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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.

The clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is variable, and novel biologic parameters need to be added to the clinical staging systems to predict an indolent or aggressive outcome. We investigated the 70-kDa zeta-associated protein (ZAP-70), CD38, soluble CD23 (sCD23), and cytogenetics in 289 patients with B-CLL. Both a shorter progression-free survival (PFS) and overall survival (OS) were observed in ZAP-70+ (P < .001), in CD38+ (P < .001) and in sCD23+ patients (P < .001 and P = .013, respectively). ZAP-70+CD38+ or ZAP-70+ patients with an unmutated IgVH status showed both a shorter PFS (P < .001) and OS (P < .001 and P < .001, respectively) as compared with ZAP-70–/CD38– or ZAP-70– patients with mutated IgVH genes. Discordant patients showed an intermediate outcome. Note, ZAP-70+ patients even if CD38– or mutated showed a shorter PFS, whereas ZAP-70– patients even if CD38+ or unmutated had a longer PFS. Furthermore, ZAP-70 positivity was associated with a shorter PFS both within normal karyotype (P < .001) and within the poor-risk cytogenetic subset (P = .02). The predictive value of ZAP-70 expression was confirmed in multivariate analysis. Thus, ZAP-70 protein determined by flow cytometry improves the prognostic significance of cytogenetics and appears to be a better predictor of outcomes than IgVH gene mutational status. On this line, we recommend and are also interested in conducting a prospective randomized trial of early intervention versus observation for ZAP-70+ patients.

We explored the safety and efficacy of bortezomib given as single agent in patients with untreated or relapsed/refractory acute myeloid leukemia (AML), unfit for conventional chemotherapy. Fourteen patients were treated with bortezomib 1.5 mg/m2 administered twice weekly for two weeks, every 3 weeks. Median age was 70 years (range 60–81) and the median number of cycles delivered was 2 (range 1–4). Of 13 evaluable patients, in 8 (61%), the administration of bortezomib resulted in an antileukemic effect as demonstrated by peripheral blood and/or bone marrow blast reduction. In 4 (50%) of these 8, a decrease by 37% of transfusion requirement was also observed . Overall median survival was 4 months (range 0.25–10). Neurotoxicity was the most frequent adverse event with 7 of 13 (54%) patients experiencing grades 3-4 peripheral neuropathy. Neurotoxicity led to treatment discontinuation in 4 (57%) of 7. In conclusion, the observed anti-leukemic activity of bortezomib indicates that there is room for designing additional studies in which combination with other chemotherapeutic agents should be considered. Clinical registration no.: EUDRACT 2006-006923-38.

Patients with neutropenia are exposed to a high risk for infections in which fever is often the unique symptom [1]. Systemic infections remain the main cause of mortality in these patients therefore, the policy for infection management is to promptly administer empirical antibiotic therapy in order to avoid the increased risk of mortality related to the treatment delay [2]. However, microbiological diagnostic tests are not sufficiently rapid, sensitive or specific to indentify the microbial causes of fever, and a considerable number of patients suffer febrile episodes over a prolonged period without a definite microbiological etiology.

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.

To date, bone marrow (BM) is the most common source of cells to use in order to assess minimal residual disease (MRD) in acute myeloid leukemia (AML). In the present study, we investigated whether peripheral blood (PB) could be an alternative source of cells for monitoring MRD in AML.Fifty patients with AML were monitored for MRD after the achievement of complete remission. Using multiparametric flow cytometry we compared the levels of MRD in 50 and 48 pairs of BM and PB after induction and consolidation, respectively.After induction and consolidation therapy, the findings in BM and PB were significantly concordant (r=0.86 and 0.82, respectively, p0.001 for both comparisons). The cut-off value of residual leukemic cells in PB which correlated with outcome was 1.5x10 (-4). Thirty-three of 43 (77%) patients with1.5x10 (-4)residual leukemic cells in PB after induction had a relapse, whereas the seven patients with lower levels did not (p=0.0002). After consolidation, 38 patients had a level of MRD1.5x10 (-4)and 31 (82%) had a relapse; nine out of the remaining ten patients, whose levels of MRD were below 1.5x10 (-4), are still relapse-free (p=0.00006). In multivariate analysis, PB MRD status at the end of consolidation was found to have a significant effect on relapse-free survival (p=0.036).These preliminary results indicate that: (i) PB evaluation can integrate BM assessment for MRD detection in patients with AML; (ii) PB MRD status at the end of consolidation therapy may provide useful prognostic information.

