Abstract

Background: Follicular lymphoma is considered incurable, although cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy can induce sequential remissions. A patient's second complete response is typically shorter than that patient's first complete response. Idiotype vaccines can elicit specific immune responses and molecular remissions in patients with follicular lymphoma. However, a clinical benefit has never been formally proven. Methods: Thirty-three consecutive follicular lymphoma patients in first relapse received six monthly cycles of CHOP-like chemotherapy. Patients who achieved a second complete response were vaccinated periodically for more than 2 years with autologous lymphoma-derived idiotype protein vaccine. Specific humoral and cellular responses were assessed, and patients were followed for disease recurrence. Statistical tests were two-sided. Results: Idiotype vaccine could be produced for 25 patients who had a second complete response. In 20 patients (80%), a humoral (13/20) and/or a cellular (18/20) idiotype-specific response was detected. The median duration of the second complete response has not been reached, but it exceeds 33 months (range = 20+ to 51+ months). None of the 20 responders relapsed while undergoing active vaccination. All responders with enough follow-up for the comparison to be made experienced a second complete response that was statistically significantly ( P <.0001) longer than both their first complete response (18 of 18 patients) and than the median duration of a CHOP-induced second complete response, i.e., 13 months (20 of 20 patients). The five nonresponders all had a second complete response that was shorter (median = 10 months; range = 8–13 months) than their first complete response (median = 17 months; range = 10–39 months). Conclusions: Idiotypic vaccination induced a specific immune response in the majority of patients with follicular lymphoma. Specific immune response was associated with a dramatic and highly statistically significant increase in disease-free survival. This is the first formal demonstration of clinical benefit associated with the use of a human cancer vaccine.

Follicular lymphoma is an indolent B-cell malignancy for which no treatment has proved consistently curative ( 1 ) . A number of conventional therapeutic options can induce relatively durable and sequential clinical complete responses in the vast majority of patients ( 2 ) . However, second and subsequent responses tend to be progressively shorter than those achieved previously ( 2 , 3 ) . In particular, most second complete responses obtained through standard chemotherapy (such as monthly treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP] regimen) last far less than 2 years, and the median duration is 13 months ( 4 , 5 ) .

Several immunotherapeutic approaches to treatment of follicular lymphoma are currently under investigation ( 6 ) . One is idiotype vaccination, which is based on the fact that the immunoglobulin on the surface of each patient's cancer cells contains a unique idiotype protein that can serve as a tumor-specific antigen ( 7 ) . Over the past 15 years, idiotype vaccines have proved capable of eliciting both humoral ( 810 ) and cellular ( 910 ) idiotype- and tumor-specific responses in a variable percentage of patients with follicular lymphoma, particularly when used to consolidate a chemotherapy-induced first complete response ( 9 , 11 ) . Some of these immune responses have been found to translate into molecular remissions, with circulating follicular lymphoma cells positive for Bcl-2/IgH rearrangements no longer detectable in patients in whom standard chemotherapy had achieved only clinical complete response ( 9 , 12 ) . These results are consistent with those of a recent study that found that tumor-associated T-cell infiltration may influence clinical progression of follicular lymphoma ( 13 ) . However, no formal proof of the clinical efficacy of idiotype vaccination has emerged ( 10 ) . Evaluation of the efficacy of a customized patient-specific intervention is a challenge for conventional randomized trial methodology because it is difficult to make valid comparisons when the experimental intervention is unique to each patient ( 14 ) .

In what is, to our knowledge, the largest and most homogeneous series of follicular lymphoma patients in first relapse treated with standard chemotherapy plus idiotypic vaccination, we investigated the association between the ability of a soluble protein idiotype vaccine to induce any idiotype- and/or tumor-specific immune response and the objective extension of relapse-free survival in follicular lymphoma patients. In addition to systematically monitoring both vaccine-induced immune responses and minimal residual disease in all vaccinated patients, we also compared the outcome of each patient's first and second complete response, so that each patient could effectively serve as his or her own matched control subject.

P ATIENTS AND M ETHODS

Study Participants

Between October 1, 2001, and June 30, 2004, 33 consecutive patients with follicular lymphoma who were experiencing their first relapse were enrolled in this open-label, phase II, single-group, multicenter study. Eligibility criteria at relapse were as follows: confirmed diagnosis of grade I–IIIa follicular lymphoma (grade IIIa was included only if the patient had presented with a lower histologic grade at the time of first diagnosis); age between 18 and 65 years; Ann Arbor stage II–IV disease; measurable disease; a harvestable lymph node that measured at least 2 × 2 cm; documentation of first clinical complete response by bilateral bone marrow biopsy and whole-body computed tomography (CT) scan or, in case of an ambiguous CT scan result, positron emission tomography (PET) scan; Karnofsky performance status of at least 70%; life expectancy of more than 1 year; and negative serology for human immunodeficiency virus, hepatitis virus B, and hepatitis virus C. All enrolled patients provided written informed consent.

Study Design

The study was carried out according to both the Helsinki Declaration and the institutional review board–approved protocol. Patients who had not previously received anthracyclines were treated with six monthly cycles of chemotherapy with CHOP. Patients who had previously received anthracyclines received six monthly cycles of chemotherapy with cyclophosphamide, mitoxantrone, vincristine, and prednisone (CNOP). Cardiac function was assessed by planar multigated radionuclide angiography whenever patients were restaged for their disease—i.e., before treatment, after three cycles of treatment, and at the end of treatment. Standard staging procedures included bone marrow biopsy and CT scan. Patients with an equivocal restaging CT scan result always underwent PET scan. Local radiation therapy (25 Gy) was administered to all patients who had a single residual location of active disease, other than bone marrow involvement, after chemotherapy. Patients who achieved a partial response—i.e., residual measurable disease and/or residual bone marrow involvement—received autologous stem cell transplantation with carmustine, etoposide, cytosine arabinoside, and melphalan (BEAM) conditioning. All treatments were administered at the centers where the patients had been enrolled.

Patients who achieved a second clinical complete response were to subsequently receive four monthly idiotypic vaccinations, followed by one boost 2 months later and then by five more boosts given every 3 months. We chose this extended vaccination schedule, whose duration was twice as long as the expected median duration of the second clinical complete response induced by monthly treatment with CHOP or a CHOP-like regimen ( 25 ) , so that idiotype vaccines would be given during the peak interval in which relapse would be expected if the vaccine were ineffective. Patients began vaccination only after they had recovered normal numbers of circulating CD3 + , CD4 + , CD8 + , and CD19 + lymphocytes after chemotherapy. Vaccinations were discontinued immediately in any patient who had a documented clinical relapse during the active vaccination period.

