Search

A total of 143 adult acute myeloid leukemia (AML) patients with available karyotype (K) and FLT3 gene mutational status were assessed for minimal residual disease (MRD) by flow cytometry. Twenty-two (16%) patients had favorable, 115 (80%) intermediate, and 6 (4%) poor risk K; 19 of 129 (15%) carried FLT3-ITD mutation. Considering postconsolidation MRD status, patients with good/intermediate-risk K who were MRD− had 4-year relapse-free survival (RFS) of 70% and 63%, and overall survival (OS) of 84% and 67%, respectively. Patients with good- and intermediate-risk K who were MRD+ had 4-year RFS of 15% and 17%, and OS of 38% and 23%, respectively (P < .001 for all comparisons). FLT3 wild-type patients achieving an MRD− status, had a better outcome than those who remained MRD+ (4-year RFS, 54% vs 17% P < .001; OS, 60% vs 23%, P = .002). Such an approach redefined cytogenetic/genetic categories in 2 groups: (1) low-risk, including good/intermediate K-MRD− with 4-year RFS and OS of 58% and 73%, respectively; and (2) high risk, including poor-risk K, FLT3-ITD mutated cases, good/intermediate K-MRD+ categories, with RFS and OS of 22% and 17%, respectively (P < .001 for all comparisons). In AML, the integrated evaluation of baseline prognosticators and MRD improves risk-assessment and optimizes postremission therapy.

Previous observational studies suggest that rituximab may be useful in the treatment of primary immune thrombocytopenia (ITP). This randomized trial investigated rituximab efficacy in previously untreated adult ITP patients with a platelet count of 20 × 109/L or less. One hundred three patients were randomly assigned to receive 40 mg/d dexamethasone for 4 days with or without 375 mg/m2 rituximab weekly for 4 weeks. Patients who were refractory to dexamethasone alone received salvage therapy with dexamethasone plus rituximab. Sustained response (ie, platelet count ≥ 50 × 109/L at month 6 after treatment initiation), evaluable in 101 patients, was greater in patients treated with dexamethasone plus rituximab (n = 49) than in those treated with dexamethasone alone (n = 52; 63% vs 36%, P = .004, 95% confidence interval [95% CI], 0.079-0.455). Patients in the experimental arm showed increased incidences of grade 3 to 4 adverse events (10% vs 2%, P = .082, 95% CI, −0.010 to 0.175), but incidences of serious adverse events were similar in both arms (6% vs 2%, P = .284, 95% CI, −0.035 to 0.119). Dexamethasone plus rituximab was an effective salvage therapy in 56% of patients refractory to dexamethasone. The combination of dexamethasone and rituximab improved platelet counts compared with dexamethasone alone. Thus, combination therapy may represent an effective treatment option before splenectomy. This study is registered at http://clinicaltrials.gov as NCT00770562.

An aberrant pattern of expression of L-selectin and intercellular adhesion molecule 1 (ICAM1) may characterise CD34+ blast cells in myelodysplastic syndromes (MDS) and secondary acute myeloid leukaemia (sAML).In a three-colour flow cytometric assay, we evaluated the expression of L-selectin and ICAM1 on CD34+ blast cells from the bone marrow (BM) of 66 MDS patients; for the purpose of comparison CD34+ blast cells of 18 sAML and CD34+ stem cells of 17 normal donors were also analysed.The ratio of L-selectin/ICAM1 expression was identified as a parameter correlated with the percentage of BM blast infiltration and the time to leukaemic progression among MDS patients. In fact, the values of L-selectin/ICAM1 ratio were inversely correlated with the BM blast infiltration (r = -0.34, P = 0.004). Furthermore, MDS patients with a baseline ratio1 had a higher leukaemic progression rate (41% vs. 19%, P = 0.008); the actuarial risk of disease progression for this subgroup of MDS patients was also higher (64% vs. 11% at 2 yr, P = 0.002). Furthermore, in two patients a decrease of the ratio was observed when overt leukaemic transformation occurred; conversely, restoration of a normal ratio was observed in two patients after a chemotherapy-induced remission.(i) L-selectin is defective in the stem cell compartment of MDS and sAML, whereas ICAM1 is overexpressed; (ii) the ratio of their expression has a prognostic role; and (iii) a ratio1 significantly predicts progression to overt leukaemia in MDS patients.

