B cells CD19+ in patients with B‐cell chronic lymphocytic leukaemia and autoimmune haemolytic anaemia

The study presents results of B and T lymphocytes population analysis in patients with chronic lymphocytic leukaemia B cells and autoimmune haemolytic anaemia (CLL‐B + AIHA). We evaluated the following groups of patients: (1) with newly recognized CLL‐B and co‐existent AIHA (untreated), (2) after short‐term treatment with corticosteroids, (3) after treatment with chemotherapy and corticosteroids. The control groups were made of patients with CLL‐B without AIHA. The populations of lymphocytes and determination of cells immunophenotype were performed by means of flow cytometry. The analysed data were obtained from 25 patients. The untreated patients with CLL‐B + AIHA presented significantly more numerous population of neoplastic cells CD19+ CD5+ in comparison with patients without AIHA. The patients with AIHA showed a reduced percentage of B CD19+ CD22+ cells in comparison with those without AIHA. Untreated patients with AIHA or after a short‐term corticosteroid treatment showed a higher ratio of the number of CD19+ CD5+ cells to the number of T CD4+ and T CD8+ lymphocytes than CLL‐B patients without AIHA. It can be presumed that the differences found may be related to the pathogenesis of the autoimmune haemolysis syndrome in patients with CLL‐B.     

CTLA-4 exon 1 polymorphism in patients with autoimmune blood disorders

CTLA-4 is a CD28 homologue that plays an important role in negative regulation of T-cell responses. Its transient expression on the surface of activated T cells antagonizes the activating signals and terminates the T-cell response. An A to G polymorphism at position 49 of the CTLA-4 first exon has recently been associated with several autoimmune disorders. In the present study we have examined the prevalence of the A and G alleles of the CTLA-4 gene in 50 patients with autoimmune hemolytic anemia (AIHA), of which 20 had idiopathic AIHA and 30 had AIHA and chronic lymphocytic leukemia (CLL), and in 60 patients with immune thrombocytopenic purpura (ITP). Control subjects were 100 healthy individuals and 100 CLL patients without clinical evidence for an autoimmune disease. The G allele was present at a significantly higher frequency among the patients with AIHA (P = 0.003), whereas no difference was observed between patients with ITP and controls. The G allele frequency was highest among CLL patients who had developed AIHA. The obtained data indicate that the G allele of CTLA-4 predisposes to the development of AIHA, particularly among patients with CLL.     

The successful treatment of refractory autoimmune hemolytic anemia with rituximab in a patient with chronic lymphocytic leukemia

The most frequent autoimmune complication in chronic lymphocytic leukemia (CLL) is autoimmune hemolytic anemia (AIHA). There are various treatment modalities; however, there is not much experience with the use of the chimeric anti-CD20 monoclonal antibody rituximab in the autoimmune complications of CLL. Here, we present our patient with CLL and AIHA whose AIHA was unresponsive to various treatment modalities. The administration of 375 mg/m(2)/day rituximab weekly for four cycles halted hemolysis and resulted in resolution of the patient's anemia. One year after therapy, the patient is well with a normal blood count. Rituximab might be preferred over other treatment modalities in the autoimmune complications of CLL because it is effective and has fewer side effects than other therapies.     

Autoimmune haemolytic anaemia emerging during Campath treatment in a patient with CD5 negative chronic lymphocytic leukaemia

Campath is being employed for the treatment of autoimmune haemolytic anemia (AIHA) whether or not associated to B cell chronic lymphoid leukaemia (CLL). CD5 negative CLL is relatively uncommon and runs an indolent course. We report a CD5 negative CLL patient who developed AIHA associated with cytomegalovirus infection reactivation whilst on treatment with Campath for progressive disease.     