Vaccine Production and Administration

Idiotype vaccine production was carried out in the Cell Therapy Area of the University Clinic of Navarra and administered to all patients at the Day Hospital of the same institution. Patient- and tumor-specific idiotype proteins were isolated from tumor cells using the rescue hybridoma method previously described ( 15 , 16 ) . The isolated idiotype proteins were coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde as described ( 8 ) . The final vaccine formulation (0.5 mg of idiotype and 0.5 mg of KLH) was administered subcutaneously, together with 125 μg of granulocyte–macrophage colony-stimulating factor (GM-CSF) as an adjuvant ( 9 , 10 ) . Over the 3 days following each vaccination, GM-CSF alone (125 μg) was injected daily at the vaccination site ( 9 ) .

Clinical Response and Toxicity Evaluation

After three and six cycles of chemotherapy and following either local radiation treatment or autologous stem cell transplantation, if administered, all patients underwent a complete restaging evaluation, as described above, at their enrolling center. Disease was restaged in all patients who received the idiotype vaccine before the first and after the fifth vaccination. Subsequently, disease status was reevaluated every 6 months, including 6 months after completion of the vaccination schedule, or at the time of any suspected relapse. Relapses were always documented by tissue biopsy. Clinical response and toxic effects were defined according to the International Working Group recommendations ( 17 , 18 ) and the National Cancer Institute Common Toxicity Criteria (NCI CTC version 2.0), respectively.

Humoral Response Evaluation

To detect vaccine-induced idiotype-specific humoral immune responses (i.e., specific humoral responses), enzyme-linked immunosorbent assay (ELISA) was used as previously described ( 811 ) . In particular, a specific humoral response was recorded whenever a fourfold increase in anti-idiotype titer was found in postvaccine serum compared with both prevaccine serum and a panel of isotype-matched, irrelevant idiotype protein controls from other patients with follicular lymphoma. Vaccine-induced anti-KLH humoral responses were also monitored by ELISA. Humoral responses were assessed after each vaccination, 6 months after scheduled vaccine completion, and at the time of any relapse. All experiments were performed by personnel blinded to both clinical data and the patient's identity.

Cellular Response Evaluation

Idiotype- and tumor-specific cellular immune responses (i.e., specific cellular responses) were assessed by up to five independent methods in which cryopreserved peripheral blood mononuclear cells (PBMCs) were used as effector cells: T-cell proliferation assay, cytokine release ELISA, interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, cytotoxicity assay, and flow cytometry detection of effector cell activation (details provided below). When idiotype protein or tumor cell availability was insufficient, fewer than five methods were used. Because no single method is currently accepted as guaranteeing the existence of a bona fide cellular response, an overall specific cellular response was considered to have occurred whenever such a response was detected by at least two independent methods at any time before the administration of the second boost. When a cellular response was detected through only one method, that response was considered to be doubtful and was categorized as “no response” for analysis. Vaccine-induced anti-KLH cellular responses were also monitored within the same analyses. Most assessments were performed after each vaccination, including 6 months after scheduled vaccine completion and at the time of any relapse. All experiments were performed by personnel blinded to both clinical data and the patient's identity. Cut points for calling a response positive were chosen a priori and were based on common standards in the field.

Proliferation assays were carried out and evaluated as previously described ( 8 , 11 ) . In brief, PBMCs obtained before the first and after each vaccination were stimulated with medium alone, with tumor-specific idiotype protein, and with a panel of isotype-matched irrelevant idiotype proteins. In addition, effector cells were also stimulated separately with both autologous tumor cells and autologous normal B cells. A response was considered positive when [ 3 H]thymidine (Amersham Biosciences, Piscataway, NJ; specific activity = 3.77 GBq/mg) incorporation of more than two times that in controls was found on at least two occasions out of five.

For cytokine release assays, idiotype- and tumor-specific release of IFN-γ, tumor necrosis factor (TNF; Th1 response; Pharmingen, San Diego, CA), and interleukin (IL)-13 (Th2 response; RayBiotech, Norcross, GA) was assessed by ELISA kits on the supernatants from the cell proliferation assays. Supernatants were removed immediately before [ 3 H]thymidine was added to the cell pellets for proliferation assays. A response was considered positive when cytokine release of more than two times that of controls was found on at least two occasions out of five.

IFN-γ release ELISpot assays were also carried out and evaluated as previously described ( 19 ) . In particular, tumor cells underwent a single 3-day cycle of in vitro preactivation with recombinant human soluble CD40 ligand trimer (sCD40Lt) (Amgen, Thousand Oaks, CA) and IL-4 (Preprotech EC, London, United Kingdom). PBMCs obtained before the first and after each vaccination were then stimulated with medium alone, with tumor-specific idiotype protein, with a panel of isotype-matched irrelevant idiotype proteins, with preactivated tumor cells, and with autologous normal B cells. Statistically significant ( P <.05) differences in the frequency of idiotype- and/or tumor-specific, IFN-γ–producing T-cell precursors between pre- and postvaccine samples were determined by the Wilcoxon signed rank test.

Cytotoxicity assays were performed as previously described ( 9 ) , with major modifications. In particular, tumor cells underwent a single 3-day cycle of in vitro preactivation with sCD40Lt and IL-4 ( 19 ) . Pre- and postvaccine PBMCs that had been stimulated for 5 days with medium containing autologous soluble idiotype protein and IL-2 (Chiron Iberica, Madrid, Spain) ( 19 ) were subsequently used as effector cells against both preactivated tumor and autologous normal B cells in a standard 4-hour chromium (Amersham Biosciences; specific activity > 9.25 GBq/mg) release assay, with triplicate samples. A response was considered positive when all the following requirements were met: mean specific lysis ( 9 ) of at least 25% at a 100 : 1 effector cell : target cell ratio; evidence of a clear titration effect; P <.05 by Student's paired t test comparing postvaccine lysis of tumor cells with that of each control, i.e., postvaccine lysis of autologous normal B cells and prevaccine lysis of both tumor and autologous normal B cells.

