Rheumatoid factor positivity rather than anti-CCP positivity, a lower disability and a lower number of anti-TNF agents failed are associated with response to rituximab in rheumatoid arthritis.

OBJECTIVES: We explored clinical factors associated with a major response to rituximab (RTX) (e.g. ACR >/=50, and European League against Rheumatism (EULAR) moderate to good response) in patients with active long-standing RA and inadequate response to anti-TNF agents or traditional DMARDs. METHODS: RTX was used in 110 RA patients in six different Italian centres. The mean disease activity score on 28 joints (DAS28) was 6.4 +/- 0.99 and the mean HAQ was 1.63 +/- 0.68 at baseline. Thirty-two patients (29.1%) underwent RTX after the failure of DMARD therapy, 37 (33.6%) had failed or were intolerant to at least two anti-TNF agents, and 41 (37.3%) had failed or were intolerant to one anti-TNF agent. Univariate and multivariate analyses were performed. RESULTS: The number of previous anti-TNF agents (P = 0.043), HAQ (P = 0.023), RF positivity (P < 0.0001) and anti-cyclic citrullinated peptide (anti-CCP) positivity (P = 0.003) were associated with ACR response >or=50 between month +4 and month +6 after starting RTX by univariate analysis. Multivariate analysis confirmed that a lower HAQ, a lower number of anti-TNF agents failed before RTX and RF positivity, but not anti-CCP positivity, were the selected variables associated with an ACR response >or=50, with an accuracy of 84% of the model. Only RF positivity correlated with EULAR moderate to good response both in the univariate and in the multivariate analysis, with an accuracy of 79% of the model. CONCLUSION: RF-positive rather than anti-CCP-positive RA patients with lower baseline disability and a lower number of previously failed TNF blockers may be the best candidates to RTX.

Long-term effects of rituximab in rheumatoid arthritis: clinical, biologic, and pharmacogenetic aspects.

Rituximab selectively targets the B-cell compartment, including rheumatoid factor-positive B cells. Short-term efficacy and safety of rituximab in rheumatoid arthritis (RA) has been established by multicenter randomized placebo-controlled studies. Results of long-term follow-up of the phase II/III clinical trials have confirmed the efficacy and safety of repeated courses of rituximab in the responders. However, mechanisms of action in humans, retreatment regimens, biologic effects on memory B cells and on immunoglobulin levels of prolonged exposure of the immune system to B-cell depletion over time, and pharmacogenetic aspects remain open and intriguing issues of rituximab therapy. Several studies are ongoing to clarify possible clinical and biologic predictors of response to rituximab in RA and in other autoimmune diseases where rituximab has been proven to be effective. Preliminary clinical and pharmacogenetic results of our cohort of RA patients managed with rituximab from the year 2000 are presented.

Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).

The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.

Aberrant expression of TRAIL in B chronic lymphocytic leukemia (B-CLL) cells.

Analysis of peripheral blood (>85% CD19+/CD5+ B) lymphocytes, obtained from 44 patients affected by B chronic lymphoid leukemia (B-CLL), showed that surface TNF-related apoptosis inducing ligand (TRAIL) was expressed in all samples and at higher levels with respect to unfractionated lymphocytes and purified CD19+ B cells, obtained from 15 normal blood donors. Of note, in a subset of B-CLL samples, the addition to B-CLL cultures of a TRAIL-R1-Fc chimera, which binds at high affinity to surface TRAIL, significantly decreased the percentage of viable cells with respect to untreated control B-CLL cells, suggesting that surface TRAIL may play an unexpected role in promoting B-CLL cell survival. In spite of the majority of B-CLL lymphocytes expressed variable surface levels of "death receptors" TRAIL-R1 and TRAIL-R2, the addition in culture of recombinant TRAIL increased (>20% vs. controls) the degree of spontaneous apoptosis in only 11/44 of the B-CLL samples, had no effect in 19/44, while it significantly increased leukemic cell survival in 14/44. Taken together, these findings suggest that an aberrant expression of TRAIL might contribute to the pathogenesis of B-CLL by promoting the survival in a subset of B-CLL cells.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

BACKGROUND: Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. METHODS: PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. RESULTS: At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. CONCLUSIONS: Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.

Clinical characteristics, prognostic factors and multidrug-resistance related protein expression in 36 adult patients with acute promyelocytic leukemia.

