Prospective, non-randomized multicenter study on the safety and efficacy of combination therapy with bendamustine and rituximab for chronic cold agglutinin disease.

Background

Chronic cold agglutinin disease (CAD) is mediated by monoclonal cold-reactive autoantibodies that bind to erythrocyte surface antigens, causing hemagglutination and complement-mediated hemolysis. Anemia is severe in one-third of patients (hemoglobin level 8.0 g/dL or lower). Cold-induced circulatory symptoms are present in more than 90% of patients and may be disabling. CAD not associated with overt lymphoma or other disease has traditionally been classified as primary or idiopathic. A monoclonal lymphoproliferative bone marrow disorder can, however, be demonstrated by flow cytometry in 90% and by histology in approximately 75% of these patients, characterized by clonal proliferation of CD20+, kappa+ B-cells.

Approximately two-thirds of the patients experience exacerbation during febrile illnesses. During steady-state CAD, a majority of patients have low levels of complement proteins C3 and C4 because of a continuous consumption. These low levels, in particular low C4 availability, seem to be rate-limiting for hemolysis and prevent full-blown activation of the complement cascade with C5 cleavage and intravascular hemolysis. During acute phase reaction, C3 and C4 levels increase due to an enhanced production, resulting in exacerbation of hemolysis.

Counseling on cold avoidance has been recommended as the treatment of choice for most patients with primary CAD. A systematic review showed, however, that in more than 70% of cases, the physician and/or the patient did not perceive such measures as sufficient). Many standard therapies used in other autoimmune diseases or indolent lymphomas are inefficient, e.g. corticosteroids, alkylating agents, interferon-α and, probably, purine analogue single agent therapy. Treatment with the chimeric monoclonal anti-CD20 antibody rituximab has been shown in prospective studies to induce remission in more than half of patients. Almost all responses were partial, and the median response duration was less than one year. In a subsequent, prospective non-randomized trial of fludarabine and rituximab combination therapy in 29 patients, 22 patients (76%) responded, 6 (21%) achieving CR and 16 (55%) achieving PR. Among 10 patients non-responsive to rituximab monotherapy, CR was observed after the combination therapy in one patient and PR in six. Median increase in Hb level was 3.1 g/dL in the responders and 4.0 g/dL among those who achieved CR. Median time to response was 4.0 months. Lower quartile of response duration was not reached after 33 months, and estimated median response duration was more than 66 months. Targeting the pathogenic B-cell clone efficiently seems important for the achievement of remission.

Though the rituximab-based therapies have been a first major achievement in therapy for CAD, some problems remain to be solved. First, a considerable number of patients do not respond to rituximab monotherapy. Second, although the use of fludarabine and rituximab in combination has resulted in high response rates and prolonged response duration, toxicity is significant and there are still some non-responders. Third, the therapeutic approaches should probably be individualized; e.g., fludarabine therapy may bring about problems in the younger patients because of late-occurring adverse events and in the older ones because of short-term hematologic toxicity.

The antineoplastic drug, bendamustine hydrochloride, combines the 2-chlorethylamine group of the nitrogen mustard derived alkylating agents with the benzimidazole ring structures of the purine analogue. Bendamustine has been shown to be highly active in a variety of lymphoproliferative disorders. The combination of bendamustine and rituximab has produced high overall and complete response rates and prolonged remissions with only moderate and manageable toxicity in Waldenström's macroglobulinemia, which is closely related to primary CAD. In an attempt to improve on the therapeutic efficacy achieved with the rituximab-based regimens and the toxicity profile of the fludarabine-rituximab combination, the investigators want to study the safety and efficacy of bendamustine and rituximab combination therapy in patients with primary CAD.

Clinical study

The study is a prospective, non-randomized multicenter trial designed to investigate the efficacy and safety of bendamustine and rituximab combination therapy in patients with CAD requiring treatment.

Because of the low prevalence of CAD, it will be unrealistic to undertake a statistically robust randomized study. The investigators aim to include 50 patients during a study period of three years.

2.1 Study objective

The objective is to assess safety and efficacy of combination therapy with bendamustine and rituximab in patients with CAD.

Primary endpoints: Rate of complete and partial responses, respectively. Secondary endpoints: Time to response; time to best response; hematological toxicity grade 4; frequency of dose reduction; response duration.

2.2. Ethics

The trial will be carried out in accordance with the Helsinki Declaration and the current statutory requirements and legislation in the participating countries. The study has been approved by the relevant Medical Research Ethics Committees, the Norwegian Medicines Agency and the relevant regulatory authorities of each participating country. Data will be recorded anonymously, but a numeric code will be assigned in order to be able to track the data. All investigators as well as the ethics committees and relevant health authorities, however, will have access to the source data. The principal investigator in each country will establish monitoring of the study by an independent monitor as required.

2.3 Study design

Prospective, non-randomized international multicenter study.

2.4 Study population

In principle, individuals aged at least 18 years can be included, although enrollment of patients younger than 40 years is unlikely due to the age-specific prevalence of the disease.

3 The drugs

3.1 Rituximab

Rituximab is a humanized, chimeric monoclonal anti-CD20 antibody. Treatment with rituximab is considered an established, not experimental therapy for CAD, and involved physicians and nurses should be familiar with its administration, therapeutic and adverse effects and safety issues. In particular, they should be able to identify and manage any infusion-related side effects.