Flow cytometry was used to simultaneously identify cytokine-producing cells (TNF-positive monocytes and T cells) and quantify the amounts of secreted IFN-γ, TNF, IL-6, IL-10, IL-4, and IL-2 as previously described ( 20 , 21 ) . In particular, PBMCs were stimulated with medium alone, with tumor-specific idiotype protein, with a panel of isotype-matched irrelevant idiotype proteins, with KLH alone, and with idiotype-KLH conjugate. An overall response was considered positive only if positive results were found in at least two consecutive postvaccine time points.

Fc Receptor Polymorphism Analysis

Polymorphisms of the Fc receptor variants CD32 at position 131 and CD16 at position 158 ( 10 ) were assessed using genomic DNA extracted from each patient's PBMCs as previously described ( 22 , 23 ) .

Minimal Residual Disease Monitoring

In vaccinated patients who were found to harbor the Bcl-2/IgH rearrangement at enrollment according to qualitative polymerase chain reaction (PCR), postvaccine minimal residual disease was monitored by both qualitative ( 24 ) and quantitative ( 25 ) PCR analysis of the Bcl-2/IgH rearrangement and by flow cytometry analysis of circulating follicular lymphoma cells ( 26 ) , as previously described. All assessments were performed on peripheral blood samples ( 9 ) by personnel blinded to both clinical data and the patient's identity. In our hands, sensitivity of flow cytometry and of qualitative and quantitative PCR were 1/10 4 –1/10 5 , 1/10 5 , and 1/10 6 cells, respectively.

Statistical Analysis

Survival and time to relapse were estimated according to the method of Kaplan and Meier. To compare the cumulative durations of the first and second complete responses, because the same patients were studied both times, adjusted log-rank tests were computed and matched by patient to take into account the paired nature of survival times ( 27 ) . Statistical analyses were performed with use of SPSS version 11.0 software. Statistical tests were two-sided.

R ESULTS

Patients

We were able to administer idiotype vaccine to 25 of the 33 patients. Reasons for not administering the vaccine were unsuccessful idiotype protein isolation (one patient), patient did not achieve a second complete response or relapsed early (three patients), or both (four patients). This level of success in producing idiotype vaccine and achieving minimal disease status is similar to that seen in prior studies of previously untreated patients with follicular lymphoma ( 9 ) . The median duration of the off-therapy period to allow full peripheral lymphocyte count recovery after prevaccine chemotherapy treatment was 4 months (range = 3–7 months). Consequently, given the vaccination schedule, the last boost was to be administered within 23–27 months from the end of chemotherapy to all patients who did not relapse during the vaccination schedule time frame, for a total duration of the active vaccination period of 26–30 months.

The main clinical features of all vaccinated patients are summarized in Table 1 . It is important to note that, for 20 of the 25 vaccinated patients, the chemotherapy administered at the time of their first relapse was not more effective than that administered at diagnosis ( 28 ) . Indeed, nine patients had also received a CHOP-like regimen at diagnosis, and for six patients the dose intensity was higher than that administered at relapse because the chemotherapy cycles were given on an every-3-week basis at first diagnosis rather than monthly. Moreover, 13 of the 25 patients had received rituximab-containing therapy at diagnosis. Finally, at the time of relapse, no patient had a lower stage, lower follicular lymphoma international prognostic index score ( 29 , 30 ) , lower histologic grade, or lower tumor burden than was present at diagnosis ( Table 1 ).

Table 1.

