Eric Legouffe

Survival and proliferation factors of normal and malignant plasma cells.

Since the first identification of interleukin (IL)-6 as a myeloma cell growth factor by Dr. Kawano’s and Dr. Klein’s groups 14 years ago, numerous studies have emphasized its major roles in the emergence of malignant plasma cells in vivo and in the generation of normal plasma cells. Four transcription factors control B-cell differentiation into plasma cells. The B-cell transcription factor pax-5 is mainly responsible for a B-cell phenotype, andbcl-6 represses the plasma cell transcription factor blimp-1 and plasma cell differentiation.bcl-6 expression is triggered by CD40 and IL-4 activation. A lack of CD40 and IL-4 activation yields a down-regulation ofbcl-6 expression, and IL-6 stimulation yields an up-regulation of blimp-1, mainly through STAT3 activation. Blimp-1 further down-regulatesbcl-6 andpax-5 expression and makes plasma cell differentiation possible. IL-6 as well as IL-10 up-regulate XBP-1. XBP-1 is another transcription factor that is involved in plasma cell differentiation and whose gene expression is shut down by pax-5.The plasma cell transcription factors blimp-1 and XBP-1 are up-regulated, and the B-cell transcription factors bcl-6 and pax-5 are down-regulated, in malignant cells compared to B-cells. Apart from the recent identification of these 4 transcription factors, the factors involved in normal plasma cell generation are mostly unknown. Regarding malignant plasma cells, 3 categories of growth factors have been identified: (1) the IL-6 family cytokines, IL-10, and interferon α that activate the Janus kinase—signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase pathways; (2) growth factors activating the phosphatidylinositol (PI)-3 kinase/AKT and MAP kinase pathways, unlike the JAK/STAT pathway (insulin-like growth factor 1, hepatocyte growth factor, and members of the epidermal growth factor family able to bind syndecan-1 proteoglycan); and (3) B-cell—activating factor (BAFF) or proliferationinducing ligand (APRIL) that activate the nuclear factor κB and PI-3 kinase/AKT pathways.BAFF and APRIL bind to BAFF receptor and TACI and are major B-cell survival factors. Recent data indicate that these various growth factors may cooperate to provide optimum signaling because they are localized together and with cytoplasmic transduction elements in caveolinlinked membrane caveolae. The identification of these myeloma cell growth factors and of the associated transduction pathways should provide novel therapeutic targets in multiple myeloma.

The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells

Syndecan‐1 is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. It is expressed only on malignant plasma cells in bone marrow samples from patients with multiple myeloma (MM). Several reports have suggested that syndecan‐1 was present only on a part of the myeloma cells. By using either IL‐6‐dependent myeloma cell lines or primary myeloma cells stained by annexin V, we report here that syndecan‐1 was rapidly lost by myeloma cells undergoing apoptosis. In the same experimental conditions, expression of other cell membrane antigens such as CD38, HLA class‐I or CD49d on apoptotic myeloma cells was not affected. In addition, we show that syndecan‐1 loss was independent of activation of the gp130 IL‐6 transducer. Dexamethasone induced a strong apoptosis of myeloma cells associated with the loss of syndecan‐1. Finally, by using freshly‐explanted tumoural samples, we show that syndecan‐1 rapidly disappeared from myeloma cells in association with induction of apoptosis. In conclusion we showed that syndecan‐1 is a marker for viable myeloma cells which is rapidly lost by apoptotic cells. These results emphasize the usefulness of anti‐syndecan‐1 antibodies to purge tumoural cells from haemopoietic grafts or to purify these cells for further manipulations for immuno or gene therapies.

C-Reactive Protein Serum Level is a Valuable and Simple Prognostic Marker in Non Hodgkin's Lymphoma

