ALBERT

Description: In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5+ B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin+ cells were actively proliferating and, in contrast to Survivin+ B cells found in normal GC, were Bcl-2+. In B-CLL BM biopsies, CD5+, Survivin+ cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

Description: Abstract 1051 Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-γ rearrangement analysis. To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC. To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded. In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR. These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INFγ and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients' CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls. In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma. Disclosures: No relevant conflicts of interest to declare.

Description: Background. The role of normal lymphocytes in the development of malignancies is investigated with increasing interest, with particular focus on lymphoproliferative disorders. A few recent reports, including a large study from our group, have shown that the lymphocyte/monocyte ratio (LMR) is often altered in diffuse large B cell lymphoma (DLB-CL) and a reduced LMR at diagnosis is associated with a poor response to therapy and a marked reduction in the overall survival expectancy. Reduction of LMR is primarily due to decreased levels of circulating lymphocytes, although an increase of circulating monocytes may also concur in reducing the LMR value. The present study was undertaken to further characterize the lymphocyte subpopulations in DLB-CL and in the other germinal-center derived Follicular Lymphoma (FL). In particular, the aim was to investigate possible abnormalities of either B or T-lymphocytes that might be present in patients with DLB-CL or FL at disease onset. Patients and Methods. Peripheral blood (PB) samples were obtained at diagnosis from 23 DLB-CL and 15 FL without leukemic involvement; among them 11/23 DLB-CL and 12/15 FL did not display disease involvement also in their bone marrow (BM). In addition, 25 PB and 20 BM were obtained as controls from age matched healthy donors or from subjects undergoing diagnostic procedures without any evidence of hematological or solid malignancy. Multicolor flow cytometry (MFC) was used, to evaluate the PB distribution and the absolute value of the main cell categories, i.e.: CD3+ T cell, CD3-CD56+ NK cells, and CD19+CD20+ B cells; among these latter, the following subsets were investigated: transitional (CD38+high/CD24+high/IgD+/CD27-), naive (CD38+/CD24+/IgD+/CD27-), memory IgD+ (CD38-/+CD24+/IgD+/ CD27+), and memory IgD- (CD38-/CD24+high/IgD-/CD27+). Similarly, MFC was used to identify B-lymphocyte subsets in BM samples (B cell precursors as CD19+CD20-CD10+ and mature B cells as CD19+CD20+CD10-). Results. The absolute number of circulating B cells was significantly reduced in both FL (median 86 cell/ul) and DLB-CL (median 72 cell/ul) compared to normal age-matched controls (median 233 cells/ul), as detailed in the Figure 1. The low absolute number of circulating B cells did not correlate with a low LMR or with bone marrow involvement. Despite the generalized reduction in absolute number, the proportion of transitional and naïve B cell subsets was similar or slightly increased in B-NHL, while the percentage of memory B cell was deeply reduced in both FL and DLB-CL compared to normal controls. The amount of BM CD19+ B cells, evaluated as percentage on the whole lymphocyte population, was similar in both FL and DLBCL (15% and 15.9% respectively) and not significantly different from normal controls (18.9%). Interestingly, the percentage of B cell precursors (CD19+CD20-CD10+) within the B cell compartment was similar in FL and normal BM (50% vs 43.1%, p=ns), while it was marginally reduced in DLB-CL (23.7% vs 43.1%, p=ns). T and NK cells were not significantly different in DLB-CL and FL and in normal controls. Conclusions. Our data suggest that the homeostasis of normal B-cell compartment is impaired in FL and DLBCL, mainly at the peripheral level. The circulating memory B cells seem to be the B-cell subset affected in FL and DLB-LC. The mechanism underlying the reduction of circulating B lymphocyte number remains unknown. It might be hypothesized that FL and DLB-CL affect the microenvironment in which normal B cell differentiation occurs, resulting in the hampered production or increased elimination of differentiating B-cells. This hypothesis seems supported by the observation that the B cell population is not significantly altered in BM. Further study should be addressed to verify the impact of B-cell reduction on the long-term outcome of DLB-CL e FL and whether increasing circulating B-cells may improve the response to therapy. In addition the results here reported should be considered in relation with the prolonged B-cell impairment associated with the widely employed chemo-immunotherapy treatments for B-cell lymphoma. Figure 1. Absolute count values of total PB B lymphocyte in normal control (dottet bar), DLBCL (vertical line bar) and FL (diagonal line bar). Figure 1. Absolute count values of total PB B lymphocyte in normal control (dottet bar), DLBCL (vertical line bar) and FL (diagonal line bar). Disclosures No relevant conflicts of interest to declare.