Clinical features of all vaccinated patients *

UPN/Isotype Stage at dgn Grade at dgn FLIPI score at dgn  First-line treatment (response)  First CR duration (mo) Age at rel/sex Stage at rel Grade at rel FLIPI score at rel  Second-line treatment (response) (off therapy)  Second CR duration (mo)  Humoral response §  Cellular response § 
Patients who mounted an idiotype-specific immune response              
1/Mk  IV M A  II 4 FMD (SD) 57/M  IV M A  IIIa 6 CHOP (PR) 50+ 
    6 ESHAP (CRu)      ASCT (CR)    
    4 RITUX (CR)      (6 mo) IdVAX    
2/Gλ IIA 6 CVP (CR) 37/M IIIA II 6 CHOP (CR) 51+ 
    IFN mnt (CR)      (3 mo) IdVAX    
3/Mλ  IV M A  6 CHOP (CR) 46/F  IV M A  II 6 CNOP (CR) 51+ − 
          (3 mo) IdVAX    
5/Mk  IV M A  6 CHOP (CR) 13 46/M  IV M A  II 6 CNOP (CR) 36 
          (3 mo) IdVAX    
8/Gλ  IV M A  II 6 FMD (CR) 18 57/F  IV M A  II 6 CHOP (CR) 41+ 
          (3 mo) IdVAX    
9/Gλ  IV M A  II 6 FMD (PR) 16 62/M  IV M A  II 6 CHOP (CR) 41+ 
    4 RITUX (CRu)      (7 mo) IdVAX    
    ASCT (CR)          
10/Mλ IIIA II 6 FMD (CRu) 20 43/M  III S A  II 6 CHOP (CR) 39+ 
    4 RITUX (CR)      (6 mo) IdVAX    
13/Gλ  IV M A  II 8 CHOP (CR) 28 63/M  IV M A  IIIa 6 CNOP (CR) 34+ +/− 
          (3 mo) IdVAX    
14/Gλ  IV M A  6 CHOP (CRu) 20 41/F  IV M A  6 CNOP (CR) 34+ 
    4 RITUX (CR)      (5 mo) IdVAX    
15/Gλ IIIA II 6 CHOP (CR) 27 33/M IIIA IIIa 6 CNOP (CR) 34+ − 
          (4 mo) IdVAX    
16/Gλ IIIA II 6 CHOP (CR) 41 62/M  IV M A  II 6 CNOP (CR) 33+ − 
          (5 mo) IdVAX    
17/Mk  IV M B  II 4 FMD (PR) 17 55/M  IV M A  II 6 CHOP (PR) 32+ 
    4 ESHAP (CR)      Local RT (CR)    
          (5 mo) IdVAX    
18/Gλ IIA II 6 FMD (CRu) 19 52/F IIIA II 6 CHOP (CR) 30+ − 
    4 RITUX (CR)      (7 mo) IdVAX    
23/Gλ IA II IF RT (CR) 16 40/M IIIA II 6 CHOP (PR) 24+ 
          Local RT (CR)    
          (5 mo) IdVAX    
24/Gλ  IV M A  II 6 RITUX-FCM (CR) 26/F  IV M A  II 6 CHOP (PR) 23+ 
          Local RT (CR)    
          (3 mo) IdVAX    
26/Mλ  IV MS A  II 6 RITUX-CVP (CR) 15 64/F  IV M A  II 6 CHOP (CR) 22+ −/+ 
          (4 mo) IdVAX    
29/Mk IIIA 6 RITUX-CVP (CR) 16 43/M  IV M,skin A  II 6 CHOP (CR) 21+ − 
          (5 mo) IdVAX    
30/Mk  IV M A  II 6 RITUX-FMD (CR) 11 46/F  IV M,S A  II 6 CHOP (CR) 21+ 
          (3 mo) IdVAX    
31/Gλ  IV M A  6 RITUX-CHOP (CR) 18 45/F  IV M A  6 CNOP (CR) 20+ − 
          (3 mo) IdVAX    
32/Mk  IV M A  8 CHOP (CR) 65 65/F  IV M A  II 6 CNOP (CR) 20+ − 
          (6 mo) IdVAX    
Patients who did not mount an idiotype-specific immune response              
4/Gλ  IV M,skin A  6 FMD (PR) 10 48/M  IV M,skin A  6 CHOP (CR)  9 skin − − 
    4 RITUX (CR)      (3 mo) IdVAX    
22/Gλ IA 3 CHOP (CRu) 39 52/M IIIA II 6 CNOP (CR) 12 − − 
    IF RT (CR)      (6 mo) IdVAX    
25/Mk IA II IF RT (CR) 23 53/M  IV M A  II 6 CHOP (CR) 10 − − 
          (4 mo) IdVAX    
27/Mλ  IV M A  II 6 RITUX-FCM (CR) 17 30/M  IV M A  IIIa 6 CHOP (PR) 13 − − 
          ASCT (CR)    
          (7 mo) IdVAX    
33/Gk IIIA II 6 FMD (CRu) 13 44/M  IV M A  II 6 CHOP (CR) − − 
    4 RITUX (CR)      (3 mo) IdVAX    
UPN/Isotype Stage at dgn Grade at dgn FLIPI score at dgn  First-line treatment (response)  First CR duration (mo) Age at rel/sex Stage at rel Grade at rel FLIPI score at rel  Second-line treatment (response) (off therapy)  Second CR duration (mo)  Humoral response §  Cellular response § 
Patients who mounted an idiotype-specific immune response              
1/Mk  IV M A  II 4 FMD (SD) 57/M  IV M A  IIIa 6 CHOP (PR) 50+ 
    6 ESHAP (CRu)      ASCT (CR)    
    4 RITUX (CR)      (6 mo) IdVAX    
2/Gλ IIA 6 CVP (CR) 37/M IIIA II 6 CHOP (CR) 51+ 
    IFN mnt (CR)      (3 mo) IdVAX    
3/Mλ  IV M A  6 CHOP (CR) 46/F  IV M A  II 6 CNOP (CR) 51+ − 
          (3 mo) IdVAX    
5/Mk  IV M A  6 CHOP (CR) 13 46/M  IV M A  II 6 CNOP (CR) 36 
          (3 mo) IdVAX    
8/Gλ  IV M A  II 6 FMD (CR) 18 57/F  IV M A  II 6 CHOP (CR) 41+ 
          (3 mo) IdVAX    
9/Gλ  IV M A  II 6 FMD (PR) 16 62/M  IV M A  II 6 CHOP (CR) 41+ 
    4 RITUX (CRu)      (7 mo) IdVAX    
    ASCT (CR)          
10/Mλ IIIA II 6 FMD (CRu) 20 43/M  III S A  II 6 CHOP (CR) 39+ 
    4 RITUX (CR)      (6 mo) IdVAX    
13/Gλ  IV M A  II 8 CHOP (CR) 28 63/M  IV M A  IIIa 6 CNOP (CR) 34+ +/− 
          (3 mo) IdVAX    
14/Gλ  IV M A  6 CHOP (CRu) 20 41/F  IV M A  6 CNOP (CR) 34+ 
    4 RITUX (CR)      (5 mo) IdVAX    
15/Gλ IIIA II 6 CHOP (CR) 27 33/M IIIA IIIa 6 CNOP (CR) 34+ − 
          (4 mo) IdVAX    
16/Gλ IIIA II 6 CHOP (CR) 41 62/M  IV M A  II 6 CNOP (CR) 33+ − 
          (5 mo) IdVAX    
17/Mk  IV M B  II 4 FMD (PR) 17 55/M  IV M A  II 6 CHOP (PR) 32+ 
    4 ESHAP (CR)      Local RT (CR)    
          (5 mo) IdVAX    
18/Gλ IIA II 6 FMD (CRu) 19 52/F IIIA II 6 CHOP (CR) 30+ − 
    4 RITUX (CR)      (7 mo) IdVAX    
23/Gλ IA II IF RT (CR) 16 40/M IIIA II 6 CHOP (PR) 24+ 
          Local RT (CR)    
          (5 mo) IdVAX    
24/Gλ  IV M A  II 6 RITUX-FCM (CR) 26/F  IV M A  II 6 CHOP (PR) 23+ 
          Local RT (CR)    
          (3 mo) IdVAX    
26/Mλ  IV MS A  II 6 RITUX-CVP (CR) 15 64/F  IV M A  II 6 CHOP (CR) 22+ −/+ 
          (4 mo) IdVAX    
29/Mk IIIA 6 RITUX-CVP (CR) 16 43/M  IV M,skin A  II 6 CHOP (CR) 21+ − 
          (5 mo) IdVAX    
30/Mk  IV M A  II 6 RITUX-FMD (CR) 11 46/F  IV M,S A  II 6 CHOP (CR) 21+ 
          (3 mo) IdVAX    
31/Gλ  IV M A  6 RITUX-CHOP (CR) 18 45/F  IV M A  6 CNOP (CR) 20+ − 
          (3 mo) IdVAX    
32/Mk  IV M A  8 CHOP (CR) 65 65/F  IV M A  II 6 CNOP (CR) 20+ − 
          (6 mo) IdVAX    
Patients who did not mount an idiotype-specific immune response              
4/Gλ  IV M,skin A  6 FMD (PR) 10 48/M  IV M,skin A  6 CHOP (CR)  9 skin − − 
    4 RITUX (CR)      (3 mo) IdVAX    
22/Gλ IA 3 CHOP (CRu) 39 52/M IIIA II 6 CNOP (CR) 12 − − 
    IF RT (CR)      (6 mo) IdVAX    
25/Mk IA II IF RT (CR) 23 53/M  IV M A  II 6 CHOP (CR) 10 − − 
          (4 mo) IdVAX    
27/Mλ  IV M A  II 6 RITUX-FCM (CR) 17 30/M  IV M A  IIIa 6 CHOP (PR) 13 − − 
          ASCT (CR)    
          (7 mo) IdVAX    
33/Gk IIIA II 6 FMD (CRu) 13 44/M  IV M A  II 6 CHOP (CR) − − 
    4 RITUX (CR)      (3 mo) IdVAX    
*