Previous uncontrolled studies have highlighted the potential activity of Rituximab in patients with idiopathic thrombocytopenic purpura (ITP) relapsed or refractory to standard treatments. To better address this effect, a prospective randomized, multicenter, phase III study comparing treatment with Dexamethasone alone (arm A) vs Dexamethasone plus Rituximab (arm B) was started in July 2005 for adult patients with ITP according to the ASH guidelines. Main inclusion criteria were: age ≥ 18 years, untreated ITP, platelet (PLT) count ≤ 20 x109/L, HIV- HCV-HbsAg negativity, informed consent. Patients randomized to arm A received a single course of Dexamethasone 40 mg po on days +1, +2, +3, +4, while patients randomized to arm B received Dexamethasone (as in arm A) in association with Rituximab 375 mg/m2 iv on days +7, +14, +21, +28. Patients in arm A who failed to achieve a sustained response (SR) could be rescued with arm B treatment. The primary objective of the study was to compare SR, i.e. PLT ≥ 50 x 109/L at month + 6 of treatment. The secondary objectives were: the initial overall (OR= PLT ≥ 50 x109/L) and complete response (CR= PLT ≥ 100 x 109/L) by day 30 after starting treatment, respectively; the toxic profile. The statistical plan considered three interim analyses, after the first 50, 100 and 150 enrolled patients, with an estimated sample size of 198 patients (99 per arm). Table 1 summarizes the main demographic data and the results of efficacy and toxicity according to an intention to treat analysis of the first interim analysis. The toxic profile was characterized by only grade 3 adverse events (AE); no patient died during the study period. 16 patients of arm A were rescued with arm B. For this group SR was 81% and no patient experienced SAE or ≥ grade 3 AE. In accordance with the initial statistical plan of the study, which stated that patients’ recruitment would ceased if a ≥ 50% difference in sustained response was demonstrated, enrolment has been stopped in June 2007 with a total number of 103 randomized patients. This preliminary report indicates a significantly higher SR for arm B of treatment with no difference in toxicity profile. A final report will be prepared when the results on the entire study group will be available. Table 1 Therapy Arm A Arm B Statistics Patients 24 26 Male/female 11/13 10/16 p = NS Age (median ± SD) 54.54 ± 18.78 48.65 ± 15.10 p = NS Initial OR 15 (62.5%) 18 (69%) p = NS Initial CR 10 (42%) 16 (61%) p = NS SR 7 (29%) 21 (81%) p = 0.0001 SAE or grade 3 AE 3 (12.5%) 2 (8%) p = NS

Objective: The aim of this study was to investigate susceptibility to spontaneous or anti-fas-induced apoptosis in peripheral blood mononuclear cells (PBMC) from HIV-positive patients before and during highly active anti-retroviral therapy (HAART). Design: A longitudinal study was performed on 12 evaluable patients on HAART. This cohort was analysed prior to and at week 2, 4, 8, 16 and 24 after beginning HAART. Variations in CD4 and CD8 cells, viral load, susceptibility to spontaneous or anti-Fas-induced apoptosis in the presence of IL-2, IL-4 or IL-12 were studied. Expression of Fas and Bcl-2 were also assessed. Methods: Levels of HIV RNA were determined by a quantitative reverse transcription-PCR assay. Apoptosis was evaluated by staining isolated nuclei with propidium iodide followed by multiparameter flow cytometry analysis. Results: Spontaneous apoptosis of PBMC was promptly inhibited after the start of treatment. Similarly, anti-Fas-induced apoptosis diminished greatly during treatment. Expression of Fas decreased significantly, while that of Bcl-2 remained statistically unchanged during the first 24 weeks of therapy. Levels of apoptosis correlated inversely to CD4 cell counts and directly to viral load in a highly significant way. Expression of Fas was directly correlated to apoptosis. Interleukin (IL)-2, but not IL-4 or IL-12, protected PBMC of HIV-positive individuals from spontaneous or anti-Fas- induced apoptosis before and during HAART. Conclusion: These results suggest that regulation of apoptosis and of Fas expression are involved in immunoreconstitution during HAART. (C) 2000 Lippincott Williams and Wilkins.