Description: Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

Description: Abstract 4640 Introduction. Lenalidomide is a novel therapeutic agent with immunomodulatory activity, that is clinically active in myelodysplastic syndrome and multiple myeloma. Recent clinical trials have shown that Lenalidomide may have clinical efficacy also in patients with Chronic Lymphocytic Leukemia (CLL). Studies so far reported have been carried out in patients with refractory or relapsed CLL. Besides its clinical efficacy, some concerns have been raised on the possible toxicity of Lenalidomide, with some unique effects, namely tumor flare reactions and tumor lysis syndrome, observed in CLL patients. A recent pilot study has been started at our Institution aimed to verify safety and efficacy of Lenalidomide given to control persistent minimal residual disease (MRD) in patients with CLL in 1st Complete Remission (CR). The selection of patients in 1st CR had a dual advantage: i. the possible use of Lenalidomide as single agent, without any other concurrent chemo-immunotherapeutic drugs that might interact in the clinical response; ii. the presence of low tumor burden, that allows to minimize the risk of severe side effects, in particular the risk of tumor lysis syndrome. So far, three patients have concluded the first year of Lenalidomide. Their clinical outcome, including treatment feasibility and monitoring of MRD, is here reported. Patients and treatment plans. Case # 1: male, aged 53 yrs. in 2002 when he had a diagnosis of B-CLL, with mutated IgH genes; he remained in watchful waiting (w/w) for two yrs., then he received a Fludarabine-based treatment supplemented with Rituximab, achieving CR in September 2005; he then received 2-yr. maintenance with Rituximab; since 2008 he was off-therapy in clinical CR; Case # 2: male, aged 59 yrs. in 2005 when he had a diagnosis of B-CLL, with mutated IgH genes; he remained in w/w for three yrs., then he received a Fludarabine-based treatment supplemented with Rituximab, achieving CR in May 2008; thereafter he was off-therapy in clinical CR; Case # 3: female, aged 38 yrs. in 2002 when she had a diagnosis of B-CLL, with un-mutated IgH genes and she received an induction treatment with Fludarabine supplemented with Rituximab, achieving CR in April 2003; she then received prolonged and intermittent maintenance treatments with Rituximab; since 2008 she was off-therapy in clinical CR. All these patients showed a slow though continuous increase of their clonal B-cell population, identified by cytofluorimetric analysis as CD19+/CD5+ve cells. Based on the increase of the MRD, the patients were placed under Lenalidomide, given at 15 mg/day for 3 weeks/month, until disease progression or discontinuation for severe toxicity. Aspirin at 100 mg. daily was added for thromboembolic prophylaxis. Lenalidomide administration was approved by the Italian National Agency of drugs (AIFA). Results. Presently, all three patients are under Lenalidomide since 12 consecutive months, according to the planned treatment. There have been no severe toxicities, in particular neither tumor flare nor symptoms related to tumor lysis have been recorded. So far, no patients showed clinical signs of disease progression. MRD has been carefully monitored by immunophenotypic analysis on both bone marrow and peripheral blood. In details, the CD19+/CD5+ve clonal B-cell population progressively decreased under Lenalidomide in two patients, with the following changes over 1 yr. before and 1 yr. after Lenalidomide, respectively: 5 to 66 cells/μ L before, then reduction to 12 cells/μ L, in case #1; 189 to 4,480 cells/μ L before, then reduction to 1,317 cells/μ L, in case #2; the clonal B-cell population is still growing (from 67 to 116 cells/μ L before, and then up to 497/μ L after Lenalidomide) in the third patient, with un-mutated B-CLL. A significant increase of the activated T-cell (CD3+/DR+) population has been observed in all three patients following Lenalidomide. Conclusions. This pilot study suggests that: i. Lenalidomide can be administered at 15 mg/day, 21 days/month, for over 1 yr. to patients with B-CLL in first CR, with reduced risks of severe side effects; ii. this schedule may be effective to control the expansion of the MRD in patients with B-CLL in clinical CR; iii. further studies on large series should be planned to verify in which patient subsets Lenalidomide might be offered, in order to reduce or even eliminate the MRD that usually persists in B-CLL patients following induction chemo-immunotherapy. Disclosures: Off Label Use: Lenalidomide in CLL in first CR to control MRD.

Description: Chronic lymphocytic leukemia (CLL) has a variable clinical course. CD38 expression and IgVH gene mutational status are independent predictors of prognosis, but their relationships and the CD38 cutoff level are unknown. Using cytofluorography, we analyzed CD38 in 148 patients, in 108 of whom we were able to evaluate IgVH mutations, make correlations with disease history, and assess cumulative survival. Three different patient groups were identified by the CD38 expression pattern: a group homogeneously CD38−, a group homogeneously CD38+, and a group characterized by a bimodal profile, because of the concomitant presence of variable proportions of 2 distinct populations, one CD38+ and one CD38−. In CD38 bimodal expression patients the CD38+ subset was significantly more represented in the bone marrow than in the peripheral blood. For IgVH mutations, 11.4% of CD38−, 84.6% of CD38+, and 68.0% of CD38 bimodal expression patients had no mutation. CD38 expression, IgVH mutational status, and traditional prognostic factors were concordant. The progression rate was 12.9% for CD38−, 75.0% for CD38+, and 63.3% for CD38 bimodal expression patients. Only 25.8% of the CD38−patients but 63.3% of the bimodal and 75.0% of CD38+patients were treated. The presence of a CD38+ population, albeit small, correlated with the development of autoimmune manifestations. The CD38− group has not yet reached the median survival, which is 183 months in the CD38+ group and 156 months in the CD38 bimodal expression group, regardless of the size of the CD38+ population. The presence of a distinct CD38+ population within the leukemic clone, rather than a numerical cutoff definition, correlates with IgVH gene mutational status and, irrespective of its size, identifies CLL patients who will have progressive disease.