We assessed by multiparametric flow cytometry the levels of minimal residual disease (MRD) in 100 adult patients with acute myelogenous leukemia (AML) achieving complete remission after intensive chemotherapy. The aim of the study was to determine the optimal threshold, in terms of residual leukemic cells, and the time point of choice, that is, post-induction (post-Ind) or post-consolidation (post-Cons), able to better predict outcome. By applying the maximally selected log-rank statistics, the threshold discriminating MRD− from MRD+ cases was set at 3.5 × 10−4 residual leukemic cells, a level that allowed the identification of distinct subgroups of patients, both at post-Ind and post-Cons time points. Post-Cons MRD− patients had a superior outcome in terms of relapse rate, overall survival (OS) and relapse-free survival (RFS) (P

The objective of this study was to investigate the expression of MDR1 and bcl-2 proteins in de novo acute myeloid leukemia (AML).The expression of MDR1 and bcl-2 was analyzed by flow cytometry in a large series of 256 consecutive cases of AML. The results were recorded as percentage of positivity and relative mean fluorescence intensity (rMFI). To determine individual protein levels, an index which equals the product of the percentage of positive cells and rMFI was generated.Using cut-offs ofor=800 and 300 of the index value for bcl-2 and MDR1 expression, respectively, we identified 4 different classes of AML: 1) double negative; 2) single positive bcl-2+/MDR1-; 3) single positive bcl-2-/MDR1+; 4) double positive. The highest incidence of double negative cases was observed in the M2 class whereas double positive cases occurred more frequently in the M4, M5 and M6 subgroups. Seventy-eight percent and 71% of M0 and M1, respectively, showed single positive bcl-2+/MDR1- expression (p = 0.00001). Twenty-eight percent of patients belonging to the double positive category achieved complete remission, whereas for double negative, single positive bcl-2+MDR1- and single positive bcl-2-/MDR1+ category, the complete remission rate was 69%, 52% and 56%, respectively (p = 0.00038). In multivariate analysis, the double positive status independently affected frequency of complete remission (p = 0.008).Bcl-2 is over-expressed in CD34+ AML; conversely, MDR1 is over-expressed in CD34- AML. However, the combined expression of the two proteins defines a subset of AML with a very poor prognosis.

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 3574 Chronic lymphocytic leukemia (B-CLL) is a very heterogeneous disease with some patients experiencing rapid disease progression and others living for years without requiring treatment and therefore it is mandatory to find new prognostic markers. CD69 overexpression which resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007) and induces increased proliferation and survival of leukemic B-lymphocytes, may reflect an aggressive and progressive clinical outcome. The primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) upon CD69 in univariate analysis; 2) to correlate CD69 with other clinical or biological prognostic factors such as age, Rai stages, lymphocyte doubling time, beta-2 microglobulin, CD38, CD49d, ZAP-70, cytogenetics by FISH and IgVH status and finally, 3) to confirm CD69 as an independent prognostic factor. We investigated 417 patients (pts), median age 66 years (range 33–89), 239 males and 178 females. With regard to modified Rai stages, 127 pts had a low stage, 272 an intermediate stage and 18 a high stage. CD69 was determined by multicolor flow cytometry, fixing the cut-off value at 30%. CD69+ pts were 111/417 (26.6%). CD69 12 months (260/337; P=0.0006), beta-2 microglobulin or Disclosures: No relevant conflicts of interest to declare.