B-cell chronic lymphocytic leukemia (B-CLL) is an indolent lymphoproliferative disorder with a progressive accumulation of small, morphologically mature B lymphocytes in the bone marrow, blood and lymphoid tissues. Alemtuzumab (MabCampath) is a IgG1 monoclonal antibody (mAb) that target the CD52 antigen, a glycosylated peptide highly expressed on B-CLL cells. This mAb is active in patients with B-CLL refractory to alkylating agents and purine nucleoside analogues. The primary adverse event so far reported is the induction of a profound and prolonged depletion of CD4 and CD8 subpopulations leading to an immunodeficient status and the development of opportunistic infections [1,2]. Recently, Epstein-Barr virus (EBV) related lymphoma have been reported in patients with T and B cell lymphoproliferative disorder after treatment with alemtuzumab [2–5]. Here, we report a case of an EBV related lymphoproliferative disorder secondary to alemtuzumab therapy for B-CLL. A 53 years old man diagnosed in January 1999 as having stage Rai II classical B-CLL, was admitted to our department in June 2007 as a consequence of disease progression. Analysis of IgVH mutational status was not performed. However, peripheral blood flow cytometry analysis revealed that tumor cells expressed ZAP-70, but were negative for CD38 expression. He was previously treated with two cycles of fludarabine 25 mg/m with no response. In March 2001, he received six doses of rituximab 375 mg/m obtaining a partial remission. In December 2005, because of disease progression associated with Coombs positive autoimmune hemolytic anemia (AHA) he was treated with rituximabþfludarabineþ cyclophosphamide (FCR) for six cycles obtaining a partial remission with resolution of the (AHA). From June 2007 to July 2007, he received 12 doses of alemtuzumab 30 mg/m, obtaining the clearance of bone marrow neoplastic cells with persistence of lymph nodes involvement. CD4 and CD8 levels prior alemtuzumab therapy were 517/mL and 1380/mL, respectively. After 4 weeks of anti CD52 therapy, treatment was interrupted because of CMV reactivation which was successfully treated with ganciclovir. In October 2007, the patient presented with fever AHA recrudescence and hepatomegaly. Laboratory analysis showed high LDH level (1299 UI/L), hypoalbuminemia (2.5 g/dL), elevated liver enzyme (ALT: 92 UI/L; AST: 87 UI/L) and anemia (Hb 8.4 g/dL). Lymphocyte count on peripheral blood was less than 40/mL. A CT scan revealed the presence of multiple enlarged lymph nodes at both sides of the diaphragm and liver enlargement. An abdominal ecography revealed multiple hypoechogenic liver lesions which were biopsied and showed large B cells CD20þ. In situ hybridisation for EBV-RNA was strongly positive and serum EBV DNA viral load showed more than 6500 copies/mL. Bone marrow evaluation didn’t show any infiltration by neoplastic cells. The diagnosis of stage IV CD20þ

Abstract 4360 In spite of significant therapeutic improvements over the last 15–20 years approximately 20% of adult patients with acute myeloid leukemia (AML) is refractory and up to 60–70% relapses following front-line chemotherapy. Refractory/relapsed AML has a very unfavorable prognosis and novel agents are needed to improve outcome for these patients. Bortezomib is a potent inhibitor of the 26S proteasome which has been found to inhibit AML blast survival and interfere with the interaction between AML progenitors and microenviromental niche. The present study was designed to explore the safety and efficacy of Bortezomib given as single agent at the unconventional dose of 1.5 mg/m2, to patients with AML, unfit for conventional chemotherapy or with relapsed/refractory disease, after previous lines of therapy. Thirteen patients have been included in the study, being 10 evaluable. Nine males and 4 females, median age 70 (range 60–78), 4 and 1 cases were de novo and secondary AML, respectively. Six and 2 patients had refractory and relapsed disease, respectively. Bortezomib 1.5 mg/m2 was given on day 1,4,8 and 11 of each 21 day cycle, for a maximum of 8 cycles. Bone marrow samples were collected at baseline and, for response evaluation, on day 1 of each cycle. Three of 13 patients died early due to pulmonary infection. Among the remainder, we observed 5 haematological improvement (HI) defined as a reduction of peripheral blast count < 25% as compared to baseline and a Hb level increase by 2 gr/dL. One patient had a stable disease lasting for 3 months, the remaining 4 patients had a rapid progressive disease. Median number of delivered cycles until progression was 3 for patients with HI, whereas patients with progression were able to receive no more than 1 course of bortezomib. Median overall survival of patients with HI/stable disease was 4 months (range 3–8) versus 2 (range 1–2) of those with progression. Of 10 evaluable patients, 6 developed peripheral neuropathy which was NCI-CTC grade 4 in 2 (neurological bladder, requiring permanent catheterization in 1 instance) and grade 1–2 in 4. In the last, neuropathy resolved promptly after bortezomib discontinuation. In conclusion, bortezomib does have anti-leukemic activity and future clinical trials should focus on its combination with conventional chemotherapy. In this context, a dose reduction would be desirable due to neurologic complications observed in our study. Disclosures: Off Label Use: Bortezomib is a potent inhibitor of the 26S proteasome which has been found to inhibit AML blast survival and interfere with the interaction between AML progenitors and microenviromental niche.The purpose was to explore the safety and efficacy of Bortezomib given as single agent in refractory/relapsed AML patients.

