Rita Fazzi

Rituximab as treatment for minimal residual disease in hairy cell leukaemia

Abstract:  Purine analogues have dramatically improved the outcome of patients affected by hairy cell leukemia (HCL), although complete eradication of disease was achieved in few cases. The purpose of this study was to evaluate the role of Rituximab in eradicating minimal residual disease (MRD) in HCL patients after a pre‐treatment with 2‐chloro‐deoxy‐adenosine (2‐CdA). Ten patients received four cycles of Rituximab after administration of Cladribrine. Before starting anti‐CD20 antibody, two patients were in complete remission, six in partial remission and two showed no significant response to Cladribrine. All cases resulted IgH‐positive. Median time from the last 2‐CdA infusion was 5.7 months. Eight of 10 patients [four in partial remission (PR), two in complete remission (CR) and two unresponsive after 2‐CdA] were evaluable for response. Two months after the end of anti‐CD20 therapy, all evaluated patients presented a complete haematological remission. Moreover, Rituximab increased percentage of molecular remission up to 100% 1 yr after the end of treatment. Interestingly, in all cases but one, including those persistently polymerase chain reaction (PCR)‐positive, semi‐quantitative molecular analyses showed MRD levels lower than those found before Rituximab administration. Toxicity was very mild. The present results not only confirm the therapeutic effect of Rituximab, but also show its relevance in eradicating MRD in HCL.

Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by Rituximab

Summary:The main aim of this paper was to compare results of Genescan and real-time PCR methods in order to detect contamination in harvests from patients with follicular lymphoma. The secondary goal was to evaluate the efficacy of Rituximab as an in vivo purging agent. A total of 23 patients had been treated with CHOP followed by either high-dose therapy (12 patients) or high-dose plus Rituximab (11 patients), both followed by autologous transplantation. Results show that 86% of harvests from patients treated whith Rituximab were PCR-negative compared to 14.3% from controls. Real-time PCR was more sensitive than Genescan PCR; quantitative analysis revealed a correlation between the amount of contamination in the harvests and relapse after transplantation. Whereas all patients reinfused with negative aphereses achieved complete remission and showed a significantly better 5-year PFS (100%) compared to those reinfused with contaminated samples (41%), a very low amount of contamination does not appear to negatively affect outcome, suggesting that determination of a cutoff in the contamination level of harvests could be useful. Results suggest that real-time PCR is superior to Genescan PCR to select transplantable harvests and confirm the ability of Rituximab as an in vivo purging tool for follicular lymphoma.

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Identification and purification of mesodermal progenitor cells from human adult bone marrow.

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Rituximab as treatment for minimal residual disease in hairy cell leukaemia: extended follow-up