Abstract 2359 Poster Board II-336 B-cell chronic lymphocytic leukemia (B-CLL) exhibits features of activated and antigen-experienced B-lymphocytes and CD69 overexpression resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007). Moreover, the recent in vitro generation of anti-CD69 monoclonal antibodies able to inhibit either tumor growth or enhance NK cell function (Esplugues, 2005) prompt us to extensively evaluate CD69 antigen in our B-CLL cases. The primary endpoints of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD69; 2) to assess the additive prognostic value of CD69 and ZAP-70 and finally 3) to confirm CD69 as an independent prognostic factor. We investigated 401 patients (pts), median age 65 years, 219 males and 182 females. With regard to modified Rai stages, 120 pts had a low stage, 263 an intermediate stage and 18 a high stage. ZAP-70 and CD69 were determined by multicolor flow cytometry, fixing the cut-off values > 20% and >30%, respectively. ZAP-70+ and CD69+ pts were 166/401 (41%) and 108/401 (27%), respectively. CD69 lower than 30% was significantly associated with low Rai stage (105/120; P12 months (258/332; P=0.00001), beta-2 microglobulin (B-2M) or < 60 years resulted to be the most significant prognostic factor (P=0.004) followed by CD38 (P=0.01), IgVH status (P=0.02) and ZAP-70 (P=0.04). In conclusion, CD69 antigen, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and its easy and rapid laboratory determination may allow us to identify early progressive pts in order to take timely therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

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.

The unbalance between anti-apoptotic (bcl-2) and pro-apoptotic mitochondrial proteins (bax) represents a critical mechanism explaining the high rate of resistance and treatment failure in acute myeloid leukemia (AML) (Del Poeta, 2003). Moreover, the availability of novel and effective pro-apoptotic compounds such as bortezomib (Attar, 2008) and gemtuzumab (Del Poeta, 2008) moved us to widely investigate the impact of mitochondrial proteins such as bcl-2, bax and 7A6 on AML prognosis. 7A6 antigen has been found to be exposed on the mitochondrial membrane during the early stages of apoptosis and is recognized by monoclonal antibody APO2.7. For this purpose, a large series of 420 non M3 AML patients (pts), median age 63 years, treated with intensive chemotherapy regimens, were tested. The aims of our research were: to correlate bax/bcl-2 and APO2.7/bcl-2 ratios, as measures of mitochondrial apoptosis, with other well-known prognostic factors such as age, cytogenetics, FLT3-ITD, P-glycoprotein; to evaluate whether mitochondrial apoptosis was able to dissect normal karyotype AML with regard to prognosis, and finally to confirm that mitochondrial apoptosis is an independent prognostic factor. Bcl-2, bax and 7A6 proteins were assessed by multicolor flow cytometry and bax/bcl-2 or APO2.7/bcl-2 ratios were obtained by dividing mean fluorescence intensity (MFI) of bax/MFI bcl-2 or MFI of APO2.7/MFI bcl-2. The thresholds of positivity were set at the median values >0.35 and >0.60, respectively. Fifty-nine percent of pts were bax/bcl-2 positive and 50% were APO2.7/bcl-2 positive. There were strict correlations between higher bax/bcl-2 or higher APO2.7/bcl-2 and FAB M2 or M4/M5 AML subgroups (p0.35 or APO2.7/bcl-2>0.60 and CD34 negativity (p

