Results of a randomized trial comparing high-dose chemotherapy plus Auto-SCT and R-FC in CLL at diagnosis.

The importance of early therapy intensification in B-cell CLL (B-CLL) patients remains to be defined. Even though several studies have been published, no randomized trials comparing directly autologous stem cell transplant (ASCT) and the accepted conventional therapy (that is, rituximab, fludarabine and CY; R-FC) have been reported so far. To assess the benefit of a first-line aggressive therapy, we designed a multicenter, randomized, phase 3 trial comparing R-FC and high-dose chemotherapy supported by ASCT in patients under 65 years of age, with stage B(II) or C B-CLL. Primary end point was CR: 96 patients were enrolled (48 in each arm). On an intent-to-treat basis, the CR rates in the ASCT and R-FC arms were 62.5% and 58%, respectively. After 5 years of follow-up, PFS was 60.4% in the ASCT arm and 65.1% in the R-FC arm, time to progression 65.8 and 70.5%, and overall survival 88% vs 88.1%, respectively. Our trial demonstrates, for the first time in a randomized manner, that frontline ASCT does not translate into a survival advantage when compared with benchmark chemoimmunotherapy in B-CLL patients; the possibility of its clinical benefit in certain subgroups remains uncertain.

CD38 increases CXCL12-mediated signals and homing of chronic lymphocytic leukemia cells.

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (P<0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (P<0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38(-) CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P=0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

Telomere length is an independent predictor of survival, treatment requirement and Richter's syndrome transformation in chronic lymphocytic leukemia.

Telomere length (TL) has been associated with outcome in chronic lymphocytic leukemia (CLL). The aim of this extensive analysis carried out on 401 CLL patients was to assess TL conclusively as a prognostic biomarker. Our study included two cohorts used as learning (191 patients) and blinded validation series (210 patients). A TL cutoff of 5000 bp was chosen by receiver operating characteristic (ROC) analysis and Youden's index in the learning series. In this series, TL< or =5000 bp was independently associated to a worse outcome for both overall survival (OS; 105.5 vs 281 months, P<0.001) and treatment-free survival (TFS; 24.6 vs 73 months, P<0.001). In the blinded validation series, TL< or =5000 bp was confirmed as an independent outcome predictor for OS (79.8 vs not reached, P<0.001) and TFS (15.2 vs 130.8 months, P<0.001). Moreover, TL< or =5000 bp independently predicted the risk of Richter's syndrome (5-year risk: 18.9 vs 6.4%, P=0.016). Within CLL subsets defined by biological predictors, TL consistently identified patient subgroups harboring unfavorable prognosis. These results demonstrate that TL is a powerful independent predictor of multiple outcomes in CLL, and contributes to refine the prognostic assessment of this disease when utilized in combination with other prognostic markers. We thus believe that this prognostic biomarker has the potential for a more widespread use in CLL.

Telomere length identifies two different prognostic subgroups among VH-unmutated B-cell chronic lymphocytic leukemia patients.

Some evidences suggest that telomere restriction fragment length (TRF-L) is an effective indicator of histopathogenesis in B-cell tumors. As histopathogenesis is relevant for B-cell chronic lymphocytic leukemia (B-CLL) prognosis, TRF-L was assessed by Southern blot in 201 patients and compared to variable immunoglobulin heave chain gene mutational status (VH-MS) and to other known prognostic features. Overall survival (OS), time to first treatment (TTFT) and progression-free survival (PFS) were evaluated. Our results indicate the following: (1) TRF-L is heterogeneous among B-CLL patients (median 6014 bp, range 1465-16 762); (2) TRF-L correlates to VH-MS (r(2)=0.1994, P<0.0001) with VH-mutated patients showing long and VH-unmutated short telomeres; however, 41% of VH-unmutated and 5% of VH-mutated patients did not show this correlation and were thus defined as 'discordant'; (3) TRF-L effectively predicts outcome in terms of TTFT, PFS and OS; (4) VH-unmutated discordant patients have a better clinical outcome than VH-unmutated concordant patients (OS P<0.01, PFS P<0.05) and similar to that of VH-mutated patients (OS, PFS P=NS). Compared to VH-unmutated concordant patients, VH-unmutated discordant patients showed no peculiarity in their immunoglobulin rearrangement nor in their flow cytometry or fluorescence in situ hybridization profile. In conclusion, TRF-L can be helpful to refine prognostication of B-CLL patients, particularly those with a VH-unmutated immunoglobulin sequence.

Feasibility of peripheral blood progenitor cell mobilization and harvest to support chemotherapy intensification in elderly patients with poor prognosis: non-Hodgkin's lymphoma.