UNLABELLED: We report on a single-center experience about the characteristics and outcome of 36 acute promyelocytic leukemia (APL) patients observed at our Department of Hematology between 1990 and 2002. The expression, of multidrug-resistance (MDR) associated proteins (PGP, LRP, MRP1) was also analyzed. There were 12 males and 24 females (median age 37 yr), 89% (32 of 36) with classic morphology, and 11% (four of 36) with a microgranular variant. Risk class (according to GIMEMA/PETHEMA): 25% (nine of 36) high risk (HR), 53% (nineteen of 36) intermediate risk (IR), 22% (eight of 36) low risk (LR). PGP, LRP, and MRP1 expression at onset and at first relapse was low. CD33 antigen expression was high in all cases. The patients were treated according to GIMEMA protocols (LAP0389 and AIDA) including ATRA in induction in 75% (27 of 36) of cases and 94% (34 of 36) achieved a complete remission (CR) after induction therapy while 6% (two of 36) died early (DDI) of hemorrhage. OUTCOME: 71% (24 of 34) of evaluable patients remain in CR at a median follow-up of 57 months (range 4-158 months) while 29% (10 of 34) relapsed at a median time of 12 months (range 8-43 months) and, of them, eight of 10 died early. The majority of patients that relapsed were in high-risk group. The overall survival (OS) of the whole population at 32 months was 66% and the DFS at 42 months was 62%. A statistically significant difference in terms of DFS was observed between HR and IR/LR patients (P = 0.04 by log-rank). DFS was not affected by age, sex, Hb levels, karyotype, and BCR isoform. At conclusion, our data confirm that despite the high rate of success with ATRA plus chemotherapy as induction (more than 90% of CR), about 30% of APL patients have a relapse (without a long-lasting second remission) and underline the importance of patient stratification in distinct risk groups at diagnosis in order to better adapt the type and intensity of treatment (risk-adapted therapy). Taking into account the high expression of CD33 and the low expression of MDR proteins in APL, new and investigational approaches like gemtuzumab-ozogamicin, with or without ATRA and other new drugs, should be strongly considered expecially in HR APL.

B-cell compartment as the selective target for the treatment of immune thrombocytopenias.

BACKGROUND AND OBJECTIVES: Rituximab is a chimeric anti-CD20 monoclonal antibody active against normal and malignant B cells. Treatment with rituximab is associated with the development of a severe (even if transient) B-cell depletion from peripheral blood and lymphatic tissues. These effects could be useful in autoimmune diseases in order to interfere with the production of pathologic antibodies. DESIGN AND METHODS: To investigate this, we treated 20 patients with rituximab 375 mg/m2 i.v. every 7 days for 4 times. These 20 patients all had active and symptomatic autoimmune thrombocytopenia that had relapsed or was refractory to standard therapies (15 had idiopathic thrombocytopenic purpura, 1 idiopathic thrombocytopenia and neutropenia, 2 thrombocytopenia and concomitant undifferentiated connective tissue disease, and 2 had thrombocytopenia and concomitant B-cell lymphoprolipherative disorders). Only treatment with steroids, if strictly necessary to maintain a safe number of platelets, was allowed during the period of rituximab administration, but only patients who reached steroid discontinuation (previously not possible) were considered responders. RESULTS: Treatment was well tolerated and no acute or delayed toxic events were recorded. Rituximab proved to be active in 13/20 patients, with 9 complete and 4 partial responses. In 10/13 (77%) the response (platelet level > 50x10(9)/L) was prompt, being achieved already after the first of the four planned infusions. After a median follow-up of 180 days (range: 60-480) 4 patients had relapsed. Age < or = 60 years was correlated with a better response rate (p=0.03). No correlation was observed between response and gender, time from diagnosis to treatment (< 12 vs > 12 months), total and CD20+ lymphocyte count, level of CD20 expression on B cells before the therapy and pharmacokinetics of the drug. INTERPRETATION AND CONCLUSIONS: Rituximab appears to be a promising immunotherapeutic agent for the treatment of autoimmune thrombocytopenias.

CD56 and PGP expression in acute myeloid leukemia: impact on clinical outcome.