In a remarkable proportion of adult AML patients (40–50%), no clonal abnormalities are found on standard cytogenetic analysis. In this group of patients, several gene mutations have been described, allowing to discriminate subgroups with distinct clinical outcome. Among these molecular signatures, FLT3, a receptor tyrosine kinase with important roles in hematopoietic stem/progenitor cell survival and proliferation, has been demonstrated to be mutated in about 1/3 of AML cases, identifying a subgroup of patients characterized by disappointing overall cure rates. Therefore, roughly 60% of patients with normal karyotype are not classifiable by this specific mutation. The purpose of the present study was to investigate, in this group of patients with no specific molecular signature the prognostic role of MRD as determined by multiparametric flow-cytometry (MPFC). We analyzed a group of 127 AML cases entered into the EORTC/GIMEMA protocols AML10/AML12 (age 61 yrs). By applying the maximally selected log-rank statistics, the threshold discriminating MRD negative from positive cases was set at 3.5 x10−4 residual leukemic cells, a level that selected, at the post-consolidation time-point, two groups of patients with distinct prognosis. Seventy-two out of 127 (56%) had a normal karyotype. Among these 72, 53 were studied for FLT3 mutational status; in 13 (25%) a mutated variant of FLT3 was detected. Among the remaining 40 patients with normal karyotype and unmutated FLT3, 39 were evaluable for the MRD status at the post-consolidation time-point: 9 patients were MRD− and 30 MRD+. These two subsets showed a distinct outcome in terms of 5-years RFS (83 vs. 23%, p=0.031). Interestingly, the FLT3−MRD+ patients shared with those FLT3 positive a similar poor prognosis (Figure 1). In recent reports, the evaluation of outcome depending on FLT3 status has been integrated with the analysis of the mutated status of other genes: FLT3-mutated cases associated frequently with Nucleophosmin (NPM1) exon-12 gene mutations but rarely with other mutations. The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the group of normal-karyotype AML without FLT3 mutations. In a subset of 44 patients of our series either the mutations were investigated; this combined analysis identified 8 out of 44 (18%) patients FLT3 unmutated/NPM1 mutated with a better outcome (5-years OS 50% and RFS 50%, respectively) as compared to FLT3 unmutated/NPM1 unmutated and FLT3 mutated patients, which was however poorer than that of FLT3 unmutated/MRD− cases (Figure 1). In conclusion, MRD determination at the post-consolidation phase may help at improving the prognostic assessment of AML patients lacking specific chromosomal and/or molecular lesions, allowing risk-adapted post remissional therapies to be designed with more accurate modalities. Figure Figure

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

The current experience in the use of multiparametric flow-cytometry for immunological detection of MRD in AML, indicates that the method is feasible, rapid and relatively sensitive. In our previous studies of AML patients with aberrant phenotype (Blood 2000, Leukemia 2003 & 2006), we found that the level of MRD after consolidation therapy was the best predictor of outcome. In fact, a MRD level higher than 3.5x10−4 residual leukemic cells after consolidation was significantly correlated with a poor outcome. The major problem with using an empirically established cut-off to define thresholds is that it may not be reproducible over different populations of patients. In order to confirm the prognostic value of the threshold of 3.5x10−4 residual leukemic cells, previously selected, we used the Maximally Selected Rank Statistic analysis to determine optimal cutpoint, yielding the best separation of AML patients into two groups with different OS and/or RFS probabilities. To do this, we evaluated the trend of standardized log-rank statistics using RFS (Fig. 1, upper panels) and OS (Fig. 1, lower panels) as dependent variables, and the values of residual leukemic cells, determined both at the post-Induction (post-Ind) and post-consolidation (post-Cons) checkpoints, as independent variable (Fig. 1A and 1B, respectively). The experimental cut-off points, identified as the absolute peak in standardized log-rank statistics plots (vertical dotted lines in Fig. 1), were 3.7x10−4 and 3.4x10−4 residual leukemic cells for post-Ind and post-Cons values, respectively. According to this data we decided to utilize the 3.5x10−4 residual leukemic cells cut-off value to discriminate MRD− from MRD+ cases both after induction and consolidation. Therefore, patients with a residual leukemic cells values below the cutoff of 3.5×10−4 will be referred to as MRD−, while those with residual leukemic cells equal or exceeding the 3.5×10−4 level will be classified as MRD+. Post-Cons MRD− patients had a superior outcome in terms of relapse rate (27% vs. 78%, P the threshold of 3.5x10−4 is valid in discriminating risk categories in adult AML; post-consolidation MRD assessment is critical to predict disease outcome regardless of the MRD status after induction. Figure 1 Figure 1.