Rh autoantigen presentation to helper T cells in chronic lymphocytic leukemia by malignant B cells

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune diseases directed against constituents of the blood, including hemolytic anemia (AIHA). We hypothesized that CLL cells predispose to hematologic autoimmunity by acting as aberrant antigen-presenting cells (APCs). Initially, it was confirmed that all studied patients with AIHA secondary to CLL harbored activated helper T (T(H)) cells specific for epitopes on the dominant red blood cell (RBC) autoantigens in primary AIHA, the Rh proteins. Rh-specific T(H) cells were also detected in a number of patients with CLL who, although they did not have AIHA, had low levels of anti-RBC antibody in their sera. Fractionation of putative APC populations from the peripheral blood of patients by negative selection showed that CD5+ CLL cells are the most effective cell type in processing and presenting purified Rh protein to autoreactive T(H) cells. This ability was confirmed using positively selected CD5+ CLL cells. Thus, our study provides the first evidence for malignant cells driving an autoimmune response by acting as aberrant APCs.     

Autoimmune haemolytic anaemia in patients with chronic lymphocytic eukaemia treated with 2-chlorodeoxyadenosine (cladribine)

Abstract: Autoimmune haemolytic anaemia (AIHA) is one of the major complications in chronic lymphocytic leukaemia (CLL). Treatment with alkylating agents and the adenosine analogue, fludarabine, might trigger the development of AIHA in CLL patients despite the reduction of leukaemic clone. The influence of 2-chlorodeoxyadenosine (2-CdA) on AIHA in patients with CLL is undefined so far. In a group of 114 patients treated at our clinics with this agent, AIHA with direct antiglobulin test (DAT) positively was observed in 25 (21.9%) patients. In 23 patients AIHA was noticed before the starting of 2-CdA and in 2 patients DAT became positive after 2-CdA treatment. In 6 patients the drug caused complete resolution of haemolysis and DAT became negative. Eight patients exhibited partial resolution of haemolysis with significant improvement in haemoglobin level but DAT test remained positive. In 11 patients there was no response to 2-CdA in relation to autoimmune haemolysis. Two patients with no previous history of haemolysis developed AIHA after 5 and 6 courses of 2-CdA therapy and their DAT became positive 1–2 months after the last course of the drug. One of them entered severe haemolytic crisis and severe thrombocytopenia and died because of haemorrhagy to the central nervous system. In the other patient AIHA was controlled by steroids and chlorambucil treatment. Our study indicates that 2-CdA may suppress autoimmunohaemolytic process in some patients with CLL and trigger the development of AIHA in others.     

A prospective study of positive/negative ex vivo B-cell depletion in patients with chronic lymphocytic leukemia

OBJECTIVE: Autologous peripheral blood stem cell (PBSC) transplantation is increasingly being used in patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have prospectively investigated the feasibility and efficacy of ex vivo double purging of PBSC grafts in an open, nonrandomized, single-center phase I/II clinical study. MATERIALS AND METHODS: Twenty consecutive patients with poor-risk CLL underwent uniform stem cell mobilization with chemotherapy and granulocyte colony-stimulating factor (G-CSF). Double B-cell depletion of the harvested PBSC products was performed using immunomagnetic CD34(+) cell selection (Isolex300i Nexell, Irvine, CA) followed by a negative step with anti-CD19/20/23/37-labeled immunomagnetic beads. The purified PBSC were reinfused after myeloablative treatment with TBI/CY. RESULTS: A total of 25 separation runs was accomplished using collection products containing 3.4% (1.1-8.1) CD34(+) cells and 1.2% (0.1-42) CD19(+)CD5(+) CLL cells. After double selection, 33% (15-67) CD34(+) cells were recovered with a purity of 98.8% (89.1-99. 8). CLL cells were undetectable by high-resolution flow cytometry in 15 of 25 final products; median purging efficacy was 5 (4.1-6) log. The CD34(+) content of the 20 final grafts was 4.6 (2.2-6.5) x 10(6)/kg. Rapid and durable engraftment developed in all cases. With a median follow-up of 20 (6-29) months, 17 patients live in complete clinical remission, two have recurrent disease, and one patient died due to pulmonary embolism five months after transplant. Persistence of the leukemic clone on the molecular level was demonstrated by dot blotting with clone-specific CDR3 probes in an additional five patients. Serious or unexpected infectious complications did not occur. CONCLUSIONS: Positive/negative purging with the Isolex system allows preparation of highly purified CD34(+) fractions and up to six log of tumor cell depletion in patients with B-CLL and can be safety reinfused after myeloablative therapy without affecting hematopoietic engraftment.     