Mobilized peripheral blood progenitor cells (PBPC) are widely employed in the management of adult patients with high-risk non-Hodgkin's lymphoma (NHL), though their use in the elderly has received little attention. This study was mounted to assess the feasibility of the mobilization, harvesting, and reinfusion of PBPC in NHL patients aged >60. Twenty patients (median age: 67, range: 61-80) with poor-prognosis NHL entered the pilot study: nine others were discarded for various reasons. Thus, the program was applicable to 69% of potential candidates. Fourteen patients were at onset and six were being treated for refractory disease or relapses. Mobilization was induced with cyclophosphamide 4 g/m(2), followed by 5 micro g/kg per day granulocyte-colony stimulating factor (G-CSF) s.c. until PBPC collection or hemopoietic recovery. Sixteen patients (80%) displayed some signs of mobilization (CD34+: >5/ micro l). Maximum mobilization varied with median circulating CD34+ cells and colony forming units-granulocyte/macrophage (CFU-GM) peaks of 17.2/ micro l (range: 8.1-210) and 1,650/ml (range: 540-62,900), respectively. A median of two leukaphereses resulted in the harvesting of a median of 6.7x10(6) (range: 0.3-33.6) CD34+/kg and 21.1x10(4) (range: 1.2-209) CFU-GM/kg. Intensified therapy with intermediate-dose melphalan, associated or not with mitoxantrone, was delivered with autologous PBPC support to 13 patients and always resulted in rapid and stable hemopoietic reconstitution. The program was well tolerated and no treatment-related deaths occurred. Twelve patients are still alive with a 5-year survival projection of 59%. In conclusion, the results demonstrate the feasibility of using autologous PBPC to support therapy intensification even in elderly patients.

Rituximab anti-CD20 monoclonal antibody induces marked but transient reductions of peripheral blood lymphocytes in chronic lymphocytic leukaemia patients.

Rituximab has been recently proposed as an effective non-chemotherapeutic option for patients with follicle centre lymphoma (FCL). However, less is known on its role in chronic lymphocytic leukaemia (CLL). We thus decided to assess its effectiveness on a panel of 7 patients with refractory or relapsed CLL. Mild (5 patients) or severe (1 patient) adverse reactions were observed during the first hours of Rituximab infusion, almost exclusively at the first course. Symptoms rapidly subsided with temporary drug withdrawal and low dose steroids. All patients could receive the whole scheduled treatment. A striking reduction of peripheral blood (PB) lymphocyte counts was observed in all patients (median 93%; range 57-99%). However, Rituximab was poorly effective towards nodal and splenic disease. Patients required additional treatment after a median time of 70 d (range: 20-180 d). Our data show that Rituximab delivery in CLL patients is feasible and has an acceptable toxicity, although it probably does not represent an ideal treatment option when delivered using schedules originally designed for FCL patients. However, responses observed at PB level suggest that Rituximab has an activity which is not negligible and deserves further investigation in CLL. Future approaches will be directed to the development of alternative schedules which may include dose intensification, combination of Rituximab and chemotherapy, and in vivo purging of peripheral blood progenitor cell harvests for autografting procedures.

Fludarabine ability to down-regulate Bcl-2 gene product in CD5+ leukaemic B cells: in vitro/in vivo correlations.

CD5+ B-chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl-2 family of genes. Fludarabine (9-beta-D-arabinofuranosyl-2-fluoradenine, F-ara-A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F-ara-A-induced apoptosis might be related to Bcl-2 modifications and to evaluate in vitro/in vivo correlations. Peripheral blood lymphocytes from eight B-CLL and four leukaemic MCL were cultured in the presence of different concentrations of F-ara-A +/- methylprednisolone (MP). F-ara-A down-regulated the expression of Bcl-2 in 5/12 cases. mRNA down-regulation was maximal at 48 h; protein down-regulation was prominent after 48 h. Both events were dose-dependent. The amount of apoptosis was significantly higher in the samples treated with F-ara-A than in those exposed to MP alone. In the seven remaining cases, no Bcl-2 down-regulation was observed after exposure to F-ara-A and the degree of F-ara-A-induced apoptosis overlapped that induced by MP. The in vivo outcome after treatment with three to six courses of F-ara-A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl-2 down-regulation and prominent apoptosis after exposure to F-ara-A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl-2 modulation after exposure to F-ara-A, two had a PR, but the other three did not show any in vivo clinical response.

Osteoclast precursors circulate in the peripheral blood of patients with aggressive multiple myeloma.

