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Background Primary Sjogren9s syndrome (pSS) is an autoimmune lymphoproliferative systemic disease with a higher risk of non-Hodgkin B cell lymphoma (NHL) evolution. In SS, salivary gland (SG) epithelium plays a crucial role in initiating and perpetuating the autoimmune response. Thymic stromal lymphopoietin (TSLP) is both an epithelial and lymphopoietic cytokine involved in the maintenance of immune tolerance at interfaces between body and environment and in the regulation of lymphocytes homeostasis. Objectives To study TSLP in serum and SG biopsies of pSS patients stratified by the lymphoproliferative histopathologic status, from fully benign lesions (fbSS) to myoepithelial sialoadenitis (MESA) and to NHL, in order to evaluate a possible role of TSLP in SS pathogenesis and in lymphoma evolution. Methods Serum TSLP levels were determined by ELISA in pSS patients (n=30: 12 fbSS, 10 MESA, 8 NHL) and in controls (healthy blood donors - HD n=20; non-autoimmune sicca without SS - nSS n=10). TSLP was also studied by immunohistochemistry in SG biopsies of the same patients and nSS controls. Correlations with clinical and histopathologic parameters were performed. Of note, sequential samples were also included from three patients evolving from fbSS to NHL. Results TSLP serum levels were significantly higher in pSS compared to nSS (p=0.03) and HD (p=0.0002), with a progressive significant increase from fbSS to MESA (p=0.004) and finally to NHL (NHL vs fbSS p Of note, in the affected tissue, TSLP showed an opposite pattern of expression than in the serum. Concerning the salivary epithelium, a declining expression of TSLP was shown from fbSS (expression was similar to nSS) to MESA and finally to NHL. Strikingly, however, the three pSS patients evolving to lymphoma were the only ones showing a low TSLP epithelial expression also at baseline. Concerning the glandular lymphoid infiltrate, the TSLP expression again decreased with progression to NHL. Again, the three pSS patients evolving to lymphoma were the only ones showing a low TSLP inflammatory expression also at baseline. Conclusions Serum TSLP increases in pSS and correlates with lymphoma progression. Discrepancies are however observed between serum levels and tissue TSLP expression: while the baseline TSLP tissue expression is evident and similar to controls, tissue TSLP decreases with lymphoma progression. TSLP likely represents an additional growth factor and biomarker for pSS pathogenesis and lymphoproliferation. Disclosure of Interest None declared

Objective To conduct a long-term, prospective, randomized controlled trial evaluating rituximab (RTX) therapy for severe mixed cryoglobulinemia or cryoglobulinemic vasculitis (CV). Methods Fifty-nine patients with CV and related skin ulcers, active glomerulonephritis, or refractory peripheral neuropathy were enrolled. In CV patients who also had hepatitis C virus (HCV) infection, treatment of the HCV infection with antiviral agents had previously failed or was not indicated. Patients were randomized to the non-RTX group (to receive conventional treatment, consisting of 1 of the following 3: glucocorticoids; azathioprine or cyclophosphamide; or plasmapheresis) or the RTX group (to receive 2 infusions of 1 gm each, with a lowering of the glucocorticoid dosage when possible, and with a second course of RTX at relapse). Patients in the non-RTX group who did not respond to treatment could be switched to the RTX group. Study duration was 24 months. Results Survival of treatment at 12 months (i.e., the proportion of patients who continued taking their initial therapy), the primary end point, was statistically higher in the RTX group (64.3% versus 3.5% [P < 0.0001]), as well as at 3 months (92.9% versus 13.8% [P < 0.0001]), 6 months (71.4% versus 3.5% [P < 0.0001]), and 24 months (60.7% versus 3.5% [P < 0.0001]). The Birmingham Vasculitis Activity Score decreased only after treatment with RTX (from a mean ± SD of 11.9 ± 5.4 at baseline to 7.1 ± 5.7 at month 2; P < 0.001) up to month 24 (4.4 ± 4.6; P < 0.0001). RTX appeared to be superior therapy for all 3 target organ manifestations, and it was as effective as conventional therapy. The median duration of response to RTX was 18 months. Overall, RTX treatment was well tolerated. Conclusion RTX monotherapy represents a very good option for severe CV and can be maintained over the long term in most patients.