Treatment of pure red cell aplasia and autoimmune haemolytic anaemia in chronic lymphocytic leukaemia with Campath-1H

A patient with chronic lymphocytic leukaemia (CLL) progressive on fludarabine therapy and life-threatening anaemia related to immune haemolysis and pure red cell aplasia was treated with Campath-1H. The patient had sustained complete remission of both CLL and anaemia, but died of recurrent sepsis and cachexia 10 months after completion of the treatment. Campath-1H (alemtuzumab), a humanised anti-CD52 monoclonal antibody, is a potent therapeutic agent against advanced CLL and immune cytopenias. It could be indicated in the treatment of severe immune complications of CLL unresponsive to corticosteroids. Prolonged immunosuppression is a serious side-effect leading to severe infectious complication.     

Autoimmune cytopenia does not predict poor prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is characterized by an acquired immune defect that can cause autoimmune complications, including anemia and thrombocytopenia. We conducted an observational study of the epidemiology, clinical presentation and significance of autoimmune complications of CLL/SLL in 132 patients from a large population (>45,000 veterans), in which at least 90% of patients with CLL/SLL have been previously identified. Over a period of 12.5 years, 12 patients (9.1%) had autoimmune complications; of these, 6 (4.5%) had autoimmune hemolytic anemia (AIHA), 5 (3.8%) had immune thrombocytopenia (ITP), and 1 (0.8%) had pure red blood cell aplasia (PRBA). All 6 cases of AIHA had a positive direct immunoglobulin test for IgG and C3d. In 6 patients, CLL/SLL was an incidental finding at the time of presentation with autoimmune cytopenia. Nine out of 10 patients responded to immunosuppressive therapy, which was complicated by serious infection in 7 cases, one of which was fatal. The major cause of mortality in patients with autoimmune complications of CLL/SLL was secondary malignancy. Survival of patients with immune cytopenia was not significantly different from CLL/SLL patients without immune cytopenia. Among patients with anemia or thrombocytopenia, mortality was significantly higher in those with bone marrow failure compared to an autoimmune etiology. We show that in a non-referred population with a high incidence of CLL/SLL, autoimmune cytopenia can occur early in the natural history of the disease. These data suggest that the Rai and Binet classifications for CLL need to be modified for patients with autoimmune cytopenia. .     

Restricted immunoglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia

Between 10% and 25% of chronic lymphocytic leukemia (CLL) patients have episodes of autoimmune hemolytic anemia (AIHA) during the course of their disease. The anti-erythrocyte autoantibodies in most cases are polyclonal and express a different heavy chain isotype than the malignant clone, indicating that they are secreted by normal autoreactive B lymphocytes. To further investigate the pathogenesis of the AIHA in CLL, we analyzed the lg heavy (H) chain variable region genes expressed by leukemic cells from CLL patients with and without AIHA. Two VH genes were preferentially expressed by the leukemic cells in the CLL cases with AIHA and were present in 9 of the 12 investigated cases. The 51p1/DP-10 gene was expressed in 5 of these cases and was absent in the control group of 12 consecutive CLL cases without AIHA, whereas the DP-50 gene was present in 4 CLL-AIHA cases and only once in the control CLL group. A strikingly similar H-chain CDR3 region that contained a single reading frame of the DXP4 DH gene segment, and N- encoded proline at the DH/JH boundary, and a tyrosine-rich region encoded by the JH6 gene segment was observed in four CLL-AIHA cases. The preferential expression of two VH gene segments and a particular CDR3 region by the leukemic cells of patients with AIHA suggests that the antibodies produced by the CLL cells are directly involved in the pathogenesis of the hemolytic anemia.     