Osteolysis resulting in extensive bone damage is a major clinical manifestation of patients with multiple myeloma (MM). The mechanisms of bone resorption in MM are incompletely understood. The final pathway is the generation of activated osteoclasts within bone marrow (BM) microenvironment. To investigate the mechanisms of bone resorption in MM we established an experimental system that, including bone marrow (BM) stromal cells and bone slices, closely mimicks in vitro the in vivo BM microenvironment. Peripheral blood mononuclear cells (PBMC) from nine patients with MM, three monoclonal gammopathy of undetermined significance (MGUS), and nine normal controls were cultured in this system. PBMC from patients with aggressive and bone devastating MM gave rise to multi-nucleated cells with the morphology and phenotype of osteoclasts. These cells induced bone resorption in vitro which was inhibited by the addition of calcitonin. No bone resorption was observed in cultures of PBMC from patients with MM and limited bone damage, with MGUS and from normal subjects. These findings indicate that patients with aggressive MM have a population of circulating precursors that develop into functionally active osteoclast-like cells once they come in contact with the BM microenvironment. These cells may contribute to the wide-spread and generalized bone erosion observed in the patients.

Characterization of bone marrow stromal cells from multiple myeloma.

We have cultured multiple myeloma (MM) bone marrow (BM) stromal cells that are able to sustain the in vitro growth of monoclonal B-cells. Our aim was to evaluate which adhesion molecules are expressed and which extracellular matrix proteins are produced by these cells and whether they differ from the stromal cells that can be grown under the same experimental conditions from the BM of monoclonal gammopathies of undetermined significance (MGUS) and of normal donors. MM BM stromal cells that support malignant B-cell development have a striking proliferative ability that is absent in MGUS and normal donors of the same age group and are formed by four major different cell populations. Two kinds of HLA-DR+, CD10+ fibroblast-like cells can be recognized through the expression (or the lack) of alpha-smooth muscle actin isoform; further, macrophages and osteoclasts can be identified. Fibroblast-like cells that express alpha-smooth muscle actin isoform, often organized along stress fibers in a periodic fashion, may be considered as myofibroblasts. Fibroblast-like cells react strongly with antibodies to CD54 (ICAM-1), integrin beta 1, beta 3, beta 5 and some of associated alpha chains. Integrin beta 1 is diffusely exposed on the surface while beta 3 is clustered in focal contacts in association with vinculin. A still undetermined subpopulation of fibroblasts is highly positive for alpha v beta 5 that is clustered at focal contacts as shown by association with stress fiber termini and by interference reflection microscopy. A major difference between MM and normal donor BM stromal cells involves lower deposition and simpler organization of the extracellular matrix proteins (fibronectin, laminin, collagen type IV) deposited by MM fibroblast-like cells. CD14+ macrophages from MM, MGUS and normal donor BM are CD11a+ (alpha L), CD11b+ (alpha M), CD11c+ (alpha X), CD54+ (ICAM-1), CD56+ (N-CAM), beta 1 and beta 2 (CD18) integrin positive. The integrin beta 1 is diffusely expressed on the surface, while beta 2 is concentrated in podosomes. MM osteoclasts show a weak diffuse staining with CD54 and CD56 MoAbs; beta 1 integrin has a diffuse surface expression, while beta 3 integrin is concentrated in the podosomes. Normal donor osteoclasts are CD54- and the staining with CD56 is barely visible. These findings lead us to suggest that the microenvironment provided by MM BM may be significantly different from that of normal BM indicating its potential role in controlling the local proliferation and differentiation of malignant B-lineage cells.

Cytokines involved in the progression of multiple myeloma.

We have investigated which of the cytokines that are relevant in the in vitro growth of multiple myeloma (MM) malignant plasma cells are actually produced in vivo by MM patients. To this end, we have measured the levels of IL-1 beta, IL-3, IL-4, IL-6, IL-7, IL-8 and tumour necrosis factor-alpha (TNF-alpha) both in sera and in the supernatant of bone marrow (BM) stromal cell cultures from patients with MM and monoclonal gammopathy of undetermined significance (MGUS). The significance of our findings is three-fold. First, IL-6 and IL-8 are produced by MM BM stromal cells, while IL-1 beta, TNF-alpha, IL-4 and IL-7 are not. Second, IL-3 is the only cytokine consistently raised in serum samples: we have also detected low levels of serum IL-6 in a minority of cases, usually in advanced stage of the disease. Third, MM BM stromal cells are active IL-6 and IL-8 producers, while both normal and MGUS BM stromal cells are low producers, thus suggesting that in the BM of MM a number of environmental cells, that would normally be quiescent, are instead activated and that, in MM, activated BM stromal cells play an active role in supporting the progressive expansion of the B cell clone.

The nature of the B lymphocyte in B-chronic lymphocytic leukemia.

We have analyzed phenotypic, functional, and molecular properties of B-chronic lymphocytic leukemia (B-CLL) cells as compared to normal B cell differentiation stages and/or subsets. The possibility that the target B cell population transformed by the I primary oncogenic event(s) belongs to the normal CD5+ B cell subset from B mantle zone of secondary follicles is highly likely on phenotypic grounds. Though the genes responsible for the primary oncogenic event are presently unknown, a number of functional and molecular findings indicate that the end-product of their transforming activity is a cell frozen in the G0 phase of the cell cycle. This cell has several abnormalities that prevent an appropriate mitogenic response and presents a pattern of apoptosis-related gene expression that hinders apoptotic death. Pivotal to this apoptosis-escaping capacity is the expression of Bcl-2. We suggest that the increased expression of Bcl-2 together with an asynchronism between the expression of Bcl-2, c-myc, and APO1/Fas gene products shift the cellular balance away from apoptosis thereby helping the progressive accumulation in G0 of malignant CD5+ B cells.

Bone marrow microenvironment and the progression of multiple myeloma.

The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1 beta and M-CSF activating BM stromal cells to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-1 beta, TNF-beta, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population.

In vitro growth of human multiple myeloma: implications for biology and therapy.

In vitro data allow presentation of a plausible scenario for the in vivo growth, progression, and dissemination of human multiple myeloma (MM) that involves the interactions between the monoclonal B-cell clone and the bone marrow (BM) microenvironment. A large series of adhesion and extracellular matrix molecules allow trapping of circulating plasma cell precursors within the BM, and a battery of locally released cytokines promote their growth and final differentiation. Malignant B cells establish close contacts with BM stromal cells and release a host of cytokines that recruit and activate BM stromal cells and also T lymphocytes to produce other cytokines. All these cytokines might conceivably act in concert in a self-perpetuating mechanism of mutual help between malignant plasma cells and BM stromal cells to favor the progressive expansion of the malignant clone through a sort of an "avalanche effect." Also, most cytokines produced by malignant B cells, stromal cells, and activated T lymphocytes, including IL-1 beta, TNF-beta, M-CSF, IL-3, and IL-6, have osteoclast-activating properties, thus explaining why the expansion of the B-cell clone is matched by the activation and numeric increase of osteoclasts.

'Role of bone marrow stromal cells in the growth of human multiple myeloma.

We have verified the hypothesis that multiple myeloma (MM) may be disseminated by circulating clonogenic cells that selectively home to the bone marrow (BM) to receive the signal(s) leading to proliferation, terminal differentiation, and production of the osteoclast activating factors. Long-term cultures of stromal cells have been developed from the BM of nine patients with MM. These cells were mostly fibroblast-like elements, interspersed with a proportion of scattered macrophages and rare osteoclasts. BM stromal cells were CD54+, produced high levels of interleukin-6 (IL-6) and measurable amounts of IL-1 beta, and were used as feeder layers for autologous peripheral blood mononuclear cells (PBMC). After 3 weeks of cocultures, monoclonal B lymphocytes and plasma cells, derived from PBMC, developed and the number of osteoclasts significantly increased. Both populations grew tightly adherent to the stromal cell layer and their expansion was matched by a sharp increase of IL-6 and by the appearance of IL-3 in the culture supernatant. These data attribute to BM stromal cells a critical role in supporting the growth of B lymphocytes, plasma cells, and osteoclasts and the in vivo dissemination of MM.

Integrin distribution and cytoskeleton organization in normal and malignant monocytes.

In this work we have mapped by double-label immunofluorescence the cellular distribution of integrins and their relationship with cytoskeletal proteins in normal and malignant monocytes. In normal monocytes, CD18 and CD11c are concentrated at specific adhesion sites, named podosomes, together with actin, vinculin, and talin, while CD11a, CD11b, CD29/beta 1, CDw49d/alpha 4 and CD54/ICAM-1 retain a diffuse distribution on the cell surface without a selective pattern of localization. U-937 and fresh leukemic monoblasts under standard culture conditions do not adhere and do not form podosomes, but, when treated with TPA, they promptly adhere to substrate, form podosomes and focal adhesions in different cells and display the same integrin/cytoskeleton relationship as normal mature monocytes. Further, in these cells CD18, CD11a, CD11c, ICAM-1, and talin, but not vinculin, co-localize in homotypic cell junctions, thus showing a close relationship between integrins and talin. These observations provide morphological evidence that, in cells of the monocytic lineage, podosome formation is acquired upon differentiation and different integrins are selectively localized at different adhesion sites.