Hairy cell leukaemia (HCL) is a rare chronic lymphoproliferative disorder accounting for about 2% of adult leukaemias. Malignant cells present features of mature activated B-lymphocytes with bright expression of light-chain-restricted surface immunoglobulin (sIg), expression of numerous clonally related heavy-chain isotypes, IGHV mutation and co-expression of mature B cell CD markers. Treatment is indicated in the presence of significant cytopenia, symptomatic splenomegaly, recurrent infection or systemic symptoms. In recent years, therapeutic advances have brought about drastic changes in HCL prognosis. Purine analogues, especially 2-chlorodeoxyadenosine (Cladribine), drastically improved the survival rate, although complete eradication of disease can be rarely achieved (Piro et al, 1990; Tallman et al, 1996). Minimal residual disease (MRD) can be detected either by polymerase chain reaction (PCR) or immunohistochemical techniques on bone marrow samples of patients in complete haematological remission after purine analogue therapy. The persistence of MRD could thus explain the high relapse rate observed in HCL patients after long-term follow-up from the end of induction therapy (Wheaton et al, 1996). Rituximab could represent one attractive option in relapsed cases, because hairy cells typically express high levels of CD20 (Juliusson et al, 1994; Hoffman & Auerbach, 2000; Hagberg & Lundholm, 2001). The optimal schedule has not yet been defined, but some data suggest that extended dosing may improve patient outcome (Thomas. et al, 2003). Moreover, Rituximab could play an interesting role even in eradicating MRD (Cervetti et al, 2004; Ravandi et al, 2006). In our Institution, between May 2002 and November 2006, 27 HCL patients (25 male and 2 female; median age 60 years, range 39–72 years) were treated with anti-CD20 after a pretreatment with Cladribrine. Patients were considered eligible for the study if in partial remission or with persistence of MRD after induction. Other inclusion criteria were: age >18 years; life expectancy >6 months; absence of renal, hepatic and respiratory failure; written informed consent. Baseline clinical characteristics of patients are summarized in Table I. Cladribrine was started if patients presented at least one of the following indications: neutropenia (absolute neutrophil count, <1Æ5 · 10/l), anaemia (haemoglobin level, <100 g/l), thrombocytopenia (platelet count, <100 · 10/l), symptomatic splenomegaly, constitutional symptoms or documented repeated infections. In 17 patients older than 60 years, Cladibrine was administered IV at a dose of 5 mg/m/weekly for a total of six cycles, whereas in 10 younger patients, Cladribine was infused at the dose of 5 mg/m/d for five consecutive days. All patients received acyclovir, fluconazole and cotrimoxazole as antiinfective prophylaxis until 8 weeks after completion of Cladribine administration. Two months after the end of treatment, patients were evaluated for early response. Response criteria were those defined by the National Cancer Institute Working Group. At the time of enrolment in the study, overall response after Cladribrine was 89% [complete remission (CR) 26%, partial remission (PR) 63%; three cases were unresponsive]. The median time between the last Cladribine infusion and the beginning of this protocol was 4Æ3 months (range 1Æ5– 113 months). Minimal residual disease was evaluated by repeating PCR assays. PCR analyses were performed on DNA extracted from mononuclear cells separated by Ficoll/Hypaque gradient, as previously reported (Cervetti et al, 2004). By this method, all cases were proven to be still PCR-positive after induction. After demonstration of persistent MRD or detectable clinical disease, patients were treated by Rituximab (375 mg/m once a week for four doses). Statistical calculations were performed using the Statistical Package for the Social Sciences (spss) for Windows, release 15

Selective Culture of Mesodermal Progenitor Cells

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Bendamustine with or without rituximab in the treatment of relapsed chronic lymphocytic leukaemia: an Italian retrospective study

To retrospectively assess the efficacy of bendamustine alone and with rituximab (R–B), 109 patients with relapsed chronic lymphocytic leukaemia (CLL) were enrolled in 24 Italian centres. The median age was 66 years (range 39–85). Forty‐three percent of patients had relapsed and 57% were resistant (median previous therapies = 3; range 1–8). Twenty‐two patients received bendamustine alone and 87 patients received R–B (median B dosage: 100 mg/m2 per day, range 90–130 mg/m2 per day). The overall response rate was 69·6% (complete response 28·6%; partial response 41%), and was significantly higher in patients treated with R–B (P = 0·014) and in those responsive to the previous treatment (P = 0·04). After a median follow‐up of 7·9 months (range 1–148), the median progression‐free survival was 16 months and the median duration of response was 13 months. Median overall survival (OS) was 16·8 months for the whole cohort; patients not responding to the treatment had a significantly worse outcome than those who attained a response (P = 0·0001). In multivariate analysis, only resistant disease status at start of bendamustine treatment (HR 3·2, 95% CI 1·4–7·3, P = 0·006) had an independent prognostic value for OS. Toxicity was manageable and mostly haematological. In conclusion, in our experience R–B was an effective and well‐tolerated treatment for relapsed/refractory CLL patients, producing a remarkable high CR rate and mild toxicity.

Mesenchymal cells inhibit expansion but not cytotoxicity exerted by gamma–delta T cells

Background  Multipotent mesenchymal stromal cells (MSCs) exert a relevant immunosuppressive activity by inhibiting T‐ and B‐lymphocytes, natural killer (NK) cells and dendritic cell expansion. Nevertheless, a possible activity on gamma/delta T cells has still not been evaluated.

Good manufacturing practice-grade fibrin gel is useful as a scaffold for human mesenchymal stromal cells and supports in vitro osteogenic differentiation

BACKGROUND: Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle‐shaped cells capable of self‐renewal and can be induced to differentiate mainly into osteo‐, chondro‐, and adipogenic‐progeny types. Several biomaterials are currently available and, among them, fibrin‐based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis

A high expression of Wilms’ tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon.