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Minimal residual disease (MRD) was monitored in 80 patients with acute lymphoid (ALL, n=44) or myeloid (AML, n=36) leukemia, undergoing allogeneic haemopoietic stem cell transplantations. MRD markers were IgH-VDJ and TCR gene re-arrangement for ALL, and Wilm's Tumor (WT1) expression for AML. The overall cumulative incidence (CI) of MRD was positive in 45% and the CI of hematologic relapse was 24% (36% in MRD+ vs. 16% in MRD patients, p=0.03). The median interval from transplant to first MRD positivity was 120 days and to hematologic relapse 203 days. Patients were divided in 3 MRD groups: MRD (n=44), MRD+ given donor lymphocyte infusions (DLI) (n=17) and MRD+ not given DLI (n=19): leukemia relapse rates in these 3 groups were 16%, 6% and 63%, respectively (p

Cunninghamella bertholletiae infection occurs most frequently in neutropenic patients affected by haematological malignancies, is associated with an unfavourable outcome. We report a case of rhino-mastoidal fungal infection in a leukaemic patient. Bioptical tissue cultures yield the isolation of a mould with typical properties of Cunninghamella species. Liposomal amphotericin B (L-Amb) therapy combined with surgical intervention brought the lesion to recovery. Nevertheless, the patient died 14 days after bone marrow transplantation (BMT) from bacterial sepsis. Mastoiditis was documented at CT-scan. The conditioning regimen probably caused the reactivation of the Cunninghamella infection that led to the patient’s fatal outcome; fungal hyphae were detected after autopsy of brain and lung tissue.

Summary We studied N-ras and Ki-ras point mutations respectively at codons 12–13 and 12 in 15 patients with myelodysplastic syndromes (MDS) using the polymerase chain reaction (PCR) method for DNA amplification, and slot blot hybridization to allele specific oligonucleotide (ASO) probes. We analysed peripheral blood and bone marrow samples collected at diagnosis and repeatedly during the chronic phase of the disease to define when the activation occurred and in which haemopoietic cell populations, in order to establish possible relationships between clinical and molecular features. In three cases the N-ras oncogene was mutated at codon 12 in every cell population, both at diagnosis and throughout the chronic phase. Point mutations were not seen at the 12 codon of the Ki-ras oncogene. In patients lacking activated ras oncogene at diagnosis, mutations were not discovered during the entire period of observation. Therefore in our cases disease progression and leukaemic transformation did not correlate with the presence of the activated N-ras. Our data suggest that ras activation occurs early in the pathogenesis of MDS and involves a haemopoietic progenitor with multiple differentiative capacity, without however conferring an apparent proliferative advantage on its progeny.

Summary We report the final results of a prospective multi-centre trial testing the combination of chemotherapy (fludarabine, cytosine arabinoside and idarubicin; FLAI) followed by low-dose gemtuzumab ozogamicin (GO), for induction treatment of patients with CD33+ acute myeloid leukaemia (AML). Forty-six consecutive patients were treated: the median age was 66 (range: 60–80) years; the karyotype was unfavourable in 12 patients (26%), intermediate in 33 (71%) and favourable in one (3%). Eleven major infectious complications were recorded. There was one early death. Of the 45 evaluable patients, 24 achieved a complete response (CR; 52%), 66% and 33% in good-intermediate/poor karyotype patients. Median duration of CR was 7 (3–24) months. The cumulative incidence of relapse was 37% with an actuarial 2-year survival of 54%. These results were compared with 47 patients matched for age and karyotype who received FLAI, without GO. The proportion of patients achieving CR was comparable. However, patients with de novo AML receiving GO (n = 26) had a significantly lower risk of relapse at 2 years when compared with patients not receiving GO (n = 35) (40% vs. 80%, P = 0·01) and significantly better overall 2-year survival (40% vs. 14%P = 0·02). Patients with secondary AML had comparable outcome whether or not they received GO. This GO-based induction chemotherapy has a good toxicity profile. In keeping with a recent prospective randomised trial, the addition of GO seems to prolong disease-free survival.

To the editor: Hematologic relapse after an allogeneic hemopoietic stem cell transplantation (HSCT) in patients with acute leukemia is associated with a poor outcome, despite further cell therapy in the form of donor lymphocyte infusions (DLIs).[1][1] This has been taken as evidence that acute

Background: Acute Myeloid Leukemia (AML) is an incurable disease characterized by a highly unstable genome, resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. However, the mechanisms whereby tumor cells adapt to genomic instability are largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage repair (DDR) machinery. SIRT6 is an important regulator of cellular stress response and genomic integrity. Here we investigated the role of this NAD+ -dependent deacetylase in regulating ongoing DNA damage observed in AML patients. Methods: SIRT6 mRNA level was determined by RT-qPCR in AML patients (n=100) diagnosed at the Hematology Department of University of Genoa (Italy), compared with normal bone marrow derived CD34+ cells (n=5). Correlation studies with clinical and molecular characteristics of these patients were also performed. A panel of different AML cell lines and primary cells, both sensitive and resistant to conventional and novel anti-AML therapies, was used in the study. The anti-leukemic effect of DNA-damaging agents (DDAs) including idarubicin, Ara-C and fludarabine was evaluated in presence of SIRT6 depletion/inhibition by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results: AML patients were grouped into lower and higher SIRT6 expressers according to its median mRNA level. Patients with lower expression had a higher incidence of FLT3-ITD (p=0.034, Wilcoxon signed rank test). No significant association was observed with respect to mutations of NPM1, nor with WT1 and BAALC expression. SIRT6 expression correlated also with adverse clinical outcome in term of event free and overall survival (p=0.035 and p=0.025, respectively; unpaired t test). Based on these data, we evaluated SIRT6 role in biology of AML. We found higher SIRT6 protein level in AML cell lines carrying FLT3-ITD mutation (MOLM-14 and MV 4-11) compared to cell lines harboring other mutations (OCI-AML3, THP-1, KG, NB4, HL60, Nomo1 and U937). Targeting SIRT6 by specific shRNAs weakly reduced AML cell survival compared with control-scrambled cells, by impairing DNA repair efficiency. Indeed, a restricted effect of SIRT6 impairment on DNA damage proteins (H2AX, RAD51, 53BP1, RPA32) was measured. We next examined the therapeutic relevance of SIRT6 inhibition in AML by testing effects of its depletion in combination with genotoxic agents. Remarkably, SIRT6 depletion conferred increased sensitivity of AML cells to idarubicin, Ara-C and Fludarabine. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represents an innovative strategy to increase chemosensitivity of AML cells. Further mechanistic studies revealed that SIRT6 acts as a genome guardian in leukemia cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair. Conclusion: Genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

BACKGROUND AND AIMS The prognosis of Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) patients has improved since the introduction of tyrosine kinase inhibitors (TKI). The inclusion of TKIs in standard ALL protocols allows a great increase in complete molecular responses, but at the price of non negligible toxicities and high rates of toxic deaths. On the other and TKI monotherapy as induction treatment allows to rapidly achieve complete hematologic remission (CR) but only a minority of patients achieve a complete molecular response with high risk of relapse. On the other hand, In the last years we tested a combination of Fludarabine, Cytarabine, Daunoxome (FLAD) with or without TKIs (mainly Dasatinib) as salvage regimen in relapsed-refractory ALL, with acceptable toxicity and good efficacy. We decided to apply the same schedule in newly diagnosed Ph+ ALL as consolidation treatment after a two months TKI (Dasatinib) monotherapy induction on a minimal residual disease condition. MATERIALS AND METHODS FLAD regimen consisted with a three-days administration of Fludarabine 30 mg/sqm followed four hours later by Cytarabine 2000 mg/sqm and Daunoxome 100 mg/sqm. TKI were suspended during chemotherapy administration and were re-administrated starting from day 5. G-CSF was given to all patients from day 4 to complete hematological recovery. FLAD was administrated for up to two cycles; all patients with available donor proceeded to allogeneic bone marrow transplantation (allo-BMT) after FLAD. Minimal residual disease (MRD) was evaluated in all patients after each FLAD either by RQ-PCR for VDJ rearrangements, multicolor flow cytometry (MFC) and RQ-PCR for BCR/Abl. Ten Ph+ ALL have been treated with FLAD + TKIs from January 2008 to December 2014: six patients received FLAD as salvage regimen, two of them in post allo-BMT setting, whereas four patients were treated frontline, after hematological CR was obtained with Dasatinib + steroids induction. All frontline patients proceeded to allo-BMT after two FLAD. Median age for frontline patients was 50 years (range 29-58), median follow-up was 20 months. RESULTS As salvage regimen, 5/6 patients achieved hematological CR after FLAD, with three patients achieving also MFC MRD negativity and clearance of VDJ and BCR/Abl transcript. All patients who did not receive subsequent BMT relapsed, whereas of the two transplanted patients one is still in CR after a follow-up of 38 months. In the frontline setting, all patients received 70 days induction of Dasatinib + Steroids and achieved CR with complete hematological recovery. BCR/Abl transcript could be detected in all patients on BM samples on day 33 and on day 70 (Fig. 1), two patientshad MFC MRD positivity both on day 33 and on day 70, whereas two patients achieved MFC MRD negativity on day 33. FLAD was very well tolerated, with negligible non hematological toxicity, with a median duration of ANC Two patients achieved BCR/Abl negativity after first FLAD. All patients achieved molecular complete response after the second course (Fig. 1). No patient experienced relapse, whereas one patient died in CR on day +289 after allo-BMT due to myocardial viral infection. CONCLUSIONS FLAD has a very good efficacy in adult Ph+ ALL, with an acceptable toxicity profile. Deep responses have been observed in relapsed patients, and all newly diagnosed patients who received FLAD as consolidation regimen had achieved molecular CR before allo-BMT. Achieving complete hematological response with Dasatinib + steroids allowed us to safely administer two FLAD courses. Figure 1. BCR/abl on bone marrow samples at different timepoints for each of the four patients receiving FLAD as consolidation therapy Figure 1. BCR/abl on bone marrow samples at different timepoints for each of the four patients receiving FLAD as consolidation therapy Disclosures Off Label Use: Use of liposomal daunorubicin in the treatment of ALL.

Leukemia relapse remains a significant problem in patients with AML undergoing an allogeneic stem cell transplant(HSCT). Wilms Tumour 1 (WT1) expression has been shown to be a sensitive marker of minimal residual disease (MRD), both in patients after induction chemotherapy, as well as in patients undergoing an allogeneic HSCT. Hypotheses. The present study had 2 hypotheses: (1) WT1 expression in marrow cells of AML patients post-HSCT, will predict leukemia relapse and (2) WT1 based pre-emptive immunotherapy (IT) such as abrupt cyclosporin discontinuation and/or donor lymphocyte infusion (DLI), will prevent leukemia relapse. Patients. Bone marrow WT1 expression, was monitored in 207 patients with acute myeloid leukemia (AML) before and monthly after an allogeneic HSCT, until day +150, and then at every other outpatient access. Eligible for IT were patients without acute or chronic GvHD, with increased WT1 expression and a a marrow in hematologic remission. The trigger for IT was 180 WT1 copies in a first group of 122 patients (group A): this was based on the fact that WT1 expression in normal bone marrow is up to 180 copies . In a subsequent group of 85 patients (group B) the cut off for IT, was 100 copies, due to the fact that a first analysis of group A had shown 100 copies to be an earlier predictor of relapse (BJH 2013; 160: 503). DLI were given in escalating doses, starting at 1x105 CD3+ cells/kg in alternative donor grafts and at 1x106/kg in HLA identical grafts. DLI were escalated ½ log every month, in the absence of GvHD, to a maximum dose of 1x107/kg. Sixtyfour patients were eligible for IT, but only 35 received IT: reasons for non intervention were ongoing GHD, unavailable donor and delay in WT1 results. Results-Hypothesis N.1. Following transplantation, WT1 expression, was highly predictive of leukemia relapse: 12 relapses in 99 patients with WT1 < 100 copies /104 abl (12%); 19 relapses in 55 patients with WT1 between 101 and 180 copies (35%) and 37 relapses in 53 patients with WT1 >180 copies (70%) (p Results-Hypothesis N.2. 35 patients received pre-emptive immune intervention, 17 in group A and 18 in group B. The latter had more patients beyond first remission at transplant (56% vs 23%) , more myeloablative regimens (100% vs 65%) and more family haploidentical donors (72% vs 6%); age was comparable. The risk of relapse was 13/17 (76%) for group A and 3/18 (17%) for group B (p In conclusion, WT1 expression post-transplant is a strong predictor of leukemia relapse in patients with AML, and can be used to trigger pre-emptive immunotherapy, in approximately 50% of eligible patients. IT triggered at a WT1 cut-off level of 100 copies in bone marrow cells, is more effective, as compared to180 copies, in preventing leukemia relapse. Disclosures No relevant conflicts of interest to declare.

BACKGROUND AND AIMS Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for patients with high-risk acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are the most common tools to evaluate MRD. We recently reported that combining WT1 expression and MFC for MRD detection after induction therapy strongly impacts on relapse risk in AML. The aim of this study was to analyze the role of pre-BMT MRD assessment as predictor for the post-transplant relapse risk. MATERIALS AND METHODS We retrospectively analyzed the outcome of 253 consecutive AML patients receiving allo-BMT. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation . Median age at transplant was 45 years. Disease phase was CR1 in 161, CR2 in 63, and CR3 in 29 patients. One hundred eighty-two received myeloablative conditioning, whereas 71 patients received reduced intensity conditioning. Median follow-up was 59 months (95% CI 46.2 - 71.8 months). Relapse-free survival (RFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall Survival (OS) was calculated from the time of transplantation until death by any cause or last follow-up. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression. RESULTS Relapse occurred in 81 patients (32%). Three-year estimate of RFS was 63.7% (median not reached). The probability of relapse was significantly affected by disease status (first or subsequent CR, p Specifically, MFC-MRD was the strongest predictor of longer relapse free survival (p CONCLUSIONS Pre transplant MRD evaluation by WT1 and MFC on bone marrow samples is a reliable predictor of relapse risk. Patients with both negative pre-BMT MRD markers have a significantly longer RFS, while patients with both positive MRD markers display an higher risk of relapse. Identifying patients who have an higher risk of relapse could open the way to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches. Figure 1. RFS according to risk group Figure 1. RFS according to risk group Disclosures No relevant conflicts of interest to declare.

Background, methods and aims of the study. It is widely established that the prognosis of patients with acute myeloid leukemia (AML) is related to clinical (age, denovo or secondary leukemia, presence of comorbidities), cytogenetic and molecular (e.g. FLT3-ITD, NPM1 gene mutation.) factors. We have developed a Comprehensive Risk Score (CRS) identifying three prognostic subgroups with favorable (low risk karyotype or intermediate karyotype + mutations of NPM1 gene without FLT3-ITD), unfavorable (high risk cytogenetics or FLT3-ITD without NPM1 mutation or secondary AML) and intermediate (including all the other patients not belonging to previous groups) prognosis. The clinical utility of CRS was tested on a cohort of 292 AML patients included in the REGAL registry, a regional Italian registry that was established in October 2010 at the University of Genoa. Results. Analysis of age distribution confirmed the increased incidence of AML in patients older than 60 years (n. 182, 62.4%). Patients with de novo or secondary AML were 210 (71.9%) and 82, (28.1%) respectively. Patients with significant and often multiple comorbidities at diagnosis were 131 (44.8%). Seventeen patients (6%) received only supportive therapy, 217 (74.3%) intensive induction regimens (mainly FLAG-Ida and 3+7), including 96% of patients with age below 60 years and 61% of those over the age of 60 years. Therapy with hypomethylating agents (azacitidine and decitabine) was given to 31 patients (10.6%). In the whole cohort of patients 24 months projected survival (OS) was 66% among 62 patients defined as low risk according CRS (median not reached), 37,9% among 118 intermediate risk patients (median 13.8 months) and 12,7% in 112 patients included in the unfavorable risk group (median survival 4.8 months) (p =0.000, Fig. 1). In patients aged younger than 60 years, OS of favorable and intermediate risk groups were similar (77% and 72% in 39 low risk and in 44 intermediate risk patients, respectively), whereas the outcome of 27 high risk patients was worse (OS 28.2%, median survival of 10,3 months, p 0.0001). In the group of elderly patients (> 60 years) the outcome of the intermediate group was rather similar to that observed in the poor risk group (OS 16% and 7% in 74 intermediate and in 85 poor risk patients, respectively, p n.s). On the contrary OS of 23 favorable risk patients was good (49%, p = 0.0015).Twenty-nine patients have actually received an allogeneic bone marrow transplant (BMT) during first CR (66% of 44 with indication) and 14 in second or third CR. The sources of stem cells have been HLA-identical and haploidentical sibling in 45% and 50% of patients, respectively. Discussion and conclusions. Our study suggests that the predictive value of CRS is clearly affected by the therapeutic strategy. Whereas the score allows the stratification of the whole cohort of patients in three groups with different outcome, in younger fit patients a more intensive therapeutic approach, such as the FLAG-IDA regimen and a wider use of allogeneic bone marrow transplantation made possible by the use of haploidentical donors, results in the super-imposable prognosis between favorable risk and intermediate risk patients. However, the same intensive approach cannot overcome the unfavorable features of the majority of high risk patients. In the elderly group median age and comorbidities prevent patients from receiving intensive induction therapy. Therefore, intermediate and unfavorable risk patients show a similar poor outcome. In elderly patients the achievement of a good outcome is related to disease status (denovo AML), favorable cytogenetic and molecular features, good performance status and administration of intensive induction and consolidation treatment, including BMT in selected cases. Disclosures Carella: Seattle Genetics Inc.: Research Funding.

BACKGROUND AND AIMS Non high risk acute promyelocytic leukemia (APL) patients, as defined by WBC count at diagnosis, have nowadays a very good prognosis when treated with ATRA plus chemotherapy based protocols, with high rate of complete molecular remission after consolidation therapy. However, a small proportion of patients (roughly 10-15%) will eventually relapse, despite the achievement of molecular CR. In the past years, some groups showed that relapse risk could be predicted by testing for some gene alterations, such as FLT3-ITD, or by break point cluster region (BCR) analysis. However, those results were not confirmed in prospective trials. We retrospectively applied a simple 3 gene based molecular panel to low-intermediate risk acute promyelocytic patients, alongside with BCR analysis, in order to develop a risk score. MATERIALS AND METHODS Fifty-nine low/intermediate risk APL patients, treated with the Italian age-adapted AIDA protocol from January 1st 2004 to December 31st 2014 in our center were retrospectively included in this study. Median age was 47 years (range 19-88), 16 patients were older than 60 years (27%). BCR analysis was available in all patients, 41 patients showed BCR1/2, whereas 18 patients had BCR3 (30%). Molecular profile included determination of FLT3-ITD mutation, WT1 and BAALC gene expression levels and was available in 38/59 patients (64%); bone marrow sample of diagnosis is available for all other 21 patients for further analysis. Cut-off values for WT1 (20000/abl x 104) and BAALC (450/abl x 104) were arbitrarily chosen after pre-analysis and comparison with our published data. Five patients had FLT3-ITD mutation (13%), 18 (45%) patients had higher WT1 expression levels and BAALC was overexpressed in 9 (24%) patients. Pre-analysis showed that FLT3-ITD, low WT1 levels, high BAALC levels but not the presence of BCR3 were associated with higher relapse risk. A molecular score including those 3 genes was built: 16 patients had no risk factors (42%), 17 patients had one risk factor (45%), 5 had two risk factors (13%). RESULTS Fifty-four patients survived induction, all of them achieved CR, in 52 PML/RARα transcript was undetectable after last consolidation therapy. The two patients with molecular persistence of disease received further therapy and then achieved molecular CR. Nine patients (15%) relapsed after a median follow-up of 56 months, 45 patients are alive at the time of analysis (76%). Relapse risk was influenced only by the presence of at least one molecular risk factor (p Disease free survival (DFS) duration was significantly higher in patients who had none of the molecular risk factor, when compared to patients with at least one (3-years DFS of 100% and 70.8%, respectively, p Overall survival (OS) analysis led to similar results: patients with no molecular risk factors had a very good outcome when compared to patients with at least one alteration (3-years OS of 100% and 71.5%, respectively, p Again, none of the molecular alterations, if taken alone, had a significant impact on DFS or OS. CONCLUSIONS Even in our small cohort, molecular profile could identify a subset of low-intermediate APL patients at higher risk of relapse, who may take benefit from an intensified consolidation therapy, as it is currently applied to high risk patients defined by WBC at diagnosis. Figure 1. DFS according to molecular risk score Figure 1. DFS according to molecular risk score Disclosures No relevant conflicts of interest to declare.

The cytogenetic studies and molecular evaluation of a Philadelphia chromosome negative chronic myelogenous leukemia patient with trisomy 21 (100% metaphases) and trisomy 9 (50% metaphases) at diagnosis are described. Fluorescence in situ hybridization revealed an atypical location of the BCR/ABL fusion signal on 9q, which was duplicated in cells with trisomy 9 simulating a double Ph. The patient was successfully treated with Glivec (also known as Gleevec; Novartis, Basel, Switzerland) and achieved complete hematological and cytogenetic response as well as a reduction of BCR/ABL transcripts detected by real-time quantitative PCR.

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vδ1 T lymphocytes were significantly increased (100 to 300 cells/μL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/μL). We studied these Vδ1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor α or IFN-γ in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by γδ T cells and activates their cytolytic function. The Vδ1 T lymphocytes studied were able to lyse the ULBP3+ C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vδ1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vδ1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vδ1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vδ1 T lymphocytes may play a role in limiting the progression of B-CLL.

The multilineage hematological effects of the chemoprotective agent amifostine suggested administration of the drug to patients with myelodysplastic syndromes. Partial but encouraging responses were seen. The aim of this research was to further evaluate amifostine activity on the hemopoietic progenitor cells of myelodysplastic patients. We determined amifostine effects on in vitro growth of pre-treatment peripheral blood and bone marrow hemopoietic progenitor cells from myelodysplastic patients, and colony forming unit granulocyte-macrophage and burst forming unit erythroid levels in the peripheral blood of 7 myelodysplastic patients during and following a 3-week treatment with amifostine (200 mg/m2 i.v., three times a week). No variations were observed for hemoglobin, platelets, white blood cells and reticulocyte values. The peripheral blood levels of both colony forming unit granulocyte-macrophage and burst forming unit erythroid, however, showed a significant and sustained increase. In vitro a significant effect exerted by the drug was observed in single cases, but the in vitro effect did not predict the in vivo increase of peripheral blood levels of hemopoietic progenitor cells. These results appear to provide evidence that amifostine has a significant impact on early events of hemopoiesis in myelodysplastic patients.

High-dose chemotherapy (HDT) with autologous peripheral blood progenitor cell (PBPC) transplant has been increasingly used for newly diagnosed multiple myeloma (MM) in recent years. Presently available results suggest an improvement in the complete remission rate and survival as compared to conventional chemotherapy. However, there is no plateau in the survival curves, and experiments with new treatment schedules and conditioning regimens are warranted.In a non-randomised controlled trial, 20 patients underwent three-step HDT following conventional vincristine/doxorubicin/dexamethasone (VAD)-based induction. In the intensification phase patients received high-dose cyclophosphamide (HD-CY), high-dose etoposide (HD-VP), and mitoxantrone (NOV) plus melphalan (L-PAM) with haemopoietic rescue. Maintenance treatment with interferon was given until relapse. Actuarial overall survival (OS) and event-free survival (EFS) curves were plotted according to the method of Kaplan and Meier. In five of the eight patients achieving complete remission (CR), the molecular disease was monitored by polymerase chain reaction technique (PCR).Overall 18/20 (90%) patients responded, with a CR rate of 40%. After an average follow-up of 40 months, median EFS and OS are 25.5 and 44.6 months, respectively. Monoclonal cells were detectable in the post-treatment bone marrow and in the aphereses of the five CR patients monitored by PCR.The present three-step HDT regimen, including conditioning with mitoxantrone and melphalan, proved to be feasible and safe. Our results are in agreement with the hypothesis that HDT results in an increased remission rate and in prolonged survival in newly diagnosed MM, but a cure is unlikely.

Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients. Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse. Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression. Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p Conclusion: pre BMT MRD evaluation by WT1 and MFC on bone marrow samples is a reliable tool to predict relapse risk. Patients with negative pre-BMT MRD have a significantly longer DFS and OS, while MRD positive patients by both methods display a higher risk of relapse. Patients at higher risk of poor outcome should undergo a more stringent program of post BMT evaluations, in order to detect disease relapse earlier and might be candidate for pre-emptive therapeutic interventions aimed at delaying or avoiding AML reoccurrence. Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are a highly heterogeneous group of clonal disorders, with very different prognosis in given individuals, overall survival (OS) ranging from more than 10 years (y) for the more indolent conditions to only few months (m) for the forms approaching AML; beside of the well-established disease-related prognostic systems (classical IPSS or its revised form [IPSS-R], the prognostic implication of comorbidities is emerging as a relevant patient-related factor influencing clinical outcome. Aim of our study was to evaluate the clinical impact of comorbidities in a series of MDS patients whatever treated in a “real-life” setting. Methods: this retrospective cohort study involved the MDS patients consecutively registered between Jan 2011 and Dec 2013 into the Registro Ligure delle Mielodisplasie database, a regional registry established within the framework of the Italian Network of regional MDS registries. Data of 318 patients (pts) with available complete assessment of comorbidities at diagnosis were included into the study. The clinical characteristics and comorbidities were all considered into the analysis. Comorbidities were evaluated according to both hematopoietic cell transplantation-specific comorbidity index (HCT-CI) and MDS-specific comorbidity index (MDS-CI). All survival analyses were made from the date of diagnosis to last follow-up, death, or progression to AML. Unless specified, survival analyses were Cox models using continuous variables accounting for interactions. Results: Our cohort mainly consisted of older (median age 75y (range 40-98) “lower-risk” MDS pts: according to IPSS stratification, 151 (54.7%) pts were classified as low-risk, 86 (31.2%) as intermediate-1, 32 (11.6%) as intermediate-2 and 7 (2.5%) were in the high-risk group. One or more comorbidity of any grade of severity was seen in 177 (55.7%) pts at diagnosis. The more common comorbidity was cardiac (26.5%). At least a single comorbidity was present in 61.2% of pts older than 75y and in 50.6% of younger pts (p=0.07). Cardiovascular disorders were more frequent among older (32.9% for >75y vs 15.1% for ≤ 75y, p75 y (48% vs. 28.9% for < 75 y (p=0.001). A lower comorbidity score impacted on the clinical choice for active forms of therapy, while pts with an higher burden of comorbidities were preferentially treated with supportive care, even if difference did not reach significance (p=0.07). Overall survival and risk of non-leukemic death (NLD) were analyzed (median f.u. 26.9 m (range 1-220). HCT-CI did not significantly correlated with OS nor NLD (p= 0.1 and p= 0.07, respectively), while MDS-CI was found to be of prognostic significance both for OS (mean 136.6 (95%CI 116-157) m for the low-risk group, 81.3 (95%CI 61-102) m for the intermediate group and 48.1 (95%CI 30-66) m for the high-risk group, p=0.001) and for NLD (mean 159.6 (95%CI 139-180) m for the low-risk group, 96.5 (95%CI 72-121) m for the intermediate group and 49 (95%CI 31-67) m for the high-risk group (p Conclusions: a comprehensive evaluation of comorbidities according to a tailored tool such is MDS-CI helps to predict survival in patients with MDS and should be incorporate to current prognostic scores in order to better define clinical management of these patients. Disclosures No relevant conflicts of interest to declare.

Background Conventional induction therapy of acute myeloid leukemia (AML) is still largely based on the combination of cytarabine (ARA-C) and daunorubicin. In the last two decades alternative drug combinations have been tested in order to improve complete remission (CR) rate and quality of remission. Fludarabine has been shown to enhance ara-CTP accumulation in leukemic blasts and to inhibit DNA repair mechanisms, thus providing a rationale for combination with DNA damaging agents. In our institution a fludarabine containing induction regimen (FLAI-5) is being used since more than 15 years. Patients and methods Eighty-four consecutive non M3 AML patients (age 17-72 years) treated in our center between 2006 and 2013 were retrospectively analyzed. Median follow-up was 42 months. Induction regimen included fludarabine 30 mg/sqm and ARA-C 2g/sqm on days 1 to 5 plus idarubicin 10 mg/sqm on days 1-3-5, with or without gemtuzumab ozogamicin (3mg/sqm) on day 6. Patients achieving complete remission received a second course including ARA-C 2g/sqm on days 1 to 5 and IDA at an increased dose of 12 mg/sqm on days 1-3-5. Patients were stratified in prognostic risk groups according to a comprehensive score based on karyotype, de-novo or secondary disease, NPM and FLT3 status. High-risk and selected intermediate-risk patients underwent allogeneic bone marrow transplantation (BMT) in first complete remission if a donor was available. The other patients were scheduled to receive at least 2 and up to 4 courses of consolidation therapy with 4 days of ARA-C (2g/sqm, daily). Patients features, CR rate and overall survival (OS)analysis are summarized in Table 1. Results and Discussion Six patients (7%) died during first induction mainly because of infective or hemorrhagic events. Of the remaining 78 patients, 63 patients achieved CR (81%) and 15 did not respond (19%). FLAI-5 was generally well tolerated, with negligible non-hematological toxicity; median time to neutrophil and platelet recovery was 18 and 17 days, respectively. Most patients were able to receive subsequent therapy at full dose and in a timely manner; 33/63 CR patients underwent BMT whereas 21/30 (70%) non-transplanted patients received the full programmed therapy. In the whole cohort, 3 years OS was 50.9% (median 38 months). Factors affecting CR rates and OS are summarized in Tab.1. In a large randomized trial Burnett et al showed that FLAG-Ida favorably compared with other induction schedule. However the longer disease free survival and the lower relapse rate observed in the FLAG-Ida arm did not translate in a higher OS mostly due to the severe hematological toxicities following the second course. Our results are comparable to those recently published and show that the administration of fludarabine only in the first course may significantly reduce hematological toxicity therefore allowing patients to proceed along the consolidation program or undergo allo-BMT in a good performance status. Besides, we were able to increase IDA dose (10 and 12mg/sqm in first and second course, respectively, compared to 8 mg/sqm used in the FLAG-Ida combination). Our results show that FLAI-5 induction regimen followed by a second ARA-C and IDA containing course is an effective and well tolerated therapy for younger AML patients and confirm that CR rate and OS are mainly affected by comprehensive risk group, WBC count at diagnosis and NPM status. Abstract 2266. Table 1: Patients features and CR/OS analysis CR after 1st cycle/Tot.(%) p(univ.) p(multiv.) Median OS(months) OS 36 months (%) p(univ.) P(multiv.) All Patients 63/78 (81) - - 38 50.9 - - Age < 45 yrs 28/34 (82) 1.000 - NR 53.4 0.121 0.105 > 45 yrs 35/44 (80) 15 49.2 Karyotype Favorable 3/3 (100) 0.093 0.248 NR 100 0.003 0.775 Intermediate 52/61 (85) NR 60.9 Sfavorable 8/13 (62) 7 0 NPM Wild type 36/50 (72) 0.003 0.001 32 45.1 0.270 0.541 Mutated 26/26 (100) 65 66 FLT3 Wild type 48/60 (80) 0.721 - 65 56.3 0.249 0.658 FLT3 ITD 14/16 (88) 14 35 Disease Onset De novo 57/67 (85) 0.031 0.213 65 56.1 0.001 0.809 Secondary 6/11 (55) 7 21.4 WBC at diagnosis 30.000/mmc 22/31 (71) 14 39.4 Comprehensive Risk Group Good 17/17 (100) 0.009 0.983 NR 81.2 0.000 0.001 Intermediate 31/37 (84) NR 63.5 Poor 15/24 (63) 9 14.4 Mylotarg FLAI 44/54 (82) 1.000 - 34 49.1 0.676 - MY-FLAi 19/24 (79) 65 52 Figure 1: OS according to risk group Figure 1:. OS according to risk group Disclosures No relevant conflicts of interest to declare.

Backgrounds and aims Prevention and prompt treatment of invasive fungal infections (IFI) in acute myeloid leukemia (AML) patients can improve overall survival not only by reducing infection-related mortality but also allowing to comply induction regimens on time. The aim of the study was to estimate efficacy and feasibility of primary antifungal prophylaxis with posaconazole (PSZ) in patients affected by acute myeloid leukemia receiving front-line chemotherapy and to optimize our clinical practice. Materials and methods From January 2013 to May 2014, 28 AML patients undergoing intensive chemotherapy and potentially eligible for bone marrow transplantation in our institute received PSZ prophylaxis for IFI. All patients received a fludarabine, cytarabine and idarubicin containing regimen as first line treatment. We performed a retrospective analysis to evaluate the efficacy and feasibility of PSZ prophylaxis; to detect factors affecting drug exposure we analyzed each period of hospitalization as a single independent event. PSZ was given at the standard dose of 200 mg for 3 times/day, concurrently with a fat snack or with at least 100 ml of an acidic drink. Because of unpredictable absorption rates PSZ serum levels (TDM) were assessed routinely according to a validated high-performance liquid chromato-graphic (HPLC) method as described. A comparison with an historical cohort with similar features who had received fluconazole (FLC) prophylaxis was made. The use of empirical or targeted antifungal therapies and the incidence of IFI were compared. Results and discussion PSZ showed a good tolerability profile with no serious adverse events clearly related to prophylaxis occurring. A median number of 2 TDM for each period of hospitalization was performed (range 2-5). The achievement of a plasmatic PSZ concentration > 0,7 mcg/mL is considered optimal for prophylaxis efficacy; in 30/47 (64%) episodes of hospitalization and treatment, with at least two TDM, the threshold PSZ serum concentration was reached, with stable plasmatic levels. Median PSZ plasmatic value at first assessment was 0.89 mcg/mL (range 0.1-3.3). Table 1 summarizes patients features and factors that might affect PSZ plasma concentrations. The strongest negative factors affecting PSZ absorption are the discontinuation of prophylaxis and the concomitant assumption of proton pump inhibitors since their negative impact is shown both in univariate and multivariate analysis. No proven IFI were observed in our cohort with only one probable IFI occurring in a patient with refractory disease who did not reach adequate serum PSZ concentration. Table 2 summarizes the comparison with our historical cohort. The risk of experiencing IFI (proven or probable) during AML treatment is significantly higher in the FLC cohort (HR: 9.488, CI: 1,404 - 64,122). Moreover, the use of targeted or empirical antifungal therapies had been significantly higher in FLC cohort (HR: 2.7, CI: 1,212 - 6,050). Our clinical experience confirms the utility and cost-effectiveness of primary prophylaxis with PSZ in AML patients receiving intensive treatment. | | | Reached plasmatic concentration threshold (%) | p (univariate) | p (multivariate) | | ------------------------------------- | --------------- | --------------------------------------------- | --------------------- | ----------------------- | | All Hospitalizations | 30/47 (64) | - | - | | Sex | Male | 17/23 (74) | 0.227 | - | | Female | 13/24 (54) | | Disease Status | Active Disease | 13/27 (57) | 0.014 | 0.156 | | Complete Response | 17/20 (85) | | | | Mucositis | None or Grade 1 | 25/32 (78) | 0.008 | 0.228 | | Grade >=2 | 5/15 (33) | | Age | 45 years | 18/26 (69) | | Concomitant PPI | No | 28/36 (78) | 0.001 | 0.000 | | Yes | 2/11 (18) | | Concomitant Ranitidine | No | 26/42 (62) | 0.640 | - | | No | 4/5 (80) | | Concomitant Levofloxacine prophylaxis | Yes | 27/39 (69) | 0.118 | 0.042 | | No | 3/8 (38) | | Prophylaxis Discontinuation | Never | 27/36 (75) | 0.009 | 0.003 | | At least for 2 dd | 3/11 (27) | | Infectious Complications | None | 8/11 (73) | 0.722 | - | | At least one episode | 22/36 (61) | | PSZ Assumption with | Fat snack | 5/11 (46) | 0.153 | 0.150 | | Acidic drink | 25/35 (71) | Table 1: Factors affecting PSZ serum concentration | | FCZ | PSZ | p | | -------------------------------------------- | ----------- | ----------- | ----- | | All hospitalizations | 54 | 47 | - | | Median age (range) | 47 (17-72) | 47 (19-68) | 0.560 | | Median ANC

Introduction: R-CHOP is the gold standard for the treatment of elderly patients with DLBCL. However, unfit and frail patients frequently do not qualify for CHOP-based chemotherapy. Alternatives are an urgent medical need. Bendamustine plus rituximab (BR) has been established as a standard treatment of indolent lymphomas and preliminary data have shown a promising activity in DLBCL, both in the relapsing and upfront setting. Methods: Within the Fondazione Italiana Linfomi (FIL), we started a phase II study (R-BENDA frail study, EUDRACT2011-001421-24) in elderly patients (>70 years) with a newly diagnosed DLBCL not suitable for R-CHOP-based chemotherapy. All patients were evaluated according to ADL, IADL and CIRS-G and were considered FRAIL if the following criteria were meet: in patients aged 70-80 ADL8 grade 2 comorbidities; in patients older than 80 years ADL>5 or IADL>6 or 5-8 grade 2 comorbidities. Patients received bendamustine at a dose of 90 mg/m2 daily on days 1 and 2 of each 28-day cycle along with rituximab on day 1 for up to 6 cycles. Results: From February 2012 to February 2014, 49 patients were enrolled in 24 Italian centers. The majority (57%) were male and 57% had stage III-IV with 41% elevated LDH. The median age was 82. Overall, 83% of the planned cycles were delivered without dose reduction or delay; grade 3/4 neutropenia was reported in 25% of cycles followed by anemia 21%, and thrombocytopenia 20%. One case of febrile neutropenia was observed. Grade 3-4 non-hematological toxicity was mild and reported in 6% of cycles including 3 episodes of cardiovascular events and 7 other cases of different toxicities (one creatinine increase, one fatigue, one bleeding, one peripheral neurotoxicity, one hyponatriemia, one hyperglycemia and one liver toxicity). Two deaths during treatment have been observed (cardiac failure and sudden death). At the interim analysis (23 patients) the overall response rate was 56% with a complete response rate of 39%. Conclusions: Combination therapy with BR demonstrates low toxicity profile in this high risk population. The promising results on activity can encourage clinicians to considered BR for the treatment of FRAIL elderly patients with DLBCL not eligible for R-CHOP. Disclosures No relevant conflicts of interest to declare.

BACKGROUND AND AIMS In patients with myelodysplastic syndromes (MDS) several validated prognostic scores, such as IPSS and R-IPSS, are available to assess the risk of AML progression and predict overall survival (OS) as well as leukemia-free survival (LFS). A number of molecular aberrations can be identified in MDS. However, differently from AML, none of the current prognostic indexes takes into account molecular profile at diagnosis. WT1 expression has often been evaluated in acute leukemias and MDS. High WT1 expression levels on bone marrow at diagnosis have been reported to identify MDS patients who are at high risk of progression to AML. BAALC (Brain And Acute Leukemia Cytoplasmic) hyper-expression has been associated with a poor prognosis in AML patients, whereas its prognostic value in MDS is not yet clearly defined. The aim of our study was to determine if combined assessment of WT1 and BAALC expression levels at diagnosis could be predictive of leukemic evolution. MATERIALS AND METHODS We selected 86 patients with available WT1 and BAALC expression levels on BM samples at diagnosis. According to IPSS score, 22 patient were considered low-risk, 27 intermediate-1 and 28 intermediate-2 or high risk. Patients underwent different treatment schedules including supportive care, erythropoietin, hypomethylating and immunomodulating agents, according to their risk group. Median follow-up was 36 months (range 4 -121 months). Leukemia-free survival (LFS) was calculated from the diagnosis until last follow-up or documented leukemic progression as defined in literature. LFS was estimated using the Kaplan–Meier method. All Real-Time PCR were performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 1000x104 was used as cut-off value for high WT1 expression, a level of 1000x104 BAALC copy number/Abl copy number was set as cut-off for BAALC hyper-expression. RESULTS After a median time of 32 months, 43 patients died. The main cause of death was leukemic evolution (accounting for 31/43 deaths, 72%), other causes were cardiovascular events and infections (data not shown). The risk of death by any cause was significantly affected by leukemic evolution, diagnosis according to WHO classification and molecular expression profile at diagnosis. Multivariate analysis showed that leukemic evolution was an independent predictor of death (p Twenty-nine leukemic evolutions were observed. Median LFS was 34 months. The probability of leukemic evolution was significantly affected by karyotype, IPSS and R-IPSS scores, diagnosis according to WHO classification, and molecular profile at diagnosis. According to our data WT1 and BAALC combined expression levels further enhanced prognostic stratification. In IPSS Int-1, Int-2/high and in R-IPSS high risk groups, low levels of expression resulted in significantly lower probability of leukemic progression, whereas high levels predicted poor outcome. Furthermore, in patients assigned to IPSS unfavorable prognostic groups, low levels of WT1 and BAALC seemed to predict a significantly longer LFS. In the univariate analysis LFS duration was significantly affected by WT1 and BAALC expression levels (fig. 1), IPSS and R-IPSS scores, karyotype and WHO classification at diagnosis. A multivariate Cox Regression model showed that LFS duration was significantly influenced only by molecular profile at diagnosis and R-IPSS risk group (p Median OS was 32 months. In univariate analysis OS was significantly influenced by diagnosis according to WHO classification, karyotype, R-IPSS score, leukemic evolution and molecular profile expression at diagnosis. The multivariate model disclosed molecular expression profile, R-IPSS score and leukemic evolution as independent predictor of OS (p CONCLUSIONS In MDS patients combined WT1 and BAALC expression levels on bone marrow samples at diagnosis is a reliable predictor of risk of AML progression, LFS and OS. This can improve risk stratification especially in intermediate and high risk groups and may lead to a risk tailored therapy. Figure 1: LFS according to molecular profile Figure 1:. LFS according to molecular profile Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukaemias (AML) evolving from a myelodysplastic syndrome (MDS) or secondary to chemoradiotherapy frequently display unfavorable biologic characteristics. This may explain the lower remission rate obtained with conventional chemotherapy. Recently, the association of Fludarabine with intermediate dose Ara-C has produced interesting results particularly in high risk AML patients. Here, we report on 42 secondary AML patients treated with a combination of Fludarabine, intermediate dose Ara-C, G-CSF with or without an antracycline (FLANG, FLAG-IDA or FLAG). Overall, complete remissions (CR) were documented in 14 patients (33%) and partial responses (PR) in 12 (29%), while 10 patients proved resistant (24%). Six patients (14%) died early. The presence of a prognostically unfavorable karyotype had a negative impact on the CR rate (20% compared to 50% for patients with an intermediate prognosis karyotype, p 0.05). Patients treated with FLAG, FLANG and FLAG-IDA had similar CR rates. At the time of this analysis, after a mean follow-up of 12 months, the mean duration of CR is 16 months (range 3-66) and the mean survival is 11 months (range 1-67). The median time to granulocyte recovery (neutrophils0.5 x 10(9)/l) was 20 days (range 12-39) and 50 x 10(9)/l platelets were reached at a median of 26 days (range 9-56). Taken together, these Fludarabine containing regimens proved to be an effective and tolerable treatment for patients with secondary AML. Patients above 70 years of age may also benefit from this therapy, however the problem of treating patients with adverse chromosomal abnormalities still remains unresolved.

Fludarabine (25 mg/m2 for 5 d, every 4 wk, for 6 courses) was administered as first line therapy in 32 symptomatic chronic lymphoproliferative diseases. All CLL patients achieved at least partial response (5 CR, 2 nPR, 9 PR) but 44% of patients relapsed. In LG-NHLs response and relapse rate were similar. Haematological toxicity was low. VDJ rearrangement PCR analysis was performed on marrow samples at diagnosis and at the time of response evaluation. In the 3 patients who underwent high dose therapy with peripheral blood progenitor cell rescue analysis was also performed on apheresis samples and on marrow samples at the end of the procedure. Clonal VDJ rearrangement was always evident after Fludarabine therapy even in those patients who achieved histological and immunophenotypic complete remission, whereas it disappeared in 2 of 3 patients who underwent HDT. Our data confirm that Fludarabine monotherapy can reduce the neoplastic mass to a subclinical level and suggest the possibility that high dose therapy might produce true complete remission.

