João, Cristina

Abstract Mantle-cell lymphoma (MCL) is characterized by an aggressive clinical course and poor prognosis with a median survival of only 3 to 4 years. High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is an effective treatment strategy for MCL demonstrating significantly prolongs progression free survival (PFS) when compared to IFNa maintenance therapy of patients in first remission. We have previously shown that absolute lymphocyte count at day 15 (ALC-15) after ASCT is an independent prognostic factor for survival in hematologic malignancies (non-Hodgkin’s lymphoma (NHL), Hodgkin’s disease, multiple myeloma, primary amyloidosis, acute myelogenous leukemia) and breast cancer. The study of ALC-15 after ASCT as a prognosis factor in NHL included different subtypes of lymphomas. The relationship of ALC-15 after ASCT in patients with MCL has not been specifically addressed. Herein we present data evaluating the impact of early lymphocyte recovery (ALC-15) in patients undergoing ASCT for MCL. We retrospectively reviewed the medical records of 42 consecutive patients (9 females and 33 males) with MCL who underwent ASCT at the Mayo Clinic in Rochester from 1993 to 2005. Based on our previous studies, the ALC-15 threshold was set at 500 cells/μL. The median patient age at transplant was 57 years (range, 37–71) and the median follow-up after ASCT was 25 months (range, 2–106 months). The median overall (OS) and PFS times were significantly better for the 24 patients that achieved an ALC-15 ≥ 500 cells/mL compared with 18 patients with ALC-15 < 500 cells/μL (not reached versus 30 months, P < 0.01 and not reached versus 16 months, P < 0.0006, respectively). Multivariate analysis adjusting for other prognostic factors suggests that ALC-15 is an independent prognostic factor for OS and PFS. ALC-15 ≥ 500 cells/mL is associated with significantly improved clinical outcomes following ASCT in patients with MCL.

Abstract The proposed immune-mediated mechanisms of action of Rituximab (ADCC or “vaccinal” effect) suggest that the efficacy of rituximab therapy depends on the immunocompetency of the host. Therapies, such as IL-12, that stimulate the immune system may increase the efficacy of rituximab. Recently, we reported that absolute lymphocyte count (ALC), as a surrogate maker of immune recovery, is a survival prognostic factor in autologous stem cell transplantation for non-Hodgkin’s lymphoma (NHL). We hypothesize host immune competence (i.e, normal ALC counts) would predict a superior survival in NHL patients treated with rituximab-based regimens. The primary end-point of the study was to assess whether the ALC count prior to rituximab therapy predicted progression-free survival (PFS) and the second end-point was to identify which lymphocyte subset was responsible for affecting PFS. We studied 41 NHL patients that participated in our Phase I interleukin-12 plus rituximab study. These patients were selected because the Phase I study required baseline ALC count and flow cytometry analysis providing lymphocyte subset data prior to treatment. There were 20 females and 21 males in the study. The median age of the cohort was 53 years (range: 34–84). PFS was assessed from the initial date of treatment. The median follow-up was 42 months (1–84 months). Patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC < 0.9 × 109/l prior to rituximab therapy (31 months vs 4 months, p < 0.0001). The relapse rate in the ALC ≥ 0.9 × 109/l group was 55% (11/20) versus 95 % (20/21) in the ALC < 0.9 × 109/l (p < 0.0016). Patients with a CD4 count ≥ 500 cells/μl also experienced a superior PFS versus a CD4 count < 500 cells/μl (21 months vs 9 months, p < 0.0062). The relapse rate in the CD4 count ≥ 500 cells/μl group was 56% (9/16) versus 88 % (22/25) in the CD4 count < 500 cells/μl (p < 0.0216). No association with PFS was identified for CD3, CD8, CD19, CD16/56, the use of IL-12, or lymphoma type by WHO classification. CD4 count was found to be an independent prognostic factor for PFS. In the subgroup of patients with follicular lymphoma, patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC < 0.9 × 109/l prior to rituximab therapy (35 months vs 9 months, p < 0.0076). The relapse rate in the ALC ≥ 0.9 × 109/l group was 45% (5/11) versus 90 % (9/10) in the ALC < 0.9 × 109/l (p < 0.0243). Patients with a CD4 count ≥ 500 cells/μl also experienced superior PFS versus a CD4 count < 500 cells/μl (not reached vs 9 months, p < 0.0098). The relapse rate in the CD4 count ≥ 500 cells/μl group was 29% (2/7) versus 86 % (12/14) in the group with a CD4 count < 500 cells/μl (p < 0.0216). CD4 count was found to be an independent prognostic factor for PFS when compared to the Follicular Lymphoma International Prognostic Index at the time of treatment. Our data suggest the significance of an immunocompetent host in predicting the efficacy of rituximab therapy. These data support our previous report that patients with higher number of activated CD4+ cells at the lymphoma site have a better prognosis (Ansell et al, JCO2001; 19: 720–6). These findings warrant further studies to understand the role of ALC (i.e, CD4 count) in the mechanism of action of rituximab. Supported in part by CA69912