3.2 Bendamustine

Bendamustine is the experimental drug of this trial, marketed by Mundipharma/Norpharma. The drug is approved in the participating countries for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. It is a cytotoxic agent combining the 2-chlorethylamine group of the nitrogen mustard derived alkylating agents with the benzimidazole ring structures of the purine nucleoside analogues. Precautions and warnings include the possibility of myelosuppression, infections, skin reactions, caution in patients with cardiac disorders, nausea, vomiting, tumor lysis syndrome (very unlikely in primary CAD), anaphylaxis and extravasation.

4 Inclusion of patients.

Examination at inclusion

4.1 History.

Clinical and radiological examination Year of first occurrence of clinical symptoms is registered along with data on hemolytic anemia, circulatory symptoms, cold- or fever-induced exacerbation, previous therapies, lymph node enlargement and spleen enlargement (clinical assessment). Chest radiograph and abdominal ultrasonography should be done if not already performed during the last four months.

4.2 Blood tests

Hemolysis is detected and quantified based on Hb, MCV, reticulocyte count (x 109/L), LDH, bilirubin and haptoglobin. These measurements should be done twice during the last two months before treatment.

Hematological, biochemical and immunological assessments should be done once at inclusion + 2 weeks:

• WBC, leukocyte differential count, platelet count

• Iron, transferrin (or TIBC), ferritin, cobalamine and folate

• CRP

• Quantification of IgM, IgG and IgA Serum electrophoresis for detection and identification of monoclonal immunoglobulins (including immunofixation when appropriate)

• Cold agglutinin titer

• Specific direct antiglobulin test (DAT, direct Coombs' test), i.e. using polyspecific antiserum, anti-C3d and anti-IgG)

• Complement assessments (C3 and C4)

• CMV and VZV antibodies

• Serological tests for hepatitis B and C

• Freezing of 5 ml EDTA-blood for possible later DNA-based studies

  5 Therapy

Treatment schedule Day 1: Rituximab; 375 mg/m2 Day 1-2: Bendamustine; dosage: see Section 5.2 Day 29: Rituximab; 375 mg/m2 Day 29-30: Bendamustine; dosage: see Section 5.2 and 5.4 Day 57: Rituximab; 375 mg/m2 Day 57-58: Bendamustine; dosage: see Section 5.2 and 5.4 Day 85: Rituximab; 375 mg/m2 Day 85-86: Bendamustine; dosage: see Section 5.2 and 5.4 5.2 Initial bendamustine dose Patients with no previous cytotoxic therapy and no history of myelosuppression receive bendamustin at an initial dose of 90 mg/m2 day 1 and 2 (dose level A).

In patients who have been previously treated with a fludarabine-containing regimen, the initial bendamustine dose is 70 mg/m2 day 1 and 2 (dose level B).

In patients who have previously received myelosuppressive agents other than purine analogues, the choice of initial dose level A or B is based on an individualized assessment.

Bendamustine solution for infusion should be prepared according to the manufacturer's recommendations. The solution should be administered by intravenous infusion over 60 minutes. Antiemetic prophylaxis is given according to local routines or based on an individualized assessment.

Rituximab should be administered according to the manufacturer's recommendations and the routines of the participating hospital. Rapid (90 minutes) infusion is allowed during cycle 2-4 provided there has been no infusion-related adverse event during the preceding rituximab infusion.

For drug stability reasons, an in-line blood warmer should not be used for the infusion of bendamustine or rituximab. The infusion solution should have room temperature; and the patient, in particular the extremity chosen for infusion, should be kept warm during the procedure.

Dose adjustments

In case of significant toxicity, the dose of bendamustine should be reduced as specified by protocol. Any adverse event should be recorded in the appropriate CRF. For reporting of SAEs and SUSARs, see Protocol

6 Follow-up

6.1 Follow-up first 6 months after therapy

A) The following measurements must be done monthly during the first six months after the last cycle of any combination therapy:

I) All measurements listed in Paragraph 6.1.B.

II) In case of reduction of a previously elevated IgM level to the normal range, serum electrophoresis and immunofixation should be performed at the next visit.

III) Number of blood transfusions after the previous monthly visit. B.) At the fourth monthly visit after completion of the last combination therapy cycle, bone marrow biopsy and flow cytometric immunophenotyping of bone marrow aspirate are performed.

Inclusion Criteria:

1. CAD diagnosis defined by the combination of -

   1. Chronic hemolysis
   2. Cold agglutinin titer 64 or higher
   3. Positive direct antiglobulin test when performed with polyspecific antiserum, negative (or only weakly positive) with anti-IgG, and strongly positive with anti-C3d

2. The presence of a clonal B-cell lymphoproliferative disorder defined by -

   1. Monoclonal band by serum electrophoresis with immunofixation, and/or
   2. CD20 positive lymphocyte population with cellular kappa/lamda-ratio higher than 3.5 or less than 0.9, using flowcytometric immunophenotyping of bone marrow aspirates

3. Indication for therapy, i.e. significant anemia and/or considerable cold-induced circulatory symptoms

4. Written informed consent

Exclusion Criteria:

1. An aggressive lymphoma
2. Non-lymphatic malignant disease other than basal cell carcinoma of the skin. A history of probably cured cancer is not an exclusion criterion.
3. Known HIV infection
4. Acute or chronic hepatitis B or C
5. Liver failure or active parenchymal liver disease. Bilirubin levels higher than 51 mol/L (3.0 mg/dL) when due to hepatic impairment. Elevated serum bilirubin level due to hemolysis is not an exclusion criterion.
6. Pregnancy or breast-feeding
7. Patients of childbearing age who are not willing to use safe contraception during the entire study period and 6 months following its cessation
8. All contraindications to the study drugs will be regarded as exclusion criteria.
9. Age below 18 years
10. Inability to cooperate

Data are to be shared between authors before final analysis and publication.