Mixed-Type Autoimmune Hemolytic Anemia following Fludarabine Treatment in a Patient with Chronic Lymphocytic Leukemia/Small Cell Lymphoma

Background and objectives : Mixed-type autoimmune hemolytic anemia (AIHA) is a rare complication of chronic lympocytic leukemia (CLL). We report a patient with small lymphocytic lymphoma (phenotypic CLL) who developed symptomatic anemia 3 weeks after her fifth cycle of fludarabine, a T cell immunosuppressant. Materials and methods : An antibody screen and panel, direct antiglobulin test, rapid acid eluate, rabbit erythrocyte stroma (RESt) adsorption, and autoadsorption were performed. Results : Warm and cold autoantibodies were detected. Prompt treatment with corticosteroids and minimal blood transfusions led to marked improvement. Conslusion : Normally, T cells suppress polyclonal lymphocytes that produce autoantibodies. Suppression of T cells in this patient, in addition to the underlying disease process, may explain this mixed-type AIHA, the first reported case to occur following fludarabine treatment.     

Concomitant chronic lymphocytic leukemia and acute myeloid leukemia with an uncommon immunophenotype

We report a case of simultaneous diagnosis of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), in which the use of flow cytometry analysis allowed the demonstration of two different cell populations and the study of both immunophenotyping patterns with a large panel of monoclonal antibodies (MoAbs). CLL cells showed a typical immunophenotype with coexpression of B cell markers with CD5, CD23, CD43, and weak surface immunoglobulin light chain restriction expression, whereas the AML population had a very uncommon phenotype with expression of myeloid markers and CD56 and lack of expression of other natural killer (NK) antigens, CD34 and HLA-DR. After chemotherapeutic treatment of AML with two induction courses, the patient achieved complete remission of the AML with persistence of a CD19/CD5 positive population. After consolidation chemotherapy, this latter population was no longer detectable despite the presence of lymphoid nodules in a bone marrow biopsy. Six months after diagnosis, the patient relapsed with AML and died shortly afterwards. Am. J. Hematol. 56:281–287, 1997. © 1997 Wiley-Liss, Inc.     

Two-step immunoablative treatment with autologous peripheral blood CD34(+) cell transplantation in an 8-year-old boy with autoimmune haemolytic anaemia

Life-threatening haemolysis in children with autoimmune haemolytic anaemia (AIHA) occurs rarely. Many cases of severe autoimmune disease are currently treated with immunosuppressive high-dose chemotherapy and autograft. We report here a case of a child with severe AIHA who did not respond to conventional treatments, but was cured with an autologous peripheral blood CD34(+) cell transplantation. After d 16 post autograft, no further red cell transfusions were required. At 20 months post autograft, haematological complete remission persists.     

Interferon maintenance therapy for patients with chronic lymphocytic leukemia in remission after fludarabine therapy

Many patients with chronic lymphocytic leukemia (CLL) achieve remission after treatment with fludarabine chemotherapy. Most of these patients, however, later experience relapse. In addition, immunologic deficits may persist even in patients in complete remission; lymphopenia, predominantly involving the CD4 population, is universal after fludarabine therapy. We used recombinant alpha interferon (IFN-alpha) maintenance therapy in patients with CLL who achieved remission in response to fludarabine therapy to determine its effect on residual disease, assessed by either bone marrow biopsy or flow cytometry, and on immune restoration. Thirty-one patients were treated with IFN-alpha (3 x 10(6) U by subcutaneous injection three times weekly). Twenty-two patients (71%) were in complete remission (CR) and nine (29%) were in partial remission (PR). Of the 22 patients in CR, 21 (95%) had evidence of residual disease at the start of IFN-alpha therapy. Low CD4 levels were noted in 93% of patients, low IgG levels in 45%, and anergy or hypoergy in 52%. Only one patient in PR achieved a CR on IFN-alpha therapy: the only patient who had had no prior fludarabine but had been treated with chlorambucil and prednisone. All patients in CR with minimal residual disease had persistent disease after IFN-alpha treatment. There were no increases in CD4 counts or IgG levels; three patients with borderline responses to skin testing had an increase in the number of positive tests while on IFN-alpha. The time to progression was no different in patients treated with IFN-alpha than in a historical control group of patients who had received no further therapy after fludarabine. In summary, the use of IFN-alpha maintenance did not eradicate residual disease, restore immune function, or prolong remissions in patients with CLL responsive to fludarabine.     