Thirty-five aggressive non-Hodgkin's lymphomas (NHL) with marrow involvement received high-dose cyclophosphamide (7 g/m2) and G-CSF in order to collect peripheral blood progenitor cells (PBPC). Fourteen patients were in partial remission, 16 patients were in relapse (“sensitive”, 12; “resistant”, 4) and 5 patients were in refractory to conventional treatment. A good yield of PBPC was obtained in 30 patients, while a low number of CD34+ cells and of CFU-GM was seen in two cases. Two patients entered progression and one patient died. Thirty patients underwent PBPC autografting. Twenty-nine out of 35 (83%) patients entered complete remission (CR). Two patients died in CR of infection following marrow aplasia 3 and 6 months after autografting. At 3 years the probability of survival and disease-free survival (DFS) are of 62% and 51% respectively.

Background and aims Myelodisplastic syndromes (MDS) are a group of hemopoietic disorder characterized by an impaired blood cell production, morphologic dysplasia and peripheral cytopenias; they are the most common hematologic neoplastic disorder and its diagnosis relays on morphologic evaluation, associated to a karyotypic assay. In order to predict the outcome of patients affected from these disorders, knowing that the order of survival can be extremely variable, several prognostic index were developed such as International Prognostic Score Sistem (IPSS) or the most usefull WHO-Prognostic Score Sistem (WPSS). On the contrary of acute leukemia, these disorders have not a biomolecular profile evalutation of intrinsic markers able to stratify patients in different prognostic risk groups. The aim of our study is to assess the risk of leukemic evolution, in MDS patients, on the basis of the levels expression of WT1 and BAALC at disease diagnosis, and to evaluate the leukemia free survival (LFS) at 6-12-24-36 months of follow up, among the different risk category according to IPSS and irrespectively of treatment. Materials and method In five years we analized 102 patients with a diagnosis of MDS divided according to the WHO classification such as follows: 38 AR, 1 AR with del(q5), 21 aREB-1, 23 AREB-2, 3 chronic myelomonocitic leukemia, 1 RARS, 1 MDS, 11 RCMD, 1 5q- syndrome, 2 suspected MDS. According to IPSS 58 belonged to the low risk category, 21 to the intermediate-1, 23 to the intermediate-2/high risk. Cytogenetic assay showed 20 people with an abnormal karyotype, 8 of them fallen into the high risk class and 12 into the intemediate risk. Low risk and intermediate-1 patients were treated only with supportive care; high risk patients were treated with hypomethylating agents. Iron chelation were used when necessary. Lenalidomide was used in the only case of 5 q- syndrome Samples of bone marrow were analized with Real-Time quantitave PCR and levels of WT1 and BAALC expression were determinated. Molecular datas were analized with X-square Test and a significant association was recorded between overexpression of the genes evaluated (WT1 higher than 100 copy numbers and BAALC higher than 1000 copy numbers) and the probability of develop acute myeloid leukemia ( AML ). Results Nine out of 102 patients showed an isolated WT1 hyperexpression (3 of them developed an AML ), in 15 cases we reported an isolated BAALC overexpression (3 of them developed an AML ), while 13 out 18 patients ( 72% ) with combined WT1 and BAALC overexpression developed AML within an average time of 6 months; instead only 5% of patients, which expressed low levels of WT1 and BAALC, developed AML within the interval of observation. In particulary a combined high expression of WT1 and BAALC were strongly associated with an high risk to develop leukemia and a short LFS, especially in INT-1 subset. After that we calculated the LFS, divided for the risk category at 6-12-24-36 months of follow up. Patients with combined overexpression of WT1 and BAALC showed a LFS of 40% at 6 months of follow up and 0% at 24 months. Conclusion MDS have a great variable survival, and the current approach to these diseases relays on morphological evaluation, karyotypic assay and need of transfusional support; gene expression could be a promising system to predict the prognosis in these patients. Analysis of gene expression, which belong to AML evaluation, allows to divide patients in several risk groups; furthermore is not the single gene evaluation that is more predictable but a combined assay. With this method, which seems to be more realiable than IPSS, we could find that a great percentage of patients with levels of WT1>100 and BAALC >1000, indipendently from karyotypic status and treatment, developed AML and have a shorter LFS than the population with WT1 Disclosures: No relevant conflicts of interest to declare.

Background Treatment of elderly patients needs to be considered in view of patients’ physical condition and social environment in addition to diagnosis, staging and risk factors. Fludarabine was the first purine analogue with an oral formulation available for clinical use. The oral formulation offers equivalent efficacy and improved tolerability profile compared to the intravenous (IV) formulation. IV fludarabine requires several administrations that may expose patients to the risk of clinical side effects and potential logistic problems. Aims To assess whether frontline oral fludarabine and cyclophosphamide (FC) combination therapy for elderly or clinical unfit patients with B-cell chronic lymphocytic leukemia (B-CLL) and low grade non Hodgkin lymphoma (NHL), is well tolerated, effective and cost-saving. Methods Between April 2005 and March 2013, 10 elderly untreated patients (mean age 75, range 68-86) with B-CLL requiring treatment, according to ESMO guidelines, and 21 stage ≥3 indolent NHL (mean age 73, range 47 -89) received therapy with low dose oral fludarabine (25mg/mq/die) and cyclophosphamide (150mg/mq/die) from day 1 to 3. Study design consisted of 6 cycles repeated at 4 weeks intervals in the outpatient clinic. Patients received antibiotic prophylaxis with trimethoprim/sulphamethoxazole (160/800 mg once a day, 3 times a week) and allopurinole (300 mg once a day from days 0 to 4). Four follicular NHL, 6 small lymphocytic NHL, 7 marginal zone NHL, 2 indolent mantle cell NHL and 2 lymphoplasmacytic NHL were treated. Performance status was WHO £ 2 in all patients. Comorbidities, which included diabetes, hypertension and chronic heart disease, were present in 12 patients (grade ≤ 2). The median number of cycles was 4 (range 2-6). No patients reduced either the dose or the number of cycles because of hematologic and extra-hematologic toxicities. Specifically, only 2 patients experienced grade III neutropenia, treated with G-CSF. Results Definition of response was reported according to the updated IWCLL-NCI 2008 international general practice criteria, valuated after 3 and 6 cycles. Twenty seven of 31 patients (87%) obtained a clinical response (14 CR and 13 PR). Only 1 responding patient (90 years old) died after 24 months from therapy because of aplastic anemia. In particular, the response rate was 4/4 for follicular NHL (1RP, 3RC), 5/6 for small lymphocytic NHL (2 RC, 3 RP), 7/7 for marginal zone NHL (3 RC, 4RP),1/2 for lymphoplasmacytic and 2/2 mantle cell NHL (PR), 8/10 for B-CLL (3 RC, 5 RP). We consider as Event Free Survival (EFS) time to progression, adverse event with a grade >3 (CTCAE) during treatment, relapse or death. Overall survival (OS) was defined as the time from the start of treatment to death or last follow-up. Survival distributions were calculated using the method of Kaplan and Meier. With a median follow-up of 67 months (range 16-187), we observed a median EFS about 38 months and median OS has not been reached. We used Genzyme-sponsored Excel program to compare direct hospital cost of oral to IV FC (both 3 days regimen). IV treatment required 18 day service admissions with a total cost of € 7.527 while the oral therapy required 6 ambulatory visits with total cost of € 1.642 (costs including: pharmacy, nurses and physicians costs). In this analysis we did not consider social and psychological cost such as transportation, care giver loss of working hours, trauma of repeated venipunctures. Conclusions These results suggest that oral FC could be effective and well tolerated for elderly patients unfit for more aggressive treatments. The efficacy of our therapeutic regimen is comparable to intravenous FC (20 mg/m2 and 600 mg/m2) regimen used by Flinn et al. (Blood 2000) with an ORR about 90 %. Hovewer, in that study 17/60 patients experienced a grade 3 or 4 neutropenia with 1 death due to sepsis. In conclusion, most of our patients did not experience severe toxic side effects or required hospitalizations, enjoing a good quality of life. Disclosures: No relevant conflicts of interest to declare.