Abstract 2415 Poster Board II-392 We previously reported that in adults with primary immune thrombocytopenia (ITP) the addition of four weekly administrations of rituximab 375 mg/m2 to a single course of dexamethasone (40 mg daily for four days) improves the rate of sustained response (SR, i.e. platelet count 3 50 × 109/L at month 6 from the beginning of treatment) if compared with dexamethasone alone (63% vs 36%, P= 0.004, 95% C.I.: [0.079-0.455]) (ML18542 study, Zaja et al. Blood 2008; 112:Abstract 1). Moreover, the rate of SR in 27 patients receiving dexamethasone plus rituximab salvage therapy because of primary refractoriness or relapse after dexamethasone monotherapy was 56%. In order to better address the impact of therapy beyond month 6, a subsequent observational follow-up study up which lasted from month 6 up to month 36 was planned. Participating centers were asked to continue to monitor the patients enrolled in the ML18542 study at least every 4 months up to the end of year 3 from accrual, documenting the occurrence of delayed side effects, the response status and the need for further treatment. We considered as efficacy parameters the duration of response (RD), i.e. the interval between achievement of SR and relapse, the relapse rate (number of patients who lost their SR status) and the need of further specific anti ITP therapy after month 6 (TFS). Eighty out of the original 101 patients enrolled in the ML18542 study, 15 treated with the dexamethasone monotherapy arm, 41 with the dexamethasone plus rituximab arm and 24 who received dexamethasone plus rituximab salvage therapy, were systematically followed-up beyond month 6 for a median overall period of observation of 20 months (range: 4-40 months). Twenty-one patients could not be valuable, most of them because lost at follow up during the study period. As far as the safety profile, we documented one single case of Herpes Zoster reactivation at month 22 in a patient initially allocated to the dexamethasone arm who further received dexamethasone plus rituximab salvage therapy. No other infectious, delayed toxic events or deaths were registered. Fifty-three out 80 patients who achieved a SR according to the study protocol could be evaluated for long term efficacy. This group included 13 patients from the dexamethasone monotherapy arm who were observed after month 6 for a median period of 20 months (range: 12-28 months), 26 of dexamethasone plus rituximab arm observed for a median period of 24 months (range: 8-40 months), and 14 from the dexamethasone plus rituximab salvage therapy group observed for a median period of 20 months (range: 12-34). The rate of relapse in the three groups was 23% (3/13), 23% (6/26), and 14% (2/14) respectively (Fisher test, P= 0.824). The 30 months estimated probability of RD within each of the three treatment groups was 77%, 71%, and 85% (Log-rank test, P= 0.755), respectively. The time of SR loss was comprised between month 4 and 30 in the dexamethasone monotherapy arm, month 4 and 40 in the experimental arm, and month 8 and 34 in the salvage therapy arm. In these three groups, the use of further specific anti ITP therapy after month 6 was registered in 33%, 29% and 36% of patients (Fisher test, P= 1), respectively. Overall, in this period of observation 4 out 80 (5%) valuable patients underwent splenectomy. In conclusion the results of this study indicate: i) similar and very low pattern of long term toxicity between ITP patients treated either with single course of dexamethasone or dexamethasone plus rituximab; ii) no significant differences in the relapse rate, RD and TFS among patients who achieved SR after dexamethasone monotherapy or dexamethasone plus rituximab, either front line or salvage therapy, suggesting that relapse rate may be a possible surrogate of SR, rather than of the initial therapeutic choice. Disclosures: Zaja: Roche: Honoraria, Research Funding, financial support for the costs of travel to the ASH annual meeting. Off Label Use: Rituximab in ITP.