Interleukin-6 plays a central role in normal B-cell maturation and in proliferation of some B-cell malignancies including multiple myeloma and some non Hodgkins lymphomas (NHL). Furthermore, this cytokine also plays a major role in acute phase response by mediating synthesis of acute phase proteins such as C-reactive protein (CRP). In order to evaluate the exact role of CRP serum level as a simple prognostic factor, we analyzed CRP and IL-6 serum levels in 39 patients with NHL. Eleven patients had low grade NHL, 15 intermediate grade NHL, and 13 high grade NHL. Thirty percent of the patients presented detectable IL-6 serum levels (mean+/-SD: 33.6+/-95.2 U/ml, range: 0 to 500). Increased serum CRP levels were found in 42% of the patients with a mean of 29.2+/-41.97 mg/l] (range: 0 to 129). Thirty seven patients were studied for both markers. Three groups of patients were determined. One with low IL-6 and CRP serum levels (N=21), a second with high level of both markers (N=10), and the third with high serum CRP levels alone (N = 5). Only one patient had high level of serum IL-6 with no detectable CRP. The correlation of serum IL-6 and CRP levels with patient survival was investigated. Median survival in the group with low IL-6 level was not reached. 67% of patients of this group were still alive at 32 months from diagnosis. The group of patients with detectable IL-6 had a median of survival of 12 months (p<0.025). The survival of patients with a CRP<10 mg/l was not reached. 75% of patients survive at 32 months from diagnosis, whereas the group with higher CRP level reached a median survival at 8.5 months (p<0.009). As expected, on univariate analysis, there is a significant relationship between CRP and IL-6 levels (p<0.00017), and CRP levels and B symptoms (p<0.001). Furthermore there is a significant relationship between CRP and LDH levels (p<0.042).These results indicated that CRP may be considered as a valuable and easy prognostic biomarker of NHL.

Optimizing the use of anti-interleukin-6 monoclonal antibody with dexamethasone and 140 mg/m2 of melphalan in multiple myeloma: results of a pilot study including biological aspects.

Summary:Interleukin-6 (IL-6) is a major survival factor for multiple myeloma (MM) cells preventing apoptosis induced by dexamethasone (DEX) or chemotherapy. In all, 24 consecutive patients with MM in first-line therapy received DEX for 4 days, followed by melphalan (HDM: 140 mg/m2) and autologous stem cell transplantation (ASCT). The anti-IL-6 monoclonal antibody (mAb) (B-E8) was given till haematological recovery, starting 1 day before DEX. Results were historically compared to MM patients treated with HDM 140 and 200 mg/m2. Our results show (1) that B-E8 was able to fully neutralize IL-6 activity in vivo before and after HDM as shown by inhibition of C reactive protein (CRP) production; (2) no haematological toxicity; (3) a significant reduction of mucositis and fever; (4) a median event-free survival of 35 months and an overall survival of 68.2% at 5 years with a median follow-up of 72 months; and (5) the overall daily IL-6 production progressively increased on and after 7 days post-HDM, with the increased serum CRP levels. In the 5/24 patients with uncontrolled CRP production, a large IL-6 production was detected (320 μg/day) that could not possibly be neutralized by B-E8. These data show the feasibility to neutralize IL-6 in vivo with anti-IL-6 mAb in the context of HDM.

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

Enhanced graft-versus-tumor effect following dose-reduced conditioning and allogeneic transplantation for refractory lymphoid malignancies after high-dose therapy

Non-myeloablative regimens have been proven to allow engraftment following allogeneic stem cells transplantation (allo-SCT) with minimal procedure-related toxicity. Conventional allo-SCT may produce remissions in patients with relapsed and refractory lymphoid malignancies (LM) but these good results may be achieved at the cost of high treatment-related morbidity and mortality. Application of allo-SCT using less intensive regimens may temper the frequency of these complications, allowing a potent graft-versus-tumor effect (GVT). We present our data on 11 patients with LM receiving allo-SCT with a reduced regimen. Ten patients had received previous high-dose therapy, and were at high risk for toxicity, thus precluding the use of allo-SCT. A fludarabine and low-dose busulfan combination facilitated engraftment while exerting GVT. Hematological recovery was quick, and full donor T cell chimerism preceded acute GVHD. GVHD and infections were the major problems. Spontaneous acute GVHD occurred in eight patients (72%). Five patients (45%) achieved complete remission, and the GVT effect was closely associated with GVHD. These results support the concept that GVT is effective against LM in patients who have been heavily pretreated. Further studies are needed to investigate strategies to generate more specific alloreactive effects providing optimal GVT and an acceptable risk of GVHD and infections. Bone Marrow Transplantation (2001) 28, 335–339.