For all vaccinated patients, stage, grade, follicular lymphoma international prognostic index (FLIPI) score, and treatment are presented both at diagnosis and relapse for direct comparison on a patient-by-patient basis. UPN = unique patient number; dgn = diagnosis; mo = months; rel = relapse; CR = complete response; Cru = complete response unconfirmed; PR = partial response; SD = stable disease; mnt = maintenance; FMD = fludarabine, mitoxantrone, dexametasone; ESHAP = cisplatin, etoposide, cytosine arabinoside, prednisone; RITUX = rituximab; CVP = cyclophosphamide, vincristine, prednisone; CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone; CNOP = cyclophosphamide, mitoxantrone, vincristine, prednisone; ASCT = autologous stem cell transplantation; IF RT = involved field radiation treatment; FCM = fludarabine, cyclophosphamide, mitoxantrone; local RT = radiation treatment on a single residual lymph node; IdVAX = patient-specific idiotype vaccine.

The number before each treatment indicates the number of cycles of therapy.

Off therapy = time between documented, chemotherapy-induced second complete response and start of the vaccination.

§

+ = Vaccine-induced specific response; +/− = vaccine-induced nonspecific response; −/+ = vaccine-induced specific response documented by only one of five methods; − = no response.

Immune Response

A total of 20 (80%) of the 25 vaccinated patients showed a vaccine-induced idiotype- and/or tumor-specific immune response. Thirteen (52%) of the 25 patients developed and maintained a specific humoral response ( Tables 1 and 2 ), and in all 13 patients the response was seen after two to four vaccinations (data not shown). No patient developed a humoral response after having had no response to four vaccine doses (data not shown). Of the eight patients with a specific humoral response and sufficient follow-up for the response to be analyzed 6 months after the last vaccine administration, three still maintained a detectable, although declining, response (data not shown). In addition, 18 (72%) of the 25 patients—including 11 patients who showed a humoral response—developed and maintained a specific cellular response ( Table 1 ), and all 18 did so after two to four vaccine doses (data not shown). In one additional patient (UPN 26), such a response was documented by only one method ( Tables 1 and 2 ). Of the 12 patients with a specific cellular response and sufficient follow-up for the response to be analyzed 6 months after the last vaccine administration, four still maintained a detectable response.

Table 2.

Distribution of immune response detection among vaccinated patients *

     Cellular response
 
             
        Cytokines
 
          
   Humoral response
 
     IFN-γ
 
     TNF or IL-13
 
   Flow cytometry
 
  
 Polymorphisms CD32/CD16    Proliferation
 
   ELISA
 
   ELISpot
 
  ELISA
 
     
UPN  Id KLH Id KLH Tumor Id KLH Tumor Id Tumor Id KLH Tumor Id KLH Cytotoxicity 
Patients who mounted an idiotype-specific immune response                  
HH/VV − − − − +/− − − − 
RR/FV +/− ND +/− ND +/− ND − ND − 
HR/VV − − − ND − − − ND 
RR/FV +/− +/− − − +/− − ND − 
HH/FF − − − − − − − 
RR/FF − − − − − 
10 HR/VV − − − − − − 
13 HR/FV +/− − − − − − − 
14 HR/FV − − − − 
15 HR/VV − − − − − ND ND ND − − 
16 HR/FV − − − − − − − +/− − − ND 
17 HR/FV − − +/− − − − − − − − 
18 HR/VV − − +/− − +/− − − − 
23 RR/FV − − − − − − ND 
24 HH/FV − − − 
26 HH/FV − − − − − − − − − 
29 HR/FV − − − − − − − 
30 HH/FF +/− +/− − +/− − 
31 HH/VV − ND ND ND − ND ND ND − 
32 RR/FV − − − − − − − − − ND 
Patients who did not mount an idiotype-specific immune response                  
HR/FV − − − − − − − − − − − − − 
22 RR/FF − − − − − − − − − − − − − − − 
25 HR/FV − − − − − − − − − − − − 
27 HR/VV − − − − − − − − − − − − − ND 
33 RR/VV − − − − − − − − − − − 
     Cellular response
 
             
        Cytokines
 
          
   Humoral response
 
     IFN-γ
 
     TNF or IL-13
 
   Flow cytometry
 
  
 Polymorphisms CD32/CD16    Proliferation
 
   ELISA
 
   ELISpot
 
  ELISA
 
     
UPN  Id KLH Id KLH Tumor Id KLH Tumor Id Tumor Id KLH Tumor Id KLH Cytotoxicity 
Patients who mounted an idiotype-specific immune response                  
HH/VV − − − − +/− − − − 
RR/FV +/− ND +/− ND +/− ND − ND − 
HR/VV − − − ND − − − ND 
RR/FV +/− +/− − − +/− − ND − 
HH/FF − − − − − − − 
RR/FF − − − − − 
10 HR/VV − − − − − − 
13 HR/FV +/− − − − − − − 
14 HR/FV − − − − 
15 HR/VV − − − − − ND ND ND − − 
16 HR/FV − − − − − − − +/− − − ND 
17 HR/FV − − +/− − − − − − − − 
18 HR/VV − − +/− − +/− − − − 
23 RR/FV − − − − − − ND 
24 HH/FV − − − 
26 HH/FV − − − − − − − − − 
29 HR/FV − − − − − − − 
30 HH/FF +/− +/− − +/− − 
31 HH/VV − ND ND ND − ND ND ND − 
32 RR/FV − − − − − − − − − ND 
Patients who did not mount an idiotype-specific immune response                  
HR/FV − − − − − − − − − − − − − 
22 RR/FF − − − − − − − − − − − − − − − 
25 HR/FV − − − − − − − − − − − − 
27 HR/VV − − − − − − − − − − − − − ND 
33 RR/VV − − − − − − − − − − − 
*

Vaccine-induced, specific immune responses against idiotype (Id), autologous tumor cells, and keyhole limpet hemocyanin (KLH) are presented on a patient-by-patient basis, together with polymorphisms of Fc receptor variants CD32 (position 131) and CD16 (position 158). UPN = unique patient number; IFN-γ = interferon-γ; TNF = tumor necrosis factor; IL = interleukin; ELISA = enzyme-linked immunosorbent assay; ELISpot = enzyme-linked immunosorbent spot; H = histidine; R = arginine; V = valine; F = phenylalanine; tumor = autologous tumor cells; + = vaccine-induced, specific response; +/− = vaccine-induced, nonspecific response; − = no response; ND = not determined due to insufficient material.

