclonal antibody wasgiven a few days after starting steroids (mean 4 days;range 1–6 days). In all 3 patients receiving immuno-

suppression therapy, Cytoxan and vincristine werestopped before rituximab was administered.Response criteria to rituximab treatment for AIHA

were defined as follows: (a) complete response: nor-malization of hemoglobin levels, transfusion-free andabsence of clinical and laboratory signs of hemolysis;(b) partial response: rise in hemoglobin levels � 2 g/dl,transfusion-free either without or reduced trasfusionrequirement, and improvement of clinical and labora-tory signs of hemolysis.

RESULTS

Table I shows hemoglobin (g/dl) levels, platelets(PLT) � 103/L and absolute lymphocyte count(LC) � 103/L, before and after rituximab treatment.All but 3 patients (cases 6 and 8, who died of car-diac failure and sepsis, respectively, soon after thethird cycle, and case 12, who was HVC-positive andexperienced a rise in AST and ALT serum levels),completed the scheduled four cycles. First infusionside effects of rituximab were mild (fever in 1 caseand fever and chills in another case). All but 2patients (cases 8 and 10) showed an increase in Hblevels (mean value 3.6 g/dl, range 0.7–10 g/dl) inresponse to rituximab. In 3 patients the steroids andrituximab cycles were begun within a few days ofeach other. Thus, we cannot draw a firm conclusionthat any response noted in these 3 patients is onlydue to rituximab. At the same time, a reduction inabsolute lymphocyte counts and in the size of lymphnodes and spleen was observed. Nine patientsrequired packed red cell transfusions before startingrituximab. Five of them became transfusion-freeafter the second infusion while a 6th patient becametransfusion-independent after the fourth cycle. Fourpatients had converted from a positive to a negativeDAT by the end of the scheduled four rituximabdoses (cases 1, 2, 3, and 9). Only 3 patients (22%)were considered complete responders and 7 (50%)partial responders. Three patients continued with amaintenance program of rituximab with differentschedules: four weekly infusions every 6 months(case 1); one infusion every 4 weeks for 6 months(case 5); 4 weekly doses every 4 months (case 9).Two patients, both complete responders (cases 1and 9), received further rituximab infusions in orderto treat CLL, not for AIHA. Finally, at a mean fol-low-up of 17 months, 8 patients were still alive, 6 ofthem transfusion-free (Table I).

DISCUSSION

CLL is considered either a malignant disease or acomplex immunologic disorder. In fact, an autoim-

599Rituximab for CLL-Associated AIHA

American Journal of Hematology DOI 10.1002/ajh

TABLEI.

PersonalCharacteristicsofEachIndividualPatient,Responseto

4-W

eekRituxim

abTherapyforAIHA,andFollow-up

UPN

12

34

56

78

910

11

12

13

14

Gender

MF

FF

MF

MM

MM

FM

FM

Ageat

CLL/A

IHA

diagnosis

57/57

63/66

63/72

75/79

66/69

78/82

87/87

59/60

48/48

77/79

61/71

52/56

57/68

54/55

Raistage

atCLL

diagnosis

IVIII

0II

II0

IVII

III

IIIII

IVIV

0

Therapyfor

CLLbefore

AIH

A(in

sequence)

CHOP

CVP

Fludarabine

CHL

CHL

CHL

CHOP

CHL

None

CHL

CHOP

Flu-C

y

CHL

Fludarabine

CHL

CHL

Fludarabine

CHL

Fludarabine

FN

Deossico-

form

icin

None

Therapyfor

AIH

Abefore

rituxim

ab

(insequence)

MPL

1mg/kg

(69days)

MPL

1mg/kg

(29days)

MPL

1mg/kg

(210days)

MPL

1mg/kg

(22days)

MPL

1mg/kg

(15days)

MPL

1mg/kg

(44days)

Cy(23days)

MPL

1mg/kg

(40days)

Cy(20days)

MPL

1,5

mg/kg

(50days)

Cy(24days)

VCR (16days)

MPL

1mg/kg

(126days)

MPL

1,5

mg/kg

(1day)

MPL

1mg/kg

(6days)

MPL

1mg/kg

(4days)

MPL

1mg/kg

(10days)

MPL

1mg/kg

(16days)

Hgb(g/dl)pre/

post

rituxim

ab

11.7/13.6

6.6/9.6

6.3/10.2

7.8/10.8

7.4/10.4

7.4/8.1

4.2/5.6

6.4/4.8

5.0/15

5.2/5.2

5.7/9.3

5.7/9.8

6.5/12

7.5/11.5

LC

(�103/ml)pre/

post

rituxim

ab

19.2/1.4

16/1.6

1.3/1.1

16.6/0.6

78.4/8.7

38/1.0

33/8.3

92.5/3.2

98/4.1

3.0/14.3

29.7/38.3

6.7/8.8

25.3/0.8

4.0/2.8

PLT(�

103/ml)pre/

post

rituxim

ab

79/123

192/233

297/200

237/196

15/12

9/13

148/139

41/7

154/178

133/155

236/238

65/89

51/69

150/170

No.ofweekly

rituxim

ab

infusions

44

44

43

43

44

43

44

Response

to

rituxim

ab

CR

PR

PR

PR

PR

NR

NR

NR

CR

NR

PR

CR

PR

PR

DATafter

four

dosesofrituxim

ab

Negative

Negative

Negative

Positive

Positive

Positive

Positive

Positive

Negative

Positive

Positive

Positive

Positive

Positive

Sideeffectsto

rituxim

abinfusions

Fever

chills

None

None

Fever

None

None

None

None

None

None

None

None

None

None

Follow-up(m

onths

since

AIH

A

diagnosis)

49

27

17

27

22

34

315

724

24

10

13

Dead/alive

Alive

Dead

Alive

Alive

Dead

Dead

Dead

Dead

Alive

Alive

Dead

Alive

Alive

Alive

Cause

ofdeath

—CLLprog-

ression

——

Sepsis

Cardiac

failure

Cardiac

failure

Sepsis

——

CLL progression

——

—

CLLstatus

Disease

progression

—Stable,no

therapy

Stable,no

therapy

——

——

Stable,no

therapy

Refractory

disease

—Stable,no

therapy

Stable,no

therapy

Stable,no

therapy

Transfusion

status

Dependenton

transfusion

—Independent

from

transfusion

Independent

from

transfusion

——

——

Independent

from

transfusion

Dependenton

transfusion

—Independent

from

transfusion

Independent

from

transfusion

Independent

ontransfusion

Note:CR,complete

response;PR,partialresponse;NR,noresponse;Hgb,hem

oglobin;LC,lymphocytes;PLT,platelets;MPL,methylprednisolone(thedose

reported

isthattakingwhen

rituxim

abwasinitiated;thetimeintervalbetweenthe

initiationofsteroid

andtheinitiationofrituxim

abtherapyis

inparentheses);Cy,cytoxan(thetimeintervalbetweenthelast

dose

infusedandtheinitiationofrituxim

abis

inparentheses);VCR,vincristine(thetimeintervalbetweenthedose

infused

and

theinitiation

ofrituxim

ab

isin

parentheses);

Flu,fludarabine;

FN,fludarabine,

mitoxantrone;

Flu-C

y,fludarabine,

Cytoxan;CHL,chlorambucil;

CHOP,cytoxan,Adriamycin,oncovin,prednisone;

CVP,cytoxan,oncovin,

prednisone.