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.

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.

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. © 2004 Wiley-Liss, Inc.

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.

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.

The possibility that Daunoxome (DNX), a combination of daunorubicin (DNR) with a liposomal targeting system, escapes PGP was tested. Two pairs of leukaemic cell lines, each consisting of the parental non-multidrug resistance (MDR) line and of a MDR variant, were studied for cytotoxicity (MTT test) and for cellular DNR kinetic and accumulation (flow cytometry). DNX and free DNR were equally toxic against non-MDR cells, whereas the liposomal anthracycline was more toxic than the free drug against the MDR variant. Non-MDR cells accumulated DNR more rapidly when they were exposed to free DNR than to DNX, but MDR cells accumulated more DNR when they were exposed to DNX. The kinetics of DNX and free DNR were also studied in the blast cells of 41 cases of acute leukaemia and they were found to be related to blast cell PGP expression. In 15 cases with a low PGP expression intracellular DNR accumulation was faster and higher with free DNR than with DNX. In 26 cases with a high PGP expression the area under the curve was similar with DNX and free DNR, but the kinetics of intracellular DNR accumulation showed an early low plateau with free DNR and a slow and continuous increase with DNX. In MDR cell lines the ratio was more favourable to DNX than to free DNR. We conclude that liposome encapsulated DNR is partially protected from PGP and that it is worth testing for the treatment of PGP-positive acute leukaemia.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non-lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP+, 44/96 (46%) as LRP+, and 8/96 (8%) as MRP+. The more frequent MDR clusters were PGP−/LRP−/MRP− (32/96 cases, 33%) and the PGP+/LRP+/MRP− (27/96 cases, 28%) followed by PGP+/LRP−/MRP− (15/96 cases, 16%) and PGP−/LRP+/MRP− (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP− and LRP− cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA. All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

P-glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by now cytometry in 65 leukaemic patients (38 acute non-lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph-positive chronic myeloid leukaemias in blastic phase). By using the MRK-16, the LRP-56 and the MRPm6 MoAbs, 34% of the cases did not over-express any proteins (-); 24.5% over-expressed (+) only PGP. 11% only LRP, 1.5% only MRP, 24.5%, both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP - /LRP - /MRP - cases were comparable to the ones observed in normal leucocytes. With respect to the non-over-expressing cases, the PGP - /LRP + /MRP - cases showed only an impaired IDA (mean 204 ± 29; P < 0.001). The PGP + /LRP + /MRP - cases had a defect both in IDA (mean 166±47, P

The biologic and clinical importance of the multidrug resistance (MDR) that is related with the overexpression of the P170 glycoprotein (Pgp) is widely recognized. However, a major issue that has not yet been solved is the definition of the degree of Pgp expression which is associated with a significant decrease of the sensitivity of the cells to chemotherapy. For this reason we studied the leukemic cells from 83 cases of acute leukemia. Leukemic cells were fixed in PLP and treated with saponine. Pgp expression was assayed by flow cytometry, using the anti Pgp monoclonal antibody MRK-16. Results were expressed both as the number of positive cells and by the intensity of the reaction as defined by the mean fluorescence index (MFI), i.e. the ratio between the mean fluorescence intensity of the MRK-16 incubated cells and of the IgG2a incubated cells. Thus, Pgp expression was compared with the results of two in vitro tests of cell sensitivity to anthracyclines, daunorubicin (DNR) cell retention and DNR cytotoxicity. We found that it was not the number of MRK-16 positive cells, but the degree of the reaction with MRK-16 (MFI) that significantly related to the anthracycline toxicity tests. Therefore, we propose that for clinical purposes a quick and cheap determination of Pgp-related MDR in leukemic cells may be obtained by measuring the MFI with MRK-16 in a standard flow cytometry assay and that the assay may indeed be sufficient to estimate Pgp expression as well as the influence of Pgp on cell sensitivity to anthracyclines.

