The aim of this multicenter GIMEMA study was to correlate autoimmune complications (AIC) in B-cell chronic lymphocytic leukemia (B-CLL) with stage and therapy. Autoimmune hemolytic anemia (129/194 cases) and autoimmune thrombocytopenia (35/194 cases) were typically present in advanced and multi-treated disease. Age over the median, stage C and first and second line therapy were identified as independent risk factors by multivariate analysis. In contrast, non-hematologic AIC (30/194 cases) and the presence of serological markers of autoimmunity were mostly observed in early B-CLL, suggesting different pathogenic mechanisms underlying hematologic and non-hematologic autoimmune phenomena in B-CLL

A. Ambrosetti

A. Cortelezzi

A. Zanella

E.M. Pogliani

F. Mandelli

F.R. Mauro

G. Girelli

L. Laurenti

P. Pedotti

R. Calori

R.M. Agostinelli

S. Capalbo

V. Liso

W. Barcellini

R. M. Agostinelli

F. R. Mauro

E. M. Pogliani

AIR Universita degli studi di Milano

Relationship between Autoimmune Phenomena and Disease Stage and Therapy in B-Cell Chronic Lymphocytic Leukaemia

haematologica/the hematology journal | 2006; 91(12) | 1689 |Disorders of Erythropoiesis • Brief ReportThe aim of this multicenter GIMEMA study was to correlate autoimmune complications(AIC) in B-cell chronic lymphocytic leukemia (B-CLL) with stage and therapy.Autoimmune hemolytic anemia (129/194 cases) and autoimmune thrombocytopenia(35/194 cases) were typically present in advanced and multi-treated disease. Age overthe median, stage C and first and second line therapy were identified as independentrisk factors by multivariate analysis. In contrast, non-hematologic AIC (30/194 cases)and the presence of serological markers of autoimmunity were mostly observed inearly B-CLL, suggesting different pathogenic mechanisms underlying hematologic andnon-hematologic autoimmune phenomena in B-CLL.Key words: B-CLL, autoimmunity, AIHA, AITP, fludarabine.Haematologica 2006; 91:1689-1692©2006 Ferrata Storti FoundationFrom the Department ofHaematology and Molecular andGenetic Epidemiology Unit (PP),Fondazione IRCCS OspedaleMaggiore Policlinico, Mangiagalli eRegina Elena, Milan, Italy (WB, RC,AZ, AC); Haematology, Universityof Bari (SC, VL); Istituto di ClinicaMedica, University Politecnica delleMarche, Ancona (RMA); Departmentof Haematology, Blood Bank,University La Sapienza, Rome (GG);University La Sapienza, Rome(FRM, FM); Department of Clinicaland Experimental Medicine, Sectionof Haematology, University of Verona(AA); Haematology, UniversityCattolica del Sacro Cuore, Rome(LL); Haematology Unit, SanGerardo Hospital, Monza (EMP);University of Milan (AC).Correspondence:Wilma Barcellini, MD, Departmentof Haematology, Padiglione Granelli,Fondazione IRCCS OspedaleMaggiore Policlinico, Mangiagallie Regina Elena, Via F. Sforza 3520122 Milan, Italy.E-mail: wbarcel@policlinico.mi.itThe relationship between B-cell chron-ic lymphocytic leukemia (B-CLL) andthe immune system is widely recog-nised.1 The disease is characterized byimmunodeficiency, hypogammaglobuline-mia and a high prevalence of autoimmunephenomena mainly restricted to blood cellself-antigens.2,3 Autoimmune hemolytic ane-mia (AIHA) and thrombocytopenia (AITP)are known complications of B-CLL, whereasthere are only few reports on other autoim-mune diseases and on serological markers ofautoimmunity.2-4 The occurrence of autoim-mune cytopenias was commonly considereda poor prognostic factor,5 although, morerecently, this has been questioned.6,7 Reportson other autoimmune phenomena are anec-dotal.8-10 No studies exist that correlate non-hematologic autoimmune diseases and thepresence of autoantibodies with the clinicalstage of B-CLL and its therapy. The aim ofthis multicenter GIMEMA study was todetermine the presence of different autoim-mune complications (AIC) in B-CLL (hema-tologic cytopenias, other autoimmune dis-eases, and serological markers of autoimmu-nity) and their relationship with B-CLL stageand therapy.Design and MethodsCase series and diagnosis of AICThe diagnosis and staging of B-CLL wereperformed according to the National CancerInstitute criteria and the Binet staging sys-tem.11,12 AIC were recorded prospectivelybetween October 2001 and May 2003 (30cases), and retrospectively through chartreviews (164 cases) among all the B-CLLpatients regularly followed (total 3150) by 18hematology centers of the GIMEMA group.Age, gender, and B-CLL stage and therapywere recorded before the occurrence of theAIC, to avoid confounding AIHA