In acute myeloid leukemia (AML), the level of MRD as determined by multiparametric flow cytometry (MPFC) has been shown to impact on remission duration and survival. BM is the most common source to perform MRD assessment. We have previously showed that BM MRD negativity after consolidation was associated with a significantly longer relapse free survival (RFS) and overall survival (OS). However, in children with T acute lymphoid leukemia, it has been reported that PB might be used as an alternative source to BM for MRD studies. Based on this, we investigated whether PB might substitute for BM to monitor MRD in adult AML patients, showing the same prognostic value. Forty adult patients with AML were enrolled into the EORTC/GIMEMA protocols AML10/AML12 (age 61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged 1x10−4 after induction had a relapse whereas, the 3 patients with

In acute myeloid leukemia (AML) patients achieving complete remission, the levels of minimal residual disease (MRD) as determined by flow cytometry have been shown to impact on remission duration and survival. However, some issues such as the most suitable source (BM or PB) or the most appropriate timing (early or delayed evaluation) for MRD determination are still a subject of debate. In our experience, we observed that MRD negativity, as defined by a number of bone marrow residual leukemic cells (BMRLC) 61 yrs), all consisting in intensive induction and consolidation cycles, and allogeneic or autologous stem cell transplantation for patients aged < 61 years. Median age was 53 years (range 17–78), all FAB subtypes were represented with the exception of APL cases which were not included. Eighty-one of 89 patients maintained complete remission after consolidation (8/89 had early relapse after induction) and were suitable for the analysis. After consolidation cycle, 32 of 81 (39%) patients had

Abstract 2364 Poster Board II-341 The treatment goal of B-cell chronic lymphocytic leukemia (B-CLL) has already shifted from symptom palliation to the attainment of maximal disease control combining purine analogs with monoclonal antibodies. This immunochemotherapeutic approach resulted both in more complete responses (CR) and longer response duration, often remaining only a minimal residual disease (MRD) detectable by flow cytometry. We treated in first line 120 B-CLL symptomatic patients (pts), median age 62 years, with six monthly courses of intravenous or oral fludarabine at conventional doses and then, after a median time of 31 days, with four weekly doses (375 mg/sqm) of rituximab (rtx). Fourteen pts had a low Rai stage, 103 an intermediate stage and 3 a high stage. We defined as high risk pts having at least two of these markers: unmutated IgVH, CD38>30%, ZAP-70>20%, intermediate unfavorable cytogenetics (trisomy 12 or del11q or del17p). Based on NCI criteria, 92/120 (77%) pts achieved a CR, 24/120 (20%) a partial remission (PR) and 4/120 (3%) no response or progression. Ten pts underwent grade 3 (WHO) infective lung toxicity, 1 patient acute fatal B hepatitis and 2 pts progressed towards Richter's syndrome. Hematologic toxicity included mainly neutropenia (grade 3 and/or 4 in 56 pts) and thrombocytopenia (grade 3 and/or 4 in 8 pts). Fifty-four pts either in CR with B-CLL bone marrow cells >1% (MRD+, n=16 pts) or in CR MRD negative, but with B-CLL peripheral cells going up >1000/microl within 1 year after induction (n=22 pts) or in PR (n=16 pts), underwent consolidation and maintenance therapy with four monthly cycles of rtx at 375 mg/sqm followed by twelve monthly low doses of rtx (150 mg/sqm). The median follow-up duration was 50 months. All treated pts experienced a long progression-free survival from the end of induction treatment (40% at 9 years). On the other hand, global overall survival (OS) was 54% at 10 years. Nevertheless, CLL pts undergoing consolidation and maintenance therapy (n=54) showed a longer response duration vs MRD+ not consolidated pts (n=16; 75% vs 9% at 4 years; P1 year) pts (n=43) showed a very long response duration (79% at 6 years, Figure). Moreover, OS was shorter in MRD+ not consolidated pts (0% vs 79% at 15 years; P=0.0007). Noteworthy, within the high risk subset (n=48), consolidated pts (n=17) showed a longer response duration (56% vs 0% at 2.5 years, P=0.003) vs MRD+ not consolidated pts (n=11). Therefore, rituximab consolidation and maintenance immunotherapy improve response duration in B-CLL, thus potentially increasing OS, also within the high risk subset. Disclosures: No relevant conflicts of interest to declare.