Introduction the natural history of Chronic Myeloid Leukemia (CML) has changed after the introduction of first generation tyrosine kinase inhibitors (TKIs). Data from clinical trials suggest that about 30% of patients fails imatinib treatment and 18% never achieves complete cytogenetic remission (CCyR). However, CCyR and major molecular response (MMR) can be achieved with second generation TKIs in many patients. Aim We have analized 119 CML patients that have received at least one course of TKI and their outcomes. We have calculated how many patient are alive and continuing to receive first TKI therapy or patients who are alive but had to switch to alternative TKI therapy. Patients and methods From May 1987 and May 2013 among chronic phase CML patients diagnosed, we have analized 119 patients who received at least one course of first, second or third generation TKIs. At diagnosis patients characteristic were: mean age 52 years (range 17-80); comorbidities unrelated to CML were found in 55 patients (i.e. arterial hypertension, ischemic heart disease, atrial fibrillation and chronic obstructive pulmonary disease (grading 1-2). Nine patients had a past history of epythelial cancer. Additional karyotype abnormalities were present in 7 patients (5%). Subjects were stratified according to Sokal score: 32 high risk, 39 intermediate risk and 48 low risk. Thirty five patients were diagnosed in pre-TKI era. Thus, they received second line imatinib, after conventional chemotherapy. Sixty-nine patients were treated with frontline imatinib, 14 subjects received frontline nilotinib and 1 frontline ponatinib in. The median follow up is 102,5 months (range 3-312). Results Response to treatment was defined in according to 2009 European LeukemiaNet guidelines In our series 20 patient died (17%) because of: disease progression in imatinib (= 2; 1.7%), acute myocardial ischemia during imatinib therapy (= 2), respiratory failure in 1 case and second tumor in 9 subjects. Of nine additional patients, three were lost at follow up and 6 subjects died. Overall 96/119 patients are alive (80,7%). Patients receiving frontline TKI are 55 ( 42in imatinib; 12 in nilotinib and 1 in ponatinib). Patients treated with second-line TKI are 30 (19in imatinib post chemoterapy, 6 in Nilotinib and 5 in dasatinib). Subjects receiving third-line TKI are 10 (9 in dasatinib and 1 in imatinib post allograft transplant). Only 1 patient receives ponatinib as fourth-line TKI. Responses are: CCyR in 96% of alive patients of whom 81% have a molecular response (MMR or better). Switch to TKI other than imatinib (24 patients) was due to failure to achieve optimal response or loss of response according to LeukemiaNet guidelines. Moreover, three patients did not tolerate first line TKI. Nilotinib was generally well tolerated: we did not observe any cardiovascular adverse event. However we found that previous failure to imatinib predicted subsequent failure to nilotinib (=11). As previously reported, dasatinib induced three pleural effusions, resolved with medical therapy without requiring switch of therapy, except for one patient. Conclusion We noticed that Sokal score does not correlate with response to TKI therapy. Imatinib is well tolerated, only few patients discontinued because of side effects.However 24% of patients switched to second-line TKI due to loss of response especially among those patients previously treated with chemotherapy. Frontline nilotinib induced a sustained optimal response in 85,7%(=12) without adverse event. Our results about percentage of patient who switched therapy are slightly lower than those of clinical trials. This result support the notion that CML patients enrolled in clinical trials are not always fully representative of a general population referred to outpatient clinic. Disclosures: No relevant conflicts of interest to declare.

Fifty-three consecutive cases of adult CD30+ anaplastic large cell lymphoma (ALCL) have been analyzed. Thirty-six were classified as Hodgkin's disease like variety (HL) (67%) and seventeen as so-called common type (CT) (33%). All cases strongly expressed the CD30/Ki-1 antigen; the neoplastic cells expressed CD15, CD45 and EMA in 60%, 44% and 33% of cases, respectively; T. B and null phenotypes were found in 37%, 17% and 46% of cases. Bulky mediastinal, B symptoms, and extranodal disease at diagnosis were present in 36%, 49% and 25% of cases. EBV encoded latent membrane protein (LMP-1) was found in 10 cases. Of the 13 tested cases only 4 expressed a weak positivity of the CD40 molecule, in a fraction of the tumor cells; in the same cases CD21 was never found. Patients were treated with various protocols; of the 50 evaluable patients, 39 (78%) obtained a complete remission (CR), 3 (6%) a partial remission (PR) and 8 (16%) did not respond. The projected overall disease free survival (DFS) at 36 months is 70%. Only patients with advanced disease stage (III-IV) showed a statistically decreased DFS and survival. Only symptomatic and extranodal disease significantly appeared to influence survival. This study confirms the good outcome of this group of lymphomas and differs from other reports for some clinical (lower percentage of advanced stage, extranodal disease and skin infiltration) and pathological (HL/CT ratio and immunophenotype) features.

Abstract 2500 Wilms' tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity. The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results: KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion: Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures: No relevant conflicts of interest to declare.

Abstract 2535 Background and aims. Detection of minimal residual disease (MRD) has a relevant prognostic value in Acute Myeloid Leukemia (AML). MRD, when used as early treatment response assessment, allows identification of true low-risk and high-risk patients, who may profit alternative chemotherapy approach. In the present retrospective study, we evaluated the impact of MRD assessed by 4-color flow cytometry and WT1 RQ-PCR gene expression in a cohort of AML patients treated at our institution. Methods. Bone marrow samples of 50 adult AML patients (45 de novo and 5 secondary) with available karyotype (K), FLT3-ITD and NPM-A genes mutational status were assessed for MRD after induction. All included patients had a baseline WT1 expression greater than 1000 copies/Ablx104 (range 1060–346060; lab references for normal values 0–500). Fludarabine-based regimen was used as induction; one course of intermediate dose Ara-C 2g/sqm plus idarubicin, followed by 3 courses of intermediate dose Ara-C (2g/sqm) as further consolidation therapy. WT1 log reduction (DWT1) was used to assess the WT1 clearance (DWT1 = logWT1diagnosis – logWT1 post induction). A positive flow MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events - threshold of 2.5 × 10−4 residual leukemic cells. In patients submitted to bone marrow transplantation (BMT) only the first consolidation course was administered and disease free survival (DFS) was censored at the date of BMT. Results. Two (4%) patients had favorable, 40 (80%) intermediate, and 5 (10%) poor risk K (3 had no metaphases); 14 (28/%) carried FLT3-ITD mutation: among them 8 carried NPM-A mutation too, while 6 were wild type. After the first induction regimen 42 of 50 (84%) patients achieved a complete remission (CR). Patients with a negative flow MRD (32%) had 3 years DFS of 69.5%, whereas those with a positive flow MRD (68%) had a DFS of 27.3% (p = 0.032). Patients with a DWT1 > 1.5 log (65%) had a 3-years DFS of 58.3%, whereas those with a DWT1 ≤ 1.5 log (35%) had a DFS at 1 and 2-years of 13,5% and 0%, respectively (p < 0.001). All patients with a negative flow MRD had also a DWT1 > 1.5 log, whereas 12 (52%) of those who achieved a DWT1 > 1.5 log were still positive by flow MRD. Fourteen (28%) patients with a high risk (HR) profile at diagnosis (poor risk K, intermediate K with FLT3-ITDpos/NPM-Aneg, AML secondary to therapy or previous haematological disorder), 6 were no responder to induction, whereas no one of 8 patients in CR reached a negative MRD status in both test with a very poor outcome (projected DFS 4.8 months). MRD assessment using both flow and DWT1 allow to discriminate no-HR profile patients in three prognostic group: good (flow MRD neg) intermediate (flow MRD pos and DWT1 > 1.5 log) and adverse prognosis (flow MRD pos and DWT1 ≤ 1.5 log) with a projected DFS of 70.5 months, 38.2 months and 4.2 months, respectively (p < 0.001). Conclusions. DWT1 identified patients who would relapse better than flow, whereas a negative flow MRD was the best predictor of long DFS. Using both test in combination with baseline biologic parameters enabled the definition of discrete prognostic categories (Fig 1). Outcome of patients with DWT1 ≤ 1.5 log was very poor and comparable with that of patients with HR profile at diagnosis. In these patients forecast a cure is very difficult with the current treatment option and clinical trials with new drugs should be used already in up-front setting. Disclosures: No relevant conflicts of interest to declare.

It has been suggested that multiple myeloma, generally considered a neoplastic disorder of mature plasma cells, may arise from a pluripotent haemopoietic stem cell. The possibility that circulating lymphocytes derive from the same neoplastic progenitor has been tested in a large number of studies in the past few years, as proof of the interest that this subject is raising among scientists, and also of its elusiveness. We studied a group of 29 patients with plasma cell dyscrasias in order to evaluate clonality of haemopoietic cell populations. The X-linked markers hypoxantine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK) disclosed no monoclonal component in seven heterozygous women. Analysis of immunoglobulin gene rearrangement with four probes showed a germline configuration in samples from 25/29 patients. Only four bone marrow samples from subjects with aggressive disease had rearranged C mu sequence; one had rearrangement of JH and C mu.