TNF release ELISA performed in UPN 1 through 13 and UPN 16. IL-13 release ELISA performed in UPN 14 and UPN 17 through 33.

A specific cellular response against tumor idiotype protein and/or tumor cells ( Table 2 ) was documented by proliferation assay in five (26%) of 19 and 15 (79%) of 19 tested responders, respectively; by cytokine ELISA in 10 (53%) of 19 and 14 (74%) of 19 tested responders, respectively; and by ELISpot assay in two (11%) of 19 and nine (50%) of 18 tested responders, respectively. A specific cellular response was observed against tumor idiotype protein in 11 (55%) of 20 tested responders by flow cytometry and against tumor cells in five (31%) of 16 tested responders by cytotoxicity ( Fig. 1 ). Remarkably, cytotoxicity results were obtained with a traditional immunologic test that is rarely successful when naive human cancer cells are used as targets.

Fig. 1.

Cytotoxicity results of patients 13, 14, 17, 18, and 29. Peripheral blood mononuclear cells (PBMCs) isolated before and after vaccination were used as effector cells against both preactivated tumor cells and autologous normal B cells in a standard 4-hour chromium release assay. Data are reported as mean specific lysis (%) of triplicate values with 95% confidence intervals at different effector : target (E : T) ratios. UPN = unique patient number; pre = prevaccine PBMCs; post = postvaccine PBMCs; tumor = autologous tumor cells; auto B cells = autologous normal B lymphocytes.

Fig. 1.

Cytotoxicity results of patients 13, 14, 17, 18, and 29. Peripheral blood mononuclear cells (PBMCs) isolated before and after vaccination were used as effector cells against both preactivated tumor cells and autologous normal B cells in a standard 4-hour chromium release assay. Data are reported as mean specific lysis (%) of triplicate values with 95% confidence intervals at different effector : target (E : T) ratios. UPN = unique patient number; pre = prevaccine PBMCs; post = postvaccine PBMCs; tumor = autologous tumor cells; auto B cells = autologous normal B lymphocytes.

Clinical Response and its Association With Immune Response

The median duration of the first clinical complete response of the 25 vaccinated patients was 17 months (range = 5–65 months). Their current median follow-up is at least 32 months after completion of salvage chemotherapy treatment. The median duration of the second clinical complete response in the 20 vaccinated patients who met the criteria for immunologic response has not been reached, but it exceeds 33 months (range = 20+ to 51+ months).

Of the 20 patients with a documented vaccine-induced specific immune response—including 13 who, as of June 2006, have already completed the vaccination schedule and seven who are still receiving their last doses—none relapsed while receiving vaccinations ( Table 1 ), and all experienced or, as of that date, were still experiencing a continuous second complete response that is longer than the 13 months corresponding to the expected median duration of a second complete response achieved through monthly CHOP or CHOP-like chemotherapy alone ( 4 , 5 ) .

As of June 2006, only one of the 20 immune responders (UPN 5) had relapsed. This relapse came 10 months after the patient's last scheduled vaccination and 4 months after the original vaccine-induced specific immune response was no longer detectable. Nevertheless, this patient's second complete response duration was 36 months, which is nearly three times as long as the first complete response. It is interesting to note that the idiotype expressed on this patient's tumor cells at the time of the second relapse was not identical to the idiotype that was used to produce the vaccine; in particular, five silent mutations and one replacing mutation were detected within the light-chain variable region, whereas no nucleotide changes occurred within the heavy-chain variable region ( 31 ) . Of the five patients who did not respond to the vaccine, all relapsed within 13 months, which is well within the previously defined 2-year period for the highest risk of relapse ( 4 ) ( Table 1 ). In these five patients, vaccinations were discontinued immediately.

The cumulative duration of the second complete response of all vaccinated patients was statistically significantly longer ( P <.0001) than that of the first complete response ( Fig. 2, A ), even given the unfavorable contribution to this comparison of the early second relapse of the five patients with no vaccine-induced immune response. In particular, all 18 patients with a documented specific immune response and with a follow-up longer than the duration of their first complete response experienced or were, as of June 2006, still experiencing a continuous second complete response that was longer than their first complete response ( Table 1 and Fig. 2, B ). Conversely, all five patients who failed to respond to the vaccine experienced a second complete response that was shorter than their first complete response ( Table 1 and Fig. 2, B ). Finally, the cumulative length of the second complete response also was statistically significantly longer ( P <.0001) than the first complete response among all 18 immune responders who, at first relapse, had received a chemotherapy treatment that was not superior to the therapy received at diagnosis ( Table 1 and Fig. 2, C ).

Fig. 2.

Relapse-free survival curves for patients who received idiotype vaccination. A ) Relapse-free survival after the first and second clinical complete responses (CR) of all 25 vaccinated patients. B ) Relapse-free survival after the first and second CR of vaccinated patients after stratification for immune responsiveness to the vaccine. There were 20 responders and five nonresponders. C ) Relapse-free survival after the first and second complete response of the 18 immune responders who, at first relapse, had received a prevaccine treatment that would not be considered superior to the up-front therapy received shortly after diagnosis (that is, all immune responders but UPN 1 and UPN 23). P values are from adjusted log-rank tests, computed and matched by patient to take into account the paired nature of survival times. P value of ( B ) refers to the sole comparison between cumulative relapse-free survival of the 20 immune responders after achieving first and second complete response, respectively.

Fig. 2.