Introduction. Previous uncontrolled studies have highlighted the activity of rituximab in patients with idiopathic thrombocytopenic purpura (ITP) relapsed or refractory to standard treatments. To better address this effect, a prospective randomized (1:1), multicenter, phase III study comparing treatment with dexamethasone alone (arm A) vs dexamethasone plus rituximab (arm B) was conducted from July 2005 through June 2007 for adult patients with previously untreated ITP and a platelet (PLT) count ≤20 × 109/L. Material and methods. Patients randomized to arm A received a single course of oral dexamethasone 40 mg on days +1, +2, +3, +4, while patients randomized to arm B received dexamethasone (as in arm A) in association with rituximab 375 mg/m2 iv on days +7, +14, +21, +28. Patients in arm A who failed to achieve a sustained response and had a platelet count ≤20 × 109/L (from day +30 up to the end of 6 months) could receive salvage treatment with the experimental arm (dexamethasone plus rituximab). The primary objective of the study was to compare the sustained response (SR), i.e. PLT count 50 × 109/L at month + 6 of treatment. Secondary objectives were: evaluation of the safety, the initial response (PLT count 50 × 109/L) by day 30 after the initiation of treatment, the activity of salvage therapy with dexamethasone plus rituximab in patients non responding to dexamethasone monotherapy, the definition of clinical and laboratory factors predictive of response and to explore the pharmacokinetics parameters of rituximab and their potential relation with response. Results were analyzed by an intention to treat (ITT) and by a per-protocol (PP) analysis. Results. One-hundred-one patients (52 for arm A and 49 for arm B) and 64 patients (38 for arm A and 26 for arm B) represented the ITT and PP population, respectively. Demographic baseline data were in accordance to what expected for a population of ITP patients. No significant differences among the two groups of randomization were present. There was a female prevalence and the mean age was 47 and 49 years in arm A and B, respectively. Table 1 summarizes the ITT and PP efficacy results considering 3 different levels of response (i.e. PLT count 50 × 109/L, 100 × 109/L and 150 × 109/L). A significant advantage for arm B patients was documented. Table 1 Initial response Initial response Sustained response Sustained response Analysis Intention-to Treat Per-Protocol Intention-to Treat Per-Protocol Treatment Arm A Arm B P value Arm A Arm B P value Arm A Arm B P value Arm A Arm B P value Valuable patients 44 25 32 13 52 49 38 26 PLT 3 50×109/L 27% 68% .001 31% 69% .009 36% 63% .004 39% 85% Twenty-seven patients initially allocated to arm A and who failed to achieve initial response or SR received salvage treatment with the dexamethasone plus rituximab. In this group, ITT and PP SR rate were 56% and 59%, respectively. No clinical or laboratory factors predictive of SR were identified. In arm B patients the serum concentrations of rituximab levels did not correlate with the rate of response. Twelve SR patients of arm A, 27 of arm B and 19 of salvage therapy group were systematically followed up beyond month 6 for a median period of observation of 18 months (range 10–34 months). The rate of SR loss (platelets < 50 × 109/L) in these three groups was 25 % (3/12), 11% (3/27) and 10.5% (2/19). The safety profile was good with no substantial difference between the two arms of randomization. No patient died during the study period. Conclusion. The results of this study indicate that the association of dexamethasone plus rituximab improves patients outcome without worsening of the safety profile. This effect is characterized by prolongation of SR and reduction in relapse rate. The long period of relapse free survival registered in some patients suggests a possible curative effect. This treatment can be offered as an option before splenectomy, particularly in those patients where the surgical option is not well accepted or have higher risk of complications.