and AITPon B-CLL stage. First line therapy consistedof chlorambucil with or without steroids;second line treatment mostly consisted ofcyclophosphamide with or without ananthracycline or in fludarabine-based regi-mens. The diagnosis of AIHA was depend-ent on the presence of a positive directantiglobulin test (DAT), that of AITP wasmade according to the British Committee forStandards in Haematology,13 and the diagno-sis of other AIC was made according to stan-dard clinical and immunological criteria.14The same information collected for thepatients with AIC (age, gender, stage andtherapy) was also recorded for 434 other B-CLL patients (all the patients of the threemain participating centers because datawere not available for all 3,150), consideredas the control B-CLL group. This study wasperformed after obtaining informed consentfrom patients and approval from the institu-tional Human Research Committee. Theprocedures followed were in accordancewith the Helsinki international ethical stan-dards on human experimentation.Serological markers of autoimmunitySerological studies were performed in 227consecutive patients of the 434 B-CLLRelationship between autoimmune phenomena anddisease stage and therapy in B-cell chroniclymphocytic leukemiaWilma BarcelliniSilvana CapalboRosa Maria AgostinelliFrancesca Romana MauroAchille AmbrosettiRossella CaloriAgostino CortelezziLuca LaurentiEnrico Maria PoglianiPaola PedottiVincenzo LisoGabriella GirelliFranco MandelliAlberto Zanellafor the GIMEMA Chronic LymphocyticLeukemia Group| 1690 | haematologica/the hematology journal | 2006; 91(12)W. Barcellini et al.patients without AIC forming the control group. Thefollowing antibodies were detected by standard tech-niques:15 anti-nuclear (ANA), anti-smooth muscle(SMA), anti-mitochondrial (AMA), anti-parietal cells(PCA), and anti-liver-kidney-microsomal antibodies(LKM) by indirect immunofluorescence on Hep-2; anti-DNA antibodies by indirect immunofluorescence onCrithidia luciliae; anti-extractable nuclear antigen (ENA,in particular, SS-A, SS-B, Sm, RNP, Scl-70), anti-thy-roglobulin (anti-Tg), anti-thyroid peroxidase (anti-TPO),anti-neutrophil cytoplasmic (ANCA), anti-cardiolipinIgG and IgM (ACA), and anti-β-2-glycoprotein 1 IgGand IgM antibodies (β2GP1) by ELISA; rheumatoid fac-tor by standard nephelometry.Statistical analysisDifferences in distribution of stage and treatmentamong groups were evaluated using the χ2 test.Multivariate analyses were performed using a logisticmodel. Computations were performed with the SASstatistical package (8.1 Version, SAS Institute Inc., Cary,NC, USA). p values lower than 0.05 were consideredstatistically significant.Results ad DiscussionAIC in B-CLL patientsThere were 194 cases of AIC. The most frequent formof AIC was AIHA (129 cases, 66%), followed by AITP (35cases, 18%), and 30 other autoimmune diseases (16%):bullous pemphigus (n=9), Hashimoto’s thyroiditis (n=8),rheumatoid arthritis (n=4), systemic lupus erythematosus(n=1), autoimmune glomerulonephritis (n=1), autoim-mune gastritis (n=1), Sjögren’s syndrome (n=1),polymyositis-dermatomyositis (n=1), vasculitis (n=1),autoimmune polyneuropathy (n=1), ulcerative colitis(n=1), and Raynaud’s disease (n=1). As for idiopathicAIHA, the great majority of cases of B-CLL-associatedAIHA (89%) were due to warm autoantibodies(IgG+DAT); only 11% were cold hemagglutinin diseasesdue to IgM autoantibodies (anti-C’+DAT).Relationship between AIC and B-CLL stage andtherapyThe statistical analysis was conducted on the 164 retro-spectively identified cases of AIC. The small group of 30new cases was considered separately in the statisticalanalysis because these differed significantly for clinicalstage and therapy from the retrospective group. By com-paring patients with and without AIC (Table 1) we foundthat the former had a higher median age (p=0.03), weremostly in advanced disease stage and had already beentreated with first and second line therapy (p<0.001).Multivariate analysis showed that age >69 years (medianage), stage C and prior administration of first and secondline therapy emerged as independent factors significantlyrelated with the occurrence of AIC. Considering the rela-tionship between the different types of AIC andstage/therapy (Table 2), we found that the majority ofpatients with AIHA were in stage C whereas cases ofAITP were equally distributed according to stage and theTable 1. Clinical data of B-CLL patients with or without autoim-mune complications (control B-CLL).Control B-CLL