Relapse-free survival curves for patients who received idiotype vaccination. A ) Relapse-free survival after the first and second clinical complete responses (CR) of all 25 vaccinated patients. B ) Relapse-free survival after the first and second CR of vaccinated patients after stratification for immune responsiveness to the vaccine. There were 20 responders and five nonresponders. C ) Relapse-free survival after the first and second complete response of the 18 immune responders who, at first relapse, had received a prevaccine treatment that would not be considered superior to the up-front therapy received shortly after diagnosis (that is, all immune responders but UPN 1 and UPN 23). P values are from adjusted log-rank tests, computed and matched by patient to take into account the paired nature of survival times. P value of ( B ) refers to the sole comparison between cumulative relapse-free survival of the 20 immune responders after achieving first and second complete response, respectively.

We assessed idiotype vaccine–associated toxicity according to the NCI CTC. As seen in previous studies of idiotypic vaccination ( 812 ) , toxicity was always negligible (data not shown).

Fc Receptor Polymorphism

We investigated polymorphisms in the Fc receptor variants CD16 and CD32 because of their possible association with different clinical outcome after idiotypic vaccination ( 10 ) . Data on polymorphisms of both CD16 (at position 158) and CD32 (at position 131) are summarized in Table 2 . No association was seen between individual polymorphisms and either clinical outcome or immune response. We were unable to identify any other tumor or patient feature that was associated with failure to mount a vaccine-induced idiotype-specific immune response (data not shown).

Minimal Residual Disease

A total of 20 of the vaccinated patients were found to be positive for the Bcl-2/IgH rearrangement by qualitative PCR at enrollment. In these patients, minimal residual disease was measured at selected time points by three independent methods ( Fig. 3 ): flow cytometry, qualitative PCR, and quantitative PCR. Each method appeared to provide relatively reliable and consistent information for every patient. However, there was no association between minimal residual disease ( Fig. 3 ) and clinical outcome ( Fig. 1 ), and the associations among the results of the different methods to measure minimal residual disease were largely incomplete and inconclusive ( Fig. 3 ).

Fig. 3.

Evaluation of minimal residual disease in the 20 vaccinated patients with a Bcl-2/IgH rearrangement. For each patient (UPN, at left ), the first row ( bold type ) shows the number of copies of the Bcl-2/IgH rearrangement per 10 6 circulating peripheral blood mononuclear cells (PBMCs) according to quantitative polymerase chain reaction (PCR), and the second row shows the percentage of circulating tumor cells according to four-color flow cytometry. Solid symbols = positive for Bcl-2/IgH rearrangement according to qualitative PCR on circulating PBMCs; open symbols = negative for Bcl-2/IgH rearrangement according to qualitative PCR on circulating PBMCs. pre1 = before the first vaccination; pre5 = before the fifth vaccination; pre7/8 = before the seventh or eighth vaccination; F/U = at follow up 6 months after last vaccination; ND = not determined due to insufficient material.

Fig. 3.

Evaluation of minimal residual disease in the 20 vaccinated patients with a Bcl-2/IgH rearrangement. For each patient (UPN, at left ), the first row ( bold type ) shows the number of copies of the Bcl-2/IgH rearrangement per 10 6 circulating peripheral blood mononuclear cells (PBMCs) according to quantitative polymerase chain reaction (PCR), and the second row shows the percentage of circulating tumor cells according to four-color flow cytometry. Solid symbols = positive for Bcl-2/IgH rearrangement according to qualitative PCR on circulating PBMCs; open symbols = negative for Bcl-2/IgH rearrangement according to qualitative PCR on circulating PBMCs. pre1 = before the first vaccination; pre5 = before the fifth vaccination; pre7/8 = before the seventh or eighth vaccination; F/U = at follow up 6 months after last vaccination; ND = not determined due to insufficient material.

In one patient, UPN 9, Bcl-2/IgH rearrangement was no longer detectable by qualitative PCR after the fourth vaccination. This was the first time that this patient showed a molecular remission, which he had not shown even after undergoing autologous stem cell transplantation shortly after the initial diagnosis. However, when the 25 vaccinated patients are considered as a group, the presence or absence of PCR-detected Bcl-2/IgH rearrangement was not consistent with the clinical course of disease ( Table 1 and Fig. 3 ).

D ISCUSSION

Most patients with follicular lymphoma who receive CHOP or CHOP-like regimens after their first relapse will experience a second relapse or progression within 2 years from the end of this treatment ( 25 ) . In most, if not all, such patients the second complete response is shorter than the first complete response achieved with either similar chemotherapy regimens ( 25 ) or similarly effective standard chemotherapy regimens, such as those containing fludarabine and/or rituximab ( 1 , 27 ) ( Table 1 ). We found that idiotypic vaccination can induce specific humoral and cellular immune responses that dramatically change these patterns—that is, the duration of remission was statistically significantly lengthened. Indeed, this study has established, for the first time, a direct association between a vaccine-induced specific immune response and clinical benefit. Furthermore, second complete responses that are systematically longer than the first complete response are unprecedented; such an outcome has not even been observed in patients treated with the much more toxic option of high-dose chemotherapy followed by autologous stem cell transplantation ( 5 ) .

In our study design, the lengths of the first and second complete responses were compared directly within each patient. We believe that such a design is more straightforward and informative than a randomized trial comparing patients with and without customized idiotype vaccine following treatment with the same chemotherapy regimen ( 14 ) . In fact, our study design allowed us to conduct an atypical form of case–control study, in which each patient served as both a case patient and his or her matched control subject. Using a patient as his or her own control may make inferences about the biology of follicular lymphoma and the effects of treatment more reliable ( 14 ).

Another problem that human cancer immunotherapy is still facing is that of the most appropriate method to monitor cellular response because no standard test has yet been identified. Indeed, we detected common and intrinsic interassay discrepancies in the results, particularly those between idiotype- and tumor-specific T-cell responses ( Table 2 ). However, these discrepancies might reflect well-known issues, such as the better antigen-presenting capability of CD40L-activated tumor cells than PBMCs and the induction of epitope spreading (i.e., a phenomenon whereby non–cross-reacting epitopes become targets of the immune response) by vaccination ( 32 ).