Partially successful treatment of a patient with chronic lymphocytic leukemia and Hodgkin's disease: case report and literature review

Chronic lymphocytic leukemia (CLL) is rarely associated with Hodgkin's disease (HD). We report a case of nodular sclerosis HD in a patient previously diagnosed with CLL. Reed-Sternberg cells were CD15(+) and CD30(+). He was treated with dose-escalated CHOP and at relapse, mitoxantrone, vinblastine, and CCNU (MVC) with partial response to the former and complete response to the latter, although the patient died 15 months later. Data from 88 other similar cases published in the English language were analyzed. Based on the histological and clinical features at the time of transformation, these patients were divided into distinct categories for analysis. Prognosis was found to be poorer in patients with continued active CLL when compared with those with CLL in remission at the time of transformation to HD. It is suggested that these two presentations may derive from different pathogenic mechanisms.     

Immune anaemias in patients with chronic lymphocytic leukaemia treated with fludarabine, cyclophosphamide and rituximab--incidence and predictors

Immune anaemias (IA) [auto-immune haemolytic anaemia (AIHA) and pure red cell aplasia (PRCA)] are complications of chronic lymphocytic leukaemia (CLL). Fludarabine has been associated with AIHA, whereas both rituximab and cyclophosphamide have been used to treat this condition. Combining these agents with fludarabine may reduce the likelihood of AIHA. We report on the incidence, outcome and pretreatment predictors of IA in 300 patients treated with fludarabine, cyclophosphamide and rituximab (FCR). Nineteen patients (6.5%) developed IA [AIHA (5.8%), PRCA (0.7%)] on or after treatment with FCR. Most patients (82.4%) with AIHA had a negative direct antiglobulin test (DAT). Additional markers of haemolysis (indirect hyperbilirubinaemia, reticulocytosis, low haptoglobin and elevated lactate dehydrogenase levels) confirmed the presence of AIHA in these patients. The majority of patients responded to therapies including steroids, ciclosporin, i.v. immunoglobulin, etc. High pretreatment levels of beta-2 microglobulin predicted for development of IA. No haemolytic crisis was observed during FCR therapy in eight patients with AIHA prior to FCR. Thus, the incidence of IA among CLL patients treated with FCR was comparable with historical rates. The diagnosis of AIHA can be considered even if the DAT is negative. Pre-existing AIHA need not preclude front-line FCR therapy.     

Allogeneic blood or marrow transplantation for chronic lymphocytic leukaemia: timing of transplantation and potential effect of fludarabine on acute graft-versus-host disease

The outcome of allogeneic haemopoietic transplants including the rate of immune complications for patients with chronic lymphocytic leukaemia (CLL) refractory to or relapsing after chemotherapy with fludarabine was analysed.
Fifteen patients with advanced CLL who received allogeneic transplantation were prospectively analysed. All patients had previously received chemotherapy with fludarabine for 3–15 courses; 12 were refractory. The median number of circulating CD4^{^plus;} and CD8^{^plus;} lymphocytes at the time of transplant was 0.49 ×10⁹ l and 0.23 ×10⁹ l, respectively. One patient was transplanted from a one HLA-antigen mismatched unrelated donor. Three others received a one or two antigen mismatched graft and 11 had HLA-identical sibling donors. Patients received cyclosporine or tacrolimus in addition to methotrexate or methylprednisolone for prophylaxis of acute graft-versus-host disease (aGVHD).
Fourteen patients engrafted; one patient had graft failure, but recovered after therapy with intravenous immunoglobulin. 13 (87%) achieved complete remission (CR). Nine (53%) remain alive and in CR with a median follow-up of 36 (range 3–60) months. None developed visceral graft-versus-host disease. These data compared favourably to published reports in other leukaemia patients and for patients with CLL who received a comparable immunosuppressive therapy but without prior fludarabine exposure.
This data indicates that allogeneic haemopoietic transplantation can induce durable remission in patients with CLL refractory to fludarabine and that it is reasonable to delay transplantation until failure of fludarabine therapy. It also suggests that prior exposure to fludarabine may decrease the incidence of severe aGVHD, possibly through its immunosuppressive effects. Further studies are warranted to evaluate this observation.     