BACKGROUND AND OBJECTIVES: Overexpression of P-glycoprotein (PGP), a multidrug-related (MDR) protein, is one of the most important factors responsible for reduced drug sensitivity in acute myeloid leukemia (AML). Recently, we demonstrated that the presence of CD56 antigen, an isoform of the neural adhesion molecule, in AML cells is a negative independent prognostic factor for the achievement of complete remission (CR) and correlates with shorter survival. Since in our previous report we observed a more frequent PGP expression in CD56+ patients, we hypothesized that the reduced response to chemotherapy in this group of patients was due to increased PGP-mediated drug efflux. To confirm this hypothesis in this study PGP and CD56 expression on AML cells was correlated with other clinical and biological features and treatment response. DESIGN AND METHODS: Immunophenotypic analysis, including evaluation of CD56 and PGP expression, was performed using multiparameter flow cytometry on fresh and/or cryopreserved blast cells, obtained after informed consent, from bone marrow and/or peripheral blood of 143 consecutive newly diagnosed AML cases at the time of diagnosis. Samples expressing CD56 in at least 15% or more cells were considered as positive (CD56+). PGP expression was expressed as a mean fluorescence index (MFI) i.e. as the ratio of sample mean fluorescence channel and the isotypic control mean fluorescence channel. RESULTS: Overall results showed that 67/143 cases were PGP-/CD56-, 23/143 were PGP+ /CD56+, 40/143 were PGP+/CD56- and the remaining 13/143 were PGP-/CD56+. CD56+ and PGP+ on AML cells significantly reduced the CR rate (83% in the PGP-/CD56- group vs 60% in the PGP-/CD56+ group, 46% in the PGP+/CD56- group and 58% in the PGP+/CD56+ group, p = 0.002). In addition we observed a significantly higher proportion of total failures in patients expressing PGP or CD56 compared to in the group not expressing either (73% vs 27%, respectively; p = 0.0001). CD56 and PGP overexpression influenced the overall survival: in fact, the median survival of CD56+ and PGP+ patients ranged from 10 to 23 months, while the actuarial survival of CD56-/PGP- patients at 5 years is 52% (p = 0.023). INTERPRETATION AND CONCLUSIONS: Our data underline the independent negative prognostic role of PGP and CD56 expression in acute myeloid leukemia. Since the mechanism by which CD56 reduces drug sensitivity is still unknown, further investigations are required.

P-glycoprotein, lung resistance-related protein and multidrug resistance-associated protein in de novo adult acute lymphoblastic leukaemia.

P-glycoprotein (P-gp), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression, and blast cell intracellular daunorubicin accumulation (IDA) were evaluated in 95 previously untreated cases of adult acute lymphoblastic leukaemia (ALL) using flow cytometry. Forty-five out of 95 (47%) patients were P-gp positive (+), 12/66 (18%) were LRP+ and 11/66 (17%) were MRP+. Eighteen out of 66 (28%) patients showed a simultaneous multidrug resistance (MDR)-related protein expression higher than controls for more than one protein, while 24/66 (36%) cases did not overexpress any protein. Twenty-one out of 24 (87%) cases overexpressing at least one MDR-related protein had a defect in accumulating daunorubicin into their blast cells, while only 4/24 (16%) cases who did not overexpress any protein had similar features. The complete remission rates were similar in MDR-positive and -negative (-) patients but relapses within 6 months were more frequent in P-gp+ cases, and therefore the disease-free survival duration was shorter in P-gp+ than in P-gp- patients (P = 0.01). The number of MRP+ and/or LRP+ cases was too small to be able to draw any conclusion on their role in affecting or predicting therapy outcome. In conclusion, P-gp overexpression associated with a defect in daunorubicin accumulation is a frequent feature in adult ALL at onset and seems to be related to poorer therapy outcome and, consequently, a shorter disease-free survival. LRP and MRP overexpression seems to be a rare event and no conclusion can be drawn on its prognostic role.

B-cell depletion with rituximab as treatment for immune hemolytic anemia and chronic thrombocytopenia.

BACKGROUND AND OBJECTIVES: Rituximab reacts specifically with the CD20 antigen and induces B-cell depletion. This could interfere with the production of autoantibodies in some immune diseases. The objective of this study was to assess the effects of rituximab in autoimmune hemolytic anemia and thrombocytopenia. DESIGN AND METHODS: Seven patients (one with cold agglutinin disease, two with warm antibody autoimmune hemolytic anemia, four with chronic idiopathic thrombocytopenic purpura) previously refractory to conventional treatments were treated with weekly infusions of rituximab, 375 mg/m2, for 4 weeks. Only treatment with steroids, if strictly necessary, was allowed during the period of rituximab administration, but only patients who reached steroid suspension were considered responders. The pharmacokinetics of rituximab were quantified during therapy and the follow-up period. RESULTS: All patients had marked, even if temporary, B-cell depletion. Three patients, 1 with cold agglutinin disease (CAD) and 2 with chronic idiopathic thrombocytopenic purpura (ITP), had a complete hematologic response. In the patient with cold agglutinin disease a decrease in the agglutinin titer was observed. The hematologic improvement was prompt, appearing by the second or third infusion of rituximab. The response duration was CAD 96+, ITP 17+ and 13+ weeks in these 3 patients. Treatment tolerance was satisfactory and no infections or other late events were registered. Serum rituximab concentrations appeared to be similar to those calculated in a historical control group of patients with follicular non-Hodgkin's lymphoma who received rituximab as consolidation of response after first-line CHOP chemotherapy. INTERPRETATION AND CONCLUSIONS: Rituximab appeared to be active and safe in some patients with refractory autoimmune hemolytic anemia and thrombocytopenia. These results, along with data from literature, suggest that this agent may have a therapeutic role in autoimmune diseases.