mune derangement is hypothesized with the so-called paradoxical finding of an immune deficiencystatus along with an excess of autoimmune phenom-ena [4]. Moreover, warm type AIHA is the mostcommon autoimmune disease associated with CLL,which is considered the most common cause ofAIHA [4,13]. Previous studies reported AIHA as apoor prognostic indicator in CLL [14,15]. However,more recently, Mauro et al., by means of multivari-ate analysis, found that AIHA has no effect on sur-vival [5].Historically, first-line treatment of autoimmune

complications in CLL was immunosuppression bymeans of glucocorticoids. Cytoxan, vincristine, intra-venous immunoglobulins, and splenectomy have alsobeen used, mainly in cases with steroid-refractoryAIHA.More recently, the chimeric anti-CD20 monoclo-

nal antibody rituximab has been used in the manage-ment of steroid-refractory AIHA, including CLL-associated cases, and has shown significant activity.This molecule is active against normal and neoplasticB-lymphocytes that express the CD20 antigen, in-cluding B-CLL cells, despite their lower density ofCD20 compared to normal B-lymphocytes, and inother B-lymphoproliferative disorders [16]. How rit-uximab works in CLL-related AIHA is not fullyunderstood. This molecule is a human-mouse chi-meric monoclonal antibody specific for the CD20antigen, present on the surface of normal and malig-nant B-lymphocytes, and acts through complement-dependent cytotoxicity and antibody-dependent cellu-lar cytotoxicity [17]. B-cell depletion is thus inducedby rituximab. Red blood cell autoantibodies are gen-erally polyclonal, suggesting the productive role ofnormal B-cells. In other cases remnant normal B-lymphocytes seem to produce autoantibodies [18]. Ina very recent study, Hall et al. showed that malig-nant B-cells themselves were able to present Rh auto-antigen to helper T-cells in CLL. In light of this, rit-uximab-induced B-cell depletion in CLL could simul-taneously eliminate the antigen presenting B-cells aswell as the B-cells producing the pathogenic autoanti-bodies [19].Few CLL patients have been treated with rituxi-

mab for AIHA. The largest series was published in2002 by Gupta et al. [7]. They reported their experi-ence with eight CLL-related steroid-refractory AIHApatients using the so-called RCD treatment regimen.Rituximab, Cytoxan, and dexamethasone were givenat 4-week intervals for two to five cycles until thebest response was obtained. All eight patients ac-hieved remission of AIHA. Subsequenty, Zaja et al.used weekly rituximab at a dosage of 375/m2 intrave-nously for 4 weeks to treat four CLL patients with

warm AIHA [8]. Only one patient achieved completeremission. Several other authors have mentionedcases with encouraging results [9–12].We treated 14 patients with B-CLL-related warm

AIHA with a weekly rituximab infusion schedulefor 4 consecutive weeks. In our series, a responserate of 72% was observed (3 complete and 7 partialresponses). Most importantly, no significant sideeffects to rituximab were observed, even in elderlypatients (82 and 87 years old, respectively). In fact,neither one experience side effects to rituximab, butdied of cardiac failure due to severe anemia. In1 patient who responded partially and was carryingserum antibodies against the hepatitis C virus, a risein serum ALT and AST levels prevented continua-tion of the treatment after the third cycle. The rela-tionship between the rise in amino transferases andrituximab infusion is not clear in view of the factthat safe use of the anti-CD20 monoclonal antibodyhas been reported in HCV-infected patients, albeitwith an increase in viremia [20].The relationship between fludarabine and CLL-

associated AIHA is still a controversial issue. Sev-eral studies reported that AIHA is more frequentlyobserved in heavily pretreated CLL patients, whilethe incidence in subjects receiving fludarabine asfirst-line treatment is not different from that ob-served in untreated patients [21–25]. In light of this,it appears ever more evident that AIHA is mostprobably due to an immune dysregulation with lossof lymphocytic control of aberrant autoimmuneclones rather than to the direct drug action. How-ever, fludarabine appears to be effective by inducingan imbalance in the CD4 and CD8 ratio, whichcould be involved in the loss of immunological con-trol of autoimmune phenomena. As far as our pa-tients are concerned, a relationship between fludara-bine use and AIHA was found only in 1 case. More-over, he exhibited no response to rituximab.In conclusion, we have reported our experience

with rituximab-induced B-cell depletion as treatmentfor CLL-related AIHA. A response to this therapyhas been obtained in 72% of patients, with no life-threatening or relevant toxicity, even in the elderly.However, further investigation is needed in order toidentify the optimal administration schedule andduration of treatment.

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