10088 Background: The ability of the immune system to recover its components after autologous stem cell transplantation (ASCT) is known to predict survival in patients with different types of neoplasms. Also, our previous work showed that polyclonal immunoglobulin can be used to improve T cell diversity and, consequently, T cell function in animal models. The aim of this retrospective study was to understand if the recovery of the polyclonal compartment of immunoglobulin in patients with multiple myeloma (MM) at day 100 (± 40d) after ASCT is a predictor factor for survival in MM patients who were subject to ASCT. Methods: From294 patients with IgG-MM subject to ASCT at the Mayo Clinic, Rochester, MN since October, 1993 until June, 2004, 143 patients were studied. The rational to select this group of patients was the fact that they had levels of serum IgM collected at day 100 ± 40 after ASCT recorded. Progression free survival (PFS) was assessed from the day of the transplant. PFS was studied using Kaplan-Meier curves and the univariate and multivariate analysis were done using age-adjusted proportional Cox. The prognostic variables in the multivariate model were: recovered of IgM at day 100, B2 microglobulin, age, conditioning regimen and disease status at the time of transplant. Results: There were 59 females in the study. The median age of the cohort was 58 y (33–75). The median follow up of the study was 26 mo (range, 2-111). PFS was significantly higher in the group who recovered normal serum levels of IgM at D100 compared to the pts who did not. This corresponds to a survival that is 1.5 longer in the group of patients who recovered normal levels of IgM at day100 after ASCT versus the patients who did not (median: 34 to 23 mo, respectively; p=0.05). By univariate analysis, those who did not recover normal serum levels polyclonal immunoglobulin by day 100 after ASCT had a risk of relapse 65% higher than the risk of the patients who recovered. Multivariate analysis suggests that recover the normal levels of polyclonal immunoglobulin after ASCT is an independent prognostic factor for PFS for MM patients after ASCT. Conclusions: These results suggest that polyclonal immunoglobulin is a major factor promoting immunity after ASCT. No significant financial relationships to disclose.

Abstract Background: Classic reports on lymphocyte development hold that B and T cells develop independently. This concept derives in part from the observation that patients with pure B cell immunodeficiency and hypogammaglobulinemia have a normal thymus and T cell numbers. Our recent findings however challenge this concept. We found that T cell development depends not only on the interaction of T cell precursors with thymic epithelial cells but also on other cells. Here we report that those other cells are B cells. Aims: The purpose of this study was to determine whether B cells drive T cell development and TCR diversification in the thymus. Methods: We compared the number of sub-populations of thymocytes and TCR repertoire diversity in B-cell deficient and B-cell proficient mice and in B cell deficient mice following immunoglobulin (Ig) injections. Total leucocytes numbers were determined with a Coulter counter and numbers of thymocytes sub-populations were calculated by flow cytometry analysis. TCR repertoire diversity was measured by a novel method based on hybridization of TCR Vβ specific cRNA on a gene chip platform. Results: In B-cell deficient mice the number of thymocytes was four times reduced and TCR Vβ chain diversity was up to one million times lower compared with wild type mice. Numbers and diversity were restored by treatment of the mice with gamma globulin (see table). Conclusions: T cell development and diversification is driven by B cells. Mice Number of total thymocytes (mean ± standard deviation) p Value β V TCR diversity of thymocytes (median; min.-max.) p Value C57BL/6 (wild mice) 1.3 x 108 ± 5.1 x 107N=7 4.7 x 106; 1.0 x 105 − 1.1 x 108N=5 JH−/− (B cell immunodeficient mice) 3.1 x 107 ± 1.7 x107N=7 0.002 5.9 x 102; 3.6 x 102 − 1.1 x 103N=5 0.0002 JH−/− treated with Ig 3.9 x 107 ± 1.4 x106N=2 0.20 1.1 x 105; 2.7 x 100 − 7.7 x 105N=4 0.08