Autoimmune Multivariate(n=434) complications analysis(n=164) OR 95%CIAge (years) 68.3±10.3 70.3±9.6 2.78 1.75-4.43Gender M/F (%) 239/195 (55/45) 85/79 (52/48) 0.73 0.47-1.13Stage A (%) 291 (67) 53 (32) 1.00 RefStage B (%) 100 (23) 39 (24) 1.40 0.83-2.37Stage C (%) 43 (10) 72 (44) 2.87 1.64-5.04No therapy (%) 272 (63) 11 (7) 1.00 RefFirst line therapy (%) 110 (25) 80 (49) 13.33 6.70-26.51Second line therapy (%) 52 (12) 73 (44) 27.48 12.77-59.14Age is given as mean ± SD; OR: odds ratio; CI: confidence intervals.Table 2. Clinical data of the B-CLL patients with the differentautoimmune complications. Multivariate analysis of 113 patientswith AIHA.AIHA AITP Other(n=113) (n=28) autoimmunecomplications(n=23)OR CLAge (years) 71.4±9.1 4.85 2.65-8.89 68.4±11.3 67.2±9.2Gender 60/53 0.75 0.45-1.26 16/12 9/14M/F (%) (53/47) (57/43) (39/61)Stage A (%) 26 1.00 Ref 10 17(23) (36) (74)Stage B (%) 26 2.12 1.13-4.01 9 4(23) (32) (17)Stage C (%) 61 6.54 3.43-12.78 9 2(54) (32) (9)No therapy 2 1.00 Ref 0 (0) 9(%) (2) (39)First line 58 47.87 11.31- 13 9therapy (%) (51) 202.68 (46) (39)Second line 53 99.34 20.57- 15 5therapy (%) (47) 479.81 (54) (22)II line-treated 28 91.32 19.28- 0 0with (25) 432.52 (0) (0)fludarabine (%)Age is given as mean ± SD; OR: odds ratio; CL: confidence limits.haematologica/the hematology journal | 2006; 91(12) | 1691 |Autoimmunity in B-CLLother autoimmune diseases were mostly observed instage A (p<0.001). As regards chemotherapy, both AIHAand AITP were almost exclusively observed in patientswith first and second line-treated B-CLL, and the otherautoimmune diseases were equally distributed. We evalu-ated in particular the relationship between AIHA and sec-ond line therapy with fludarabine. Fludarabine-treatedpatients accounted for 28/53 and 33/52 of those with AICand control B-CLL patients, respectively. Multivariateanalysis showed that the relative risk of developing AIHAwas similar in patients treated with second line therapywith or without fludarabine. As found for all AIC, ageabove the median, disease progression (stage B and C) andtherapy intensification (first and second line) emerged asindependent risk factors for AIHA. To minimize theeffects of disease progression on the analysis, we focusedon stage A patients only. Multivariate analysis confirmedthat variables more associated with AIHAwere age abovethe median (OR 3.43, CI 1.22-9.63), first line therapy (OR15.62, CI 5.00-48.82), and second line therapy (OR 48.64,CI 10.00-239.19).Prospective studyThis group was mainly composed of stage A (54%) anduntreated (67%) patients, beingmore similar to the controlgroup of B-CLL patients without AIC. For this reason andbecause of the small number of cases the distribution ofthe different AIC among stages and treatments was notsignificant. However, even in this small group, multivari-ate analysis showed that stage C emerged as an independ-ent factor significantly related with the occurrence of AIC(OR 4.78, CI 1.61-14.17).Follow-up of clinical AICFollow-up data were available for 116/194 cases of AIC.Patients with AIHA, AITP, and the other autoimmune dis-eases recovered in 65%, 54%, and 40% of cases, respec-tively. It should be noted that 14/33 (42%) cases of refrac-tory AIHA were fatal and that death was directly due tothe hemolytic crisis in 5/14.Prevalence of serological markers of autoimmunityWe found that 93/227 (41%) of B-CLL patients, mostlyin stage A, had at least one positive test for a marker ofautoimmunity, distributed as follows: 36 ANA, 25 ACA,23 RF, 23 anti-TPO/anti-TG, 20 SMA, and 10 miscella-neous (AMA, anti-DNA, anti-PCA). Considering all posi-tive autoimmune phenomena grouped as positive serolog-ical autoantibodies only, hematologic diseases (AIHA andAITP), and non-hematologic diseases (Figure 1), there wasa statistically significant distribution with respect to theclinical stage (p<0.001). In fact, serological and clinical non-hematologic complications were mostly observed in stageA patients, whereas more than 70% of hematologic com-plications were found in patients with advanced disease(B+C stage).The results of this study showed that AIC occur inroughly 5% of patients with B-CLL. The more frequentcomplication was AIHA, as already reported,2-4 followedby AITP and other autoimmune diseases, the latter twowith a similar prevalence. AIC were associated withadvanced and multi-treated disease, as demonstrated bymultivariate analysis, which identified age over themedian, stage C and first and second line therapy