Background Belimumab, a human anti-BAFF monoclonal antibody that inhibits soluble BAFF, is effective in systemic lupus erythematosus (SLE), and leads to a partial B cell depletion. However, which subset of B cells may be the main therapeutic target of belimumab in SLE is still unclear. Objectives Primary objective of this study was to evaluate the effect of belimumab on the expression of BAFF receptor (BAFFR) on B cells through 12 months of belimumab treatment in SLE, and to correlate BAFFR expression with changes in B cell subsets. Methods Twelve consecutive patients with SLE (all female, age 44±7,8 yrs, 12,6±5,4 yrs of disease duration) undergoing belimumab therapy were studied. All patients received at least two immunosuppressors before belimumab. All patients were taking glucocorticoids at baseline [median (range): 7,5 (0–40) mg/day of prednisone or equivalent)], and 11/12 also at least one immunosuppressor. Median SLEDAI-2k score at baseline was 8,5 (range 8–15). Samples were collected and analysed at baseline (T0), month +3 (T3), +6 (T6), +9 (T9) and +12 (T12) for clinical and biological parameters. B cells subsets were characterized by multiparametric flowcytometry on a BD FACSCanto flowcytometer. The expression of BAFFR was also analysed as Mean Fluorescence Intensity (MFI). Parametric or non-parametric tests were used, as appropriate based on variable distribution. Data is reported as mean±standard error. Results A transient statistically significant increase of BAFFR MFI was observed from T0 to T3 (p=0,004) and from T0 to T6 (p=0,066) with a subsequent decrease until T12 (table 1). Interestingly, this course was similar to that observed in CD19+CD27+ cell subset (table 1), that showed a transient significant increase at T3 (p=0,014) and T6 (p=0,025) in 9/12 patients, with a subsequent slightly significant decrease from T6 to T12 (p=0,05) in all patients. Globally, a decrease of CD19+ cells was observed from T0 to T12 (p=0,19, table 1). This course is consistent with that observed in the CD27- B cell subset, that showed a decrease from T0 to T3 (p=0,14) and from T0 to T6 (p=0,14), and the decrease was persistent from T6 to T12 (p=0,6). SLEDAI-2k score decreased from T0 to T3 (p=0,007), to T6 (p=0,003) and to T12 (p=0,03) (table 1). Conclusions BAFF depletion differently acts on B cell subsets, CD19+27+ subset being able to increase BAFFR and transiently expand themselves, while CD19+27- being more sensitive to BAFF deprivation, and faster decreasing. Thus, early clinical response may be related to the changes in the CD27+/CD27- B cell ratio. Disclosure of Interest None declared

Objectives. We explored clinical factors associated with a major response to rituximab (RTX) (e.g. ACR 550, 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 ¼ 0.043), HAQ (P ¼ 0.023), RF positivity (P < 0.0001) and anti-cyclic citrullinated peptide (anti-CCP) positivity (P ¼ 0.003) were associated with ACR response 550 between month þ4 and month þ6 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 550, 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.