Implementation of transplant procedures and the availability of alternative sources of stem cells for patients without HLA identical siblings has made allogeneic stem cell transplant (ASCT) a suitable therapy for many patient affected by acute myeloid leukemia (AML); consequently, in AML patients transplant strategies must be carefully designed according to stringent risk/benefit analysis. To date, risk assessment relies on identification of baseline prognostic parameters such as karyotype, which has become critical in order to choose post-remissional therapy. Furthermore, there are strong evidences that the presence of specific gene abnormalities, such as mutations of FLT3, NPM and c-kit, allow to identify, within homogeneous cytogenetic groups, subsets of patients with distinct clinical outcome. We hypothesized that, in order to guide the post-remission decisional process, more robust informations may derive from the combined evaluation of conventional baseline and delayed prognosticators. Minimal residual disease (MRD) detection is potentially the most efficient tool to investigate the quality of remission and might represent the ideal parameter to be associated with baseline biological features for prognostic purposes. Therefore, we aimed to determine whether combined analysis of karyotype and MRD, using multiparametric flow-cytometry (MPFC), could help to refine risk assessment in adult patients with AML, allowing the most appropriate post-remissional strategy to be selected. We analyzed 132 patients with AML who had intensive chemotherapy with EORTC/GIMEMA protocols. According to MRC classification, 22 (17%), 104 (78%) and 6 (5%), respectively, had good, intermediate and poor risk karyotype. Thirteen of 107 (12%) carried a FLT3-ITD. Within good and intermediate risk categories, MRD positivity (≥ 3.5×10−4 residual leukemic cells at the post-consolidation time-point) was associated with a worse prognosis in terms relapse free (RFS) and overall survival (OS). In fact, we observed that: MRD negative good and intermediate risk categories shared the same favorable prognoses with RFS and OS of 80% vs. 69% and 84% vs 54%, respectively; MRD positive good and intermediate risk categories fared as worse as those with poorrisk karyotype or FLT3-ITD in terms of RFS (28% vs. 17% vs. 0%) and OS (42% vs. 21% vs. 17%). Using this approach the conventional cytogenetic classification that uses three categories is simplified into 2 prognostically defined groups: favorable, including MRD negative good and intermediate risk karyotype; unfavorable, including MRD positive good and intermediate risk karyotype, poor risk karyotype and FLT3-ITD positive cases (Fig. 1). Based on these observations, we believe that ASCT is recommended, not only for poor-risk karyotype or FLT3 positive AML, but also for good/intermediate risk categories not gaining MRD negativity, being this option able to provide a superior chance of prolonged RFS (Maurillo et al, JCO 2008). On the other hand, patients belonging to MRD negative good/intermediate risk categories, who can experience a 5-years survival higher than 60%, may have their life expectancy hampered by the choice of a therapeutic strategy with a disadvantageous risk/benefit ratio: for this category a standard intensification procedure (chemo and/or autologous transplant) is indicated. In conclusion, the combined assessment of baseline prognosticators (cytogenetics) and parameters inherent the quality of response (MRD), is useful to define discrete categories of risk within the respective karyotypic groups, allowing tailored therapeutic approaches to be applied according to the actual clinical risk and avoiding under or over treatments. Figure 1 Figure 1.

Autologous (AuSCT) and allogeneic stem cell transplantation (SCT) are well established post-remissional strategies for patients with Acute Myeloid Leukemia (AML). However, there is still a debate ongoing about the relative merit of each of these options in first CR. Minimal residual disease (MRD) may be a useful tool to stratify AML patients into categories of risk which can benefit from differentiated post-remissional approaches. To address this issue, we analysed retrospectively, a series of 123 patients affected with AML in whom flow-cytometry serial determinations of MRD were available, at established time-points (post-induction, post-consolidation, post-stem cell transplantation). All were entered into the EORTC/GIMEMA protocols AML10/AML12 (age 61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged the threshold of 3.5x10−4 at post-consolidation check-point is critical to predict disease outcome; MRD− patients have an excellent outcome regardless of the post-consolidation therapy; in the MRD+ group, AuSCT seems not to improve the prognosis whereas the use of SCT is associated with a superior outcome, although a larger number of patients is required to confirm this assumption. Figure Figure