asindependent risk factors. This finding is in line with therecently reported close correlation between ZAP-70expression and incidence of autoimmune cytopenias inB-CLL.16Considering only AIHA, the more frequent andpotentially fatal complication, multivariate analysisgave comparable results, adding stage B to stage C, andconfirming first and second line therapy as independentrisk factors. Also considering stage A patients only, toavoid the confounding effect of anemia of advanced andmulti-treated disease, variables more associated withthe presence of AIHA were age over the median, andfirst line and second line therapy. Interestingly, multi-variate analysis showed that patients treated with orwithout fludarabine have a comparable risk of AIHA, atvariance with the reported association between AIHAand fludarabine treatment,17,18 but in agreement withrecent observations by Catowsky et al.19The novel finding of our study is that the distributionof the different AIC differed with respect to stage andtherapy, in that AIHA and AITP were typically presentin advanced and multi-treated disease, whereas thenon-hematologic autoimmune diseases and serologicalmarkers of autoimmunity were mostly observed inearly B-CLL. Hematologists are usually familiar withAIHA and AITP, but less so with non-hematologic AIC.The latter complications, even if usually less severe thanhematologic ones, should be carefully searched for, par-ticularly in patients with early B-CLL. Although it can-not be excluded that steroids and cytotoxic drugs mayFigure 1. Distribution of autoimmune phenomena grouped intohematologic complications (AIHA and AITP, ), non-hematologicdiseases (), and serological autoantibodies only (), χ2 test:p<0.001.90%80%70%60%50%40%30%20%10%0%stage A stage B stage Csuccessfully treat non-hematologic AIC so that they areless prominent in more heavily treated patients, this dif-ferent distribution of complications is open to otherinterpretations. Hall et al.20 demonstrated that malignantB-CLL cells are able to present purified Rh protein toautoreactive T-helper cells, driving an autoimmuneresponse against erythrocytes. Autoimmunity againstblood cells might, therefore, reflect a late event in thenatural history of B-CLL, related to the accumulation ofmalignant B-CLL antigen-presenting cells. In contrast,non-hematologic AIC, as well as serological autoimmu-nity, might have a different pathogenic mechanism,involving deficient control by normally-competent Tcells, already present in early disease stage.AppendixOther participating centres and physicians in the GIMEMAstudy were: Marco Candela, Giovanni Danieli, UniversitàPolitecnica delle Marche, Ancona; Giovanni Pizzolo,Università degli Studi, Verona; Giuseppe Leone, UniversitàCattolica del Sacro Cuore, Roma; Albano Del Favero,Università degli Studi, Perugia; Francesco Zaja, RenatoFanin, Università degli Studi, Udine; Federico Chiurazzi,Bruno Rotoli, Università Federico II, Napoli; MaurizioLonginotti, Università degli Studi, Sassari; Sara Galimberti,Mario Petrini, Università degli Studi, Pisa; Luigi Gugliotta,Arcispedale Santa Maria Nuova, Reggio Emilia; NunzioFilardi, Francesco Ricciuti, Ospedale S. Carlo, Potenza;Tambone Reyes, Ospedale di Cefalù; Daniele Dini, GiuseppeTorelli, Università degli Studi, Modena; Rossella Paolini,Ospedale di Rovigo.WB designed the study and prepared the manuscript; SC con-tributed by collecting the patients’ data collection and with labora-tory work; RMA, FRM, AA, RC, LL, GG, EMP helped to collectthe patients’ data; PP conducted the statistical analysis; AC, VL,FM, and AZ contributed to discussion of the manuscript.Manuscript received April 6, 2006. Accepted September 26,2006.W. Barcellini et al.| 1692 | haematologica/the hematology journal | 2006; 91(12)References1. Caligaris-Cappio F, Hamblin TJ. B-cellchronic lymphocytic Leukemia: a birdof a different feather. J Clin Oncol1999; 17:399-408.2. Hamblin TJ, Oscier DG, Young BJ.Autoimmunity in chronic lymphocyticleukaemia. J Clin Pathol 1986;39:713-6.3. Rozman C, Montserrat E. Chronic lym-phocytic leukaemia. N Engl J Med1995;333:1052-7.4. Diehl LF, Ketchum LH. 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Relationship between autoimmune phenomena and disease stage and therapy in B-cell chronic lymphocytic leukemia

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Relationship between autoimmune phenomena and disease stage and therapy in B-cell chronic lymphocytic leukemia

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