Background Targeting B cell activating factor (BAFF) by belimumab is effective in systemic lupus erythematosus (SLE) (1), and leads to a partial B cell depletion (2,3). However, which subset of B cells may be the main therapeutic target of belimumab in SLE, either why the response under belimumab is usually delayed in time is still unclear. Objectives Our purpose was to study the effect of belimumab on different B cell subsets in SLE and in particular the expression of BAFF receptor (BAFFR) on B cells through 12 months of belimumab treatment. Methods Seven patients with SLE (all female, age 46,7±7,8 years, mean disease duration of 13,8±4,2 years) undergoing belimumab therapy from May 2013 were studied. All patients received at least two immunosuppressors before belimumab; all patients were taking glucocorticoids at baseline (10±12 mg/day), and 6/7 were also taking at least one immunosuppressor. Mean SELENA-SLEDAI score at baseline was 9,7±2,7. Samples were collected and analyzed at baseline (T0), month +3 (T3), month +6 (T6), month +9 (T9) and month +12 (T12, 5 pts) for clinical and biological parameters. B cells subsets were characterized by multiparametric flowcytometry on a BD FACSCanto flowcytometer. The expression of BAFFR was also analyzed as Mean Fluorescence Intensity (MFI). Data were reported as mean ± standard error. Results In the group of 7 SLE patients studied, a decrease of the SELENA-SLEDAI score from 9,7±2,7 at T0, to 6,1±1,9 at T3, to 5,0±1,0 at T6, to 4,4±0,8 at T9 and finally to 4,1±0,9 at T12 was documented. At T0 peripheral blood counts of CD19+ cells were low (median 46 cell/μl, range 1,1-267), possibly because of the past and concomitant immunosuppressive treatments and the chronic glucocorticoids administration. CD19+CD27- cells decreased after belimumab treatment from 60±56 cell/μl at T0 to 24±14 cell/μl at T3, and then remained low over time (21±11 cell/μl at T6, 25±19 cell/μl at T9 and 23±14 cell/μl at T12). By contrast, CD19+CD27+ cells showed a transient increase as absolute counts at T6 (from 17±13 cell/μl at T0 to 23±17 cell/μl at T6), while they markedly decreased from T6 to T12 (9±7 cell/μl). BAFFR expression remained unchanged over time during therapy as percentage of B cells (mean 97±1,7% at T0 vs. 97±2% at T9); however, a transient increase in BAFFR MFI was observed from T0 to T3 (11,6±1,9 to17±4,7), and then decreased to T12 (10,9±1,5). The count of CD27+ B cells and the mean MFI BAFFR expression showed a similar curve over time, differently from CD19+CD27- B cells (figure 1). Conclusions Hyperexpression of BAFF receptor on CD27+B cells occurs shortly after belimumab consistently with the increase in the CD27+ B cell count in the first three months of treatment. Subsequently, the BAFFR expression decrease is followed by the lowering of CD27+ B cells from T6 to T12. The intensity of BAFFR expression on CD27+ B cells may explain the pharmacodynamic of belimumab in SLE. Since belimumab acts slowly in SLE, the results observed support the hypothesis of a therapeutic effect of belimumab also on CD27+ B cells. References Furie R et al. Arthritis Rheum 2011. Stohl W et al. Arthritis Rheum 2012. Jacobi AM et al. Arthritis Rheum 2010. Disclosure of Interest None declared

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. © 2005 Wiley-Liss, Inc.

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.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.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 level50x10(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. Ageor = 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 vs12 months), total and CD20+ lymphocyte count, level of CD20 expression on B cells before the therapy and pharmacokinetics of the drug.Rituximab appears to be a promising immunotherapeutic agent for the treatment of autoimmune thrombocytopenias.

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.

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.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.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.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.

In this work three human cell lines with multidrug resistance (MDR) caused by a P-glycoprotein (PGP) overexpression, CEM VLB, HL60 DNR, LOVO DX and two cell lines with MDR associated with a multidrug related protein (MRP) or a lung resistance-related protein (LRP) overexpression named GLC4 ADR and SW1573/2R120 were tested for Amifostine protection against Daunorubicin, Doxorubicin, Idarubicin and Mitoxantrone toxicity. This class of anticancer agents was chosen because they are commonly used in the first line treatments of acute leukemias where a PGP, an LRP or an MRP overexpression often occurs even at onset. A 7-day incubation with escalating doses of anticancer agents with or without a 15 minute preincubation in Amifostine or its active metabolite WR-1065 were used. In conclusion, in none of the MDR positive and negative cell lines did Amifostine modify the toxicity of the anticancer drugs. The observation that even the WR-1065 metabolite gave no protection against Anthracyclines toxicity strengthened the data and provided confirmation for the further in vivo testing of the safety and efficacy of Amifostine in leukemias.

We analysed the expression of three drug transporter proteins [p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.