Abstract Introduction: The immunoglobulin (Ig) is one of the most diverse molecules in nature, and its therapeutic potential, by stimulation of the immune system, has been widely studied/used. Its effect is manifest at the cellular and humoral compartments. However it is still to clarify the specific effect of each fraction of the molecule in the observed immune stimulation. Aims: To develop a murine model of ASCT that mimics the autologous transplant in humans To study the independent effect of the various fractions of the immunoglobulin in the reconstitution of the immune system after ASCT. Methods: In this study, we developed, de novo, a murine model of ASCT. Four groups of transplanted animals were established (weekly injections of: Ig (n=8) or Fab fragment (n=7) or Fc fragment (n=6) or saline (control, n=7)). The evolution of the immune reconstitution was monitored weekly and compared among groups by characterization of cellular fractions by flow cytometry, serum levels of Igs by ELISA and thymic function by quantification of TRECs (“T cell receptor excision circles”). The statistical analysis of the longitudinal data was performed by a linear-mixed effects model. Results: We determined that a regimen of daily injections of G-CSF for 4 consecutive days produces a degree of mobilization of hematopoietic stem cells adequate for a donors/recipient ratio of 9 to 7, and to a standard engraftment extent. Regarding the recovery of cellular sub-populations among groups, the major differences occur up to the 6th week (w) after ASCT. As soon as 2 w after ASCT the treated groups show higher counts of CD4+ T cells compared with control mice (p<0.05). The maximum number of CD4+ T lymphocytes was attained at 2 w after ASCT for mice treated with Fab and Fc and at 9 w for mice treated with polyclonal Ig and PBS. The recovery of CD4+ T lymphocytes in the treated groups was higher compared to control (p<0.001 for Fab, p<0.001 for Fc and p=0.001 for polyclonal Ig). For CD8+ T cells, the mice treated with Fc show, during all the follow up, superior values, being statistically significant when compared to PBS treated mice (p=0.003). The differences with the other groups vary with time. Regarding the recovery of CD19+ lymphocytes, the mice treated with Fab reveal, after the 7th w, superior counts compared to all the other groups (p<0.05). Treated groups (Fab and Fc) show higher values of serum IgM compared to control, as well as an earlier recovery of levels (p<0.05). In what concerns CD4+ naïve T cells, the group treated with polyclonal Ig shows an increased recovery during the first weeks of treatment compared with the other groups (p<0.05). Conclusions and Discussion: The early treatment with polyclonal Ig or its fragments is highly beneficial for the recovery of CD4 and CD8 T lymphocytes (earlier recovery and higher cell counts). After the 6th week after transplant there is a tendency for homeostasis. The increase of CD4+ naïve T cells with Ig treatment may well correspond to an increased thymopoiesis, as suggested by our previous studies.

Abstract T cell diversity was once thought to depend on the interaction of T cell precursors with thymic epithelial cells. Recent evidence suggests, however, that diversity might arise through the interaction of developing T cells with other cells, the identity of which is not known. In this study we show that T cell diversity is driven by B cells and Ig. The TCR Vβ diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 × 102 from wild-type values of 1.1 × 108; in mice with oligoclonal B cells, the TCR Vβ diversity of thymocytes is 0.01% that in wild-type mice. Adoptive transfer of diverse B cells or administration of polyclonal Ig increases thymocyte diversity in mice that lack B cells 8- and 7-fold, respectively, whereas adoptive transfer of monoclonal B cells or monoclonal Ig does not. These findings reveal a heretofore unrecognized and vital function of B cells and Ig for generation of T cell diversity and suggest a potential approach to immune reconstitution.