De novo CD5-positive diffuse large B-cell lymphoma associated with autoimmune hemolytic anemia presenting as erythroid hypoplasia

A 54-year-old woman was admitted due to high-grade fever, cervical lymphadenopathy and general malaise in May 2003. On examination, severe anemia was noted, direct Coombs and cold hemagglutinin tests were positive and the haptoglobin level was low in the peripheral blood. However, a bone marrow examination revealed marked erythroid hypoplasia. A diagnosis was made of co-existing combined type autoimmune hemolytic anemia (AIHA) and erythroid hypoplasia. A pathologic diagnosis of de novo CD5-positive diffuse large B-cell lymphoma (de novo CD5+ DLBCL) was made based on a cervical lymph node biopsy. The patient was treated with CHOP accompanied by rituximab (R-CHOP), resulting in complete remission after 3 courses of chemotherapy. The AIHA and erythroid hypoplasia subsided after 2 courses of R-CHOP. The sera obtained during erythroid hypoplasia significantly inhibited the growth of erythroid progenitor cells (erythroid colony-forming units, CFU-E) from her bone marrow collected after recovery. We report here a patient with de novo CD5+ DLBCL associated with both AIHA and erythroid hypoplasia, suggesting that the lymphoma triggered an abnormal immunity which generated some humoral inhibitors against erythropoiesis.     

Successful treatment with fludarabine of chronic lymphocytic leukemia associated with nephrotic syndrome

We successfully treated a patient with chronic lymphocytic leukemia (CLL) associated with a nephrotic syndrome. An 82-year-old man had been diagnosed as having CLL and been under observation for a year without treatment. In January, 2001, he developed hypoprotenemia, proteinurea, and edema in the extremities and face. With the exacerbation of the symptoms, he was admitted to our hospital in March of the same year. Under the diagnosis of nephrotic syndrome with CLL, the patient underwent induction therapy for CLL with fludarabine (13 mg/m2/day for 4 days), which brought about a complete remission of CLL and the disappearance of the edema. To our knowledge, this was the first case in Japan where fludarabine was dramatically effective in treating both CLL and the nephrotic syndrome. This result indicated that fludarabine is beneficial for not only CLL but also complications like nephrotic syndrome.     

Fatal intravascular autoimmune hemolytic anemia after fludarabine treatment for chronic lymphocytic leukemia

We report the case of a patient with chronic lymphocytic leukemia (CLL) who developed fatal intravascular autoimmune hemolytic anemia (AIHA) after fludarabine treatment. He had previously received several treatments including two courses of fludarabine. The direct antiglobulin test (DAT) was negative at diagnosis but was found to be positive with anti-IgG after the first fludarabine treatment. When the patient was treated again with fludarabine nine months later, the DAT became positive with anti-IgG and anti-C3d antiglobulins after the second course of treatment. Abrupt, fatal intravascular hemolysis occurred after the third course. The occurrence of severe AIHA in CLL patients treated with fludarabine has been reported by several authors. Physicians should be aware of the risk of severe AIHA in CLL patients with a history of AIHA or positivation of the DAT during previous fludarabine administration, or in case of secondary fixation of complement to the red cell membrane occurring during fludarabine treatment.