With respect to the type of chemotherapy used before vaccination, it should be emphasized that, at the time this study was designed, it was not clear that the addition of rituximab or fludarabine might provide better clinical results in follicular lymphoma patients experiencing their first relapse. For this reason, and because of the well-known immunosuppressive side effects of both drugs ( 1 , 28 ) , we avoided their use before administering the idiotype vaccine. Given the overall results with idiotypic vaccination that we have now reported in patients with first-relapse follicular lymphoma and the recent finding ( 32 ) that patients newly diagnosed with mantle cell lymphoma are able to mount delayed idiotype-specific humoral responses despite being pretreated with a regimen including rituximab, we recommend that rituximab and fludarabine should be reserved for first-line treatment of newly diagnosed follicular lymphoma patients, with or without rituximab maintenance therapy, particularly if chemotherapy is not followed by any active immunotherapy. Indeed, with current treatment practices most newly diagnosed patients are unlikely to receive idiotypic vaccination, mainly because of the specialized nature of the methods used to generate a vaccine that is customized for each patient. However, precisely because of the immune responses induced in patients with mantle cell lymphoma mentioned above ( 32 ) , the addition of rituximab to chemotherapy might be potentially useful even in newly diagnosed follicular lymphoma patients selected for subsequent idiotypic vaccination, especially if multiple vaccinations are given over a prolonged period.

We also recommend that idiotypic vaccination not begin until both a second clinical complete response and full recovery of circulating lymphocytes have been achieved, instead of after detection of a clinical partial response or after a prespecified period after completion of chemotherapy. Our data support a 3-month interval between consecutive vaccine administrations because no patient who mounted a vaccine-induced immune response lost this response while receiving booster vaccinations, whereas seven of 12 immune responders lost the response by the sixth month after the final booster dose (data not shown). Yet, even in these seven patients, the clinical response continued well beyond the point at which an immune response could be detected. Interestingly, as of June 2006 the only patient to have relapsed after completing the vaccination program did so with a tumor clone expressing an altered idiotype protein. Thus, with the data we have so far, it is impossible to determine whether sustained, detectable vaccine-induced immune response was associated with long-term relapse-free status after the long vaccination schedule concludes.

No previous study of idiotypic vaccination in follicular lymphoma ( 10 , 11 , 33 ) was designed to address the possible role of long-term vaccination. In those trials, even patients in whom short-term vaccination induced specific immune responses continued to relapse in the absence of booster vaccine, in contrast to our findings over the 2.5-year vaccination time frame. This difference implies that as long as repeated idiotype vaccine doses both elicit and maintain specific and sustained immune responses, they may effectively keep the tumor burden below a clinically detectable level, whether or not they actually cure patients with follicular lymphoma of their disease. Indeed, it is possible that patients who lose the specific vaccine-induced response and in whom some minimal residual disease remains will ultimately relapse. However, this conclusion is also not definitive because of two major conceptual pitfalls: first, the real clinical impact of minimal residual disease in follicular lymphoma has never been fully elucidated, particularly when ever-more-sensitive PCR techniques are used to detect it ( 24 , 25 , 34 ) , and second, vaccine-induced immune memory might persist in vivo, albeit at undetectable levels, at least in some patients with minimal residual disease, possibly contributing to its maintenance. Based on our best immunologic assays, it appears that periodic booster vaccinations are required to maintain the idiotype-specific response. This finding raises the question of whether there may be some benefit to continuing booster immunizations indefinitely in patients with follicular lymphoma who maintain their clinical remission.

Our results also indicate that both specific humoral and cellular immune responses that are induced through vaccinations are associated with clinical benefit. However, it should be noted that there is no widely accepted single standardized method to assess immune response. It is unclear whether direct T-cell cytotoxicity, natural killer cell–mediated antibody-dependent cell-mediated cytoxicity, or both may actually take place in patients responding to idiotype vaccines and whether such responses are related to clinical outcome. It remains formally possible that the capacity to mount an idiotype-specific response is a surrogate for some other factor that maintains remission.

In conclusion, we have reported on what is, to our knowledge, both the largest and most homogeneous series of follicular lymphoma patients in first relapse treated with idiotypic vaccination and the first to include results of extensive vaccine boost administration. In this context, our data provide evidence that whenever an idiotype vaccine induced any sustained and specific immune response, it also provided an objective clinical benefit—i.e., a longer second complete response—to patients with follicular lymphoma and did so without relevant side effects. To the best of our knowledge, this is the first formal demonstration of clinical benefit associated with the use of a human cancer vaccine.

A PPENDIX

The study was conducted on behalf of both Grupo Español de Linfomas/Trasplante Autologo de Medula Oseo and Programa para el Estudio y Tratamiento de Hemopatias Malignas study groups.

Participating institutions and principal staff include Basurto Hospital, Bilbao, Spain: J. M. Beltran; General Hospital, Manresa, Spain: R. Salinas; Morales Messeguer Hospital, Murcia, Spain: J. Moraleda, J. Nieto, V. Vicente; Son Dureta Hospital, Palma de Mallorca, Spain: J. Besalduch, J. Rodríguez; University Clinic of Navarra, Pamplona, Spain: J. García-Foncillas, R. García-Muñóz, S. Martin-Algarra, F. Martínez-Regueira, J. Merino, J. Pardo, A. Sánchez Ibarrola; University Hospital, Salamanca, Spain: M. D. Caballero, R. García Sanz, J. M. Hernández, J. F. San Miguel; Center for Cancer Investigation, Salamanca, Spain: J. Almeida, A. C. García Montero; Txagorritxu Hospital, Vitoria, Spain: C. Menchaca; Miguel Servet Hospital, Zaragoza, Spain: P. Giraldo, P. Mayayo.

S. Inogès, M. Rodrìguez-Calvillo, N. Zabalegui, and A. Lòpez-Dìaz de Cerio contributed equally to this work.
The study was partially supported by the Union Temporal de Empresas “Fundacion para la Investigacion Medica Aplicada project”, the Red Tematica de Investigacion Cooperativa de Centros de Cáncer (C03/10) of the Instituto de Salud Carlos III, and both the Department of Health and the Department of Education of the Government of Navarra. No sponsor took part in the design, analysis, and interpretation of the data, in the writing of the manuscript, or in the decision to publish it.
M. Rodrìguez-Calvillo holds a Fondo de Investigaciones Sanitarias contract of the Spanish Ministry of Health. M. Bendandi is a Scholar in Clinical Research of the Leukemia and Lymphoma Society.
We are indebted to Professor Marta Garcia Granero (Statistic Unit, Department of Genetics, University of Navarra) for the statistical analyses and to Margarita Legarrea and Silvia Gallego for their outstanding technical work. We thank Amgen for providing the sCD40Lt.

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