Treatment of Waldenstrom's Macroglobulinemia with Very Low Doses of Alpah Interferon

Waldenströms macroglobulinemia (WM) is a differentiated B-cell malignancy which is usually less responsive to standard chemotherapy because of low-proliferating cells. Interferon alpha has been shown to possess a therapeutic action in numerous B-cell malignancies including the early stage of chronic lymphocytic leukemia, multiple myeloma, follicular lymphoma and hairy cell leukemia. Fourteen patients with progressive WM were included in a pilot study using very low dose of interferon alpha-2a (1 Million Units 3 times a week). The mean duration of treatment was 10.3 months (range 2-44). Six of 14 (42%) patients presented an increase in the hemoglobin level (> or = 0.9 g/dL) and 4/14 (28%) had a substantial decrease of the monoclonal component (> or = 20% of reduction). Only two patients presented both types of response, while the others with an increase in the hemoglobin level had a slight decrease in the monoclonal component (MC) (1 patient), a stable MC (1 patient) or a slight increase of MC (1 patient). One additional patient had a 15% decrease of the MC with a stable hemoglobin level. Response was observed within 3 months with a median duration of 6 months. Treatment was stopped for 3 patients because of flu-like symptoms (2 patients), or thrombocytopenia (1 patient). Follow up was possible in 12 patients lasting up to a maximum of 30 months after discontinuing treatment. Seven patients died, including 4 with progressive disease, two of infection and one of cardiac failure. In the view of these results, very low dose of interferon alpha may constitute a new approach for treatment of some cases of WM.

Low doses of GM-CSF (molgramostim) and G-CSF (filgrastim) after cyclophosphamide (4 g/m2) enhance the peripheral blood progenitor cell harvest: results of two randomized studies including 120 patients.

The use of a combination of G-CSF and GM-CSF versus G-CSF alone, after cyclophosphamide (4 g/m2) was compared in two randomized phase III studies, including 120 patients. In study A, 60 patients received 5 × 2 μg/kg/day of G-CSF and GM-CSF compared to 5 μg/kg/day of G-CSF. In study B, 60 patients received 2.5 × 2 μg/kg/day G-CSF and GM-CSF compared to G-CSF alone (5 μg/kg/day). With the aim to collect at least 5 × 106/kg CD34 cells in a maximum of three large volume leukapherises (LK), 123 LK were performed in study A, showing a significantly higher number of patients reaching 10 × 106/kg CD34 cells (21/29 in G+GM-CSF arm vs 11/27 in G-CSF arm, P=0.00006). In study B, 109 LK were performed, with similar results (10/27 vs 15/26, P=0.003). In both the study, the total harvest of CD34 cells/kg was twofold higher in G-CSF plus GM-CSF group (18.3 × 106 in study A and 15.85 × 106 in study B) than in G-CSF group (9 × 106 in study A and 8.1 × 106 in study B), a significant difference only seen in multiple myeloma, with no significant difference in terms of mobilized myeloma cells between G-CSF and GM-CSF groups.

Front-line high-dose chemotherapy (HDT) combined with rituximab for adults with aggressive large B cell lymphoma (DLBCL). A pilot study by the GOELAMS

6662 Background: The beneficial effect of adding rituximab to CHOP has been shown for elderly patients with DLBCL (Coiffier et al NEJM 2002). We have shown that HDT with autologous stem cell transplantation is superior to CHOP in young adults with DLCL (Milpied et al NEJM in press). The feasibility of adding rituximab to front-line HDT remains to be established. METHODS A prospective pilot trial was proposed to patients with DLBCL, with intermediate-high or high age-adjusted IPI, up to the age of 60 y.o. This program consisted of 2 courses of high-dose CHOP-like regimen, 15 days apart, with rituximab (375 mg/ m2) on d1 of each course, followed by rituximab on d 22, harvest of peripheral blood stem cells (G-CSF mobilized) on days 28,29, then rituximab on d 36 followed by a course of high-dose methotrexate with cytarabine and by a BEAM regimen starting on d 66 to 80, followed by the infusion of stem cells. RESULTS Between 04/2002 and 05/2003, 41 consecutive patients gave their informed consent and were included in that trial. Their median age was 50y.o.( 18-60 yo); 23 had WHO PS >/= 2, the LDH level was > N in 37 patients and 38 had Ann Arbor stage III or IV. The age-adjusted IPI was intermediate-high in 21 and high in 19 patients.At the time of this abstract, the program has been completed in 75% of the patients. Three patients died of toxicity of the treatment before the BEAM regimen and autologous transplantation. On an intent-to-treat basis, the response rate at the end of the treatment was CR/CRu= 64%, PR=8%, failure to achieve a response or progression= 20% and toxic death=8%. No toxic death was directly attributable to the addition of rituximab. CONCLUSIONS The addition of 4 doses of rituximab to this HDT program is feasible with no unexpected toxicity, allows the harvesting of sufficient numbers of stem cells to allow an autologous transplantation following a BEAM regimen in responding patients . Follow-up and comparison with historical control will be performed and presented [Table: see text].