Abstract 4675 The prognostic value of WT1 expression at diagnosis is still controversial. It has been retrospectively evaluated in 99 consecutive non pretreated non M3 AML patients who had undergone a complete prognostic work up at diagnosis and had received intensive chemotherapy. Biological markers were evaluated on fresh marrow samples collected at diagnosis. WT1 expression was evaluated using TaqMan Gene Expression Assays as described. All patients received induction therapy with combination of fludarabine, Ara-C and anthracycline ± low dose gemtuzumab ozogamicin (n. 59) or with a conventional combination of Ara-C and anthracycline (n. 40) A conventional post-induction chemotherapy including intermediate dosage Ara-C was administered to all responding patients. Univariate comparisons between patients in CR vs non CR were performed using chi-square analysis or Fisher's exact test for categorical variables and t-test for continuous variables. P values < 0.05 were considered statistically significant. Analyses were performed using SPSS. The prognostic impact of WT1 expression was evaluated using quartiles as cut off point and selecting the one with the lowest p value. The event free survival and OS were calculated using the Kaplan Meier method. Non CR after the first induction course, relapse and death due to any cause were considered events. OS and EFS duration were calculated from start of treatment. The impact of multiple predictor variables was assessed by multivariate analyses according to the Cox regression model for OS and EFS while for the evaluation of RC was used the Logistic regression model. Median age of patients was 59 years (range 17-81). Cytogenetic alterations were prognostically favorable in 3 patients and belonged to the intermediate prognostic group in 77 patients (normal karyotype in 75 patients and +8 in two). Nineteen patients had a poor prognosis cytogenetics. For statistical analyses we considered two karyotipic groups: unfavorable (19 patients) and not unfavorable (80 patients). CRs were 60/99 (60%), of which 40 in 51 patients aged 60 or less (78%) and 20 in 48 older than 60 years (41%). Twenty-six patients relapsed, 54 are alive, 45 have died, with a median follow up of 360 days (range 20-2300). In Table 1 are reported clinical indicators of outcome being patients grouped according to the percentile of WT1 expression with the lowest p value (75th). Statystical analysis showed that all WT1 quartiles were balanced for other prognostic factors, such as cytogenetics, BAALC expression, FLT3 and NPMA and B mutations, age, blast count and therapy. The lack of consense on the role of WT1 level at diagnosis in the prognostic stratification indicate that further clinical studies are required. The clear correlation between the level of WT1 transcript and the tumor burden explains why WT1 is used in the follow up of leukemic patients as universal marker of residual disease, also in patients with specific chimeric products. On the contrary, the biological explanation of the prognostic impact of WT1 transcript level at diagnosis remains uncertain. Over the years WT1 gene has been considered as an oncogene or a tumor suppressor gene. In our experience the protective influence of high WT1 expression cannot be explained with an association with good prognosis biological features (such as mut NPM and / or low BAALC). The positive prognostic value of high WT1 expression might be implicated either with WT1 antioncogenic function, or with the stimulating effect of WT1 oncogene on leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.Table 1WT1 2400 N./N.pts (%)p univ,p multiv.*RR (95% CI)CR (all karyotypes)41/ 75 (54)19/24 (82)0,0260.063.364 (0.927-12.202)CR (int/good karyot.)36/59 (61)19/210.010,0276.649 (1.240-35.645)CR (denovo AML int kar)31/45 (69)14/15 (98)0.020,03412.557 (1.218-129.446)CR (denovo, N.K.)26/40 (65)15/16 (94)0.0250.0413.430 (1.111-162.318)EFS at 24 months (all karyotypes)8%6%0.0020.050.486 (0.235-1.007)EFS at 24 months (int / good karyot.)9%64%0.0010.0230.360 (0.150-0.866)EFS at 24 months (de novo, N.K.)5%70%0.0010.0070.227 (0.077-0.671)OS (all karyot)15%55%0,110,660.837 (0.371-1.890)OS (int/good kar.)18%63%0,050,180.507 (0.186-1.381)Table 1 legend: * for multivariate analysis age, karyotype, FLT3, NPM mutation, BAALC expression, denovo/secondary disease were considered. Disclosures: No relevant conflicts of interest to declare.

We decided to use a different approach, taking advantage of a well-established technique for clonal analysis, in which restriction fragment length polymorphisms (RFLPs) of X-chromosome genes are used in conjunction with methylation patterns to determine the clonal composition of a cell population

14621 Imatinib mesylate (IM) is a powerful and selective p210 Bcr-Abl tyrosine kinase inhibitor that has been demonstrated to be effective for the treatment of CML. An Italian multicenter study was...

A new deficient variant of glucose-6-phosphate dehydrogenase (G6PD) causing severe congenital nonspherocytic hemolytic anemia (CNSHA) is described. The variant enzyme, characterized by slow electrophoretic mobility, extreme in vivo and in vitro lability, high Km for G6P and strongly acidic pH optimum, appears to be unique, and has been designated G6PD Genova. Investigation of an obligate heterozygote using various cytochemical, biochemical and recombinant-DNA techniques showed G6PD mosaicism in the erythrocytes and leukocytes. Therefore, the presence of a disadvantageous mutation at one Gd locus did not determine selection in favor of the normal allele in the heterozygote's hemopoietic cells.

Imatinib mesylate (IM) is a powerful and selective p210 Bcr-Abl tyrosine kinase inhibitor that has been demonstrated to be effective for the treatment of CML. An Italian multicenter study was performed to investigate the safety, efficacy, tolerance and compliance of IM in newly diagnosed CML patients. From February 2000 through February 2007, we collected 339 CML patients in different phases of disease and previously treated with Interferon-alpha and/or other therapies. One hundred fifty-two untreated Ph+ CML patients received Imatinib at diagnosis at a dose of 400 mg orally per day; 88 were male and 64 female with a median age of 50 (range, 17–80) years. Complete hematological remission was achieved in 94% of patients. The estimated rate of complete cytogenetic remission (CCR) was 69% after 12 months; with a median follow up of 38 months the rate of CCR raised to 86,8%. Levels of Bcr-Abl transcripts had fallen by at least 3 log in 59% of cytogenetic remitters patients at 12 months; after a median follow up of 38 months, 26% of these patients achieved complete molecular remission (CMR). The most commonly reported adverse events after IM were edema (including peripheral and periorbital edema) (50%), muscle cramps (37,7%), diarrhea (40,7%), fatigue (38,8%); moreover in 8 (5,2%) patients grade 3–4 events were observed consisting of pancitopenia and/or elevated liver enzymes. Eight (5,2%) patients died: 2 patients for progressive disease after allografting, 2 patients of cardiac infarction, 1 patient of severe necrotic fascitis, 1 patient of metastatic colon cancer and 2 patients of blastic crisis. In summary, at 84 months 144/152 (94,7%) patients are alive and 125 (86,8%) of them are still receiving IM. Nineteen (13%) patients discontinued Imatinib: 7 patients for adverse events grade 3–4 and 12 patients for progressive disease. These data are comparable to those of IRIS study and confirm the safety and efficacy of IM in newly diagnosed patients.

Tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. It has been recently reported that in the latter case, tumor cells are refractory to imatinib antiproliferative effect in vitro and the response to the drug in vivo is due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, 20 CML patients, prior and during treatment with imatinib mesylate, underwent bone marrow (BM) aspirates every 6 months, including: morphologic and phenotypic analysis, cytogenetic and biomolecular evaluation, compared to peripheral blood. Plasma from BM and peripheral blood was also recovered for cytokyne-chemokine dosage. We report that in 12 out of 20 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed upon treatment with imatinib mesylate, with >10% (range 10–16%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, while a smaller fraction of IgM+CD126+CD20– (3–4%) or IgD+CD126+CD20- (2–3%) cells was also found. The lasting 8 patients had

Background. Hematologic relapse after an allogeneic HSCT for acute leukaemia is associated with poor outcome: in our Unit, 5 year survival for acute lymphoblastic leukaemia (ALL) relapsing after an allograft is 10% and for acute myeloid leukaemia (AML) this figure is 15%. Most relapses (79%) occur within the first year after transplant. Aim of the study. Prospectively assess minimal residual disease (MRD) after allogeneic HSCT to test (a) incidence of MRD positivity, (b) predictive value on hematologic relapse and (c) whether MRD positivity can be used to initiate treatment with donor lymphocyte infusions (DLI). Patients and methods. Seventy three patients with ALL (n=29) or AML (n=44) received an allogeneic HSCT, most of them after a conventional (CY-TBI) conditioning regimen. Re-arrangement for JH regions and fusion genes (bcr/abl) (RQ-PCR) were used to monitor MRD in ALL, whereas Wilms Tumour 1 (WT1) expression (RQ-PCR) was used in AML. Monitoring was done on bone marrow samples taken pre-transplant, and on day+30, +60, +90, +120, +150, +180. At the time of a positive MRD patients would be eligible for DLI in escalating doses, starting at 10^6/kg CD3+ cells for matched related donors, increasing by ½ log every 30 days up to a maximum dose of 5x10^7/kg CD3+ cells. For unrelated and 1 antigen family mismatched transplants the dose of DLI was reduced by 1 log. Number of MRD+ patients post-transplant. Overall 30 patients of 73 patients (41%) were MRD positive after transplant, 66% for ALL and 25% for AML. Predictive value of MRD positivity on relapse. Hematologic relapse occurred in 15 of 30 MRD+ patients and in 2 of 43 MRD negative patients (p=0.0001). For ALL patients these figures were 10/19 vs 2/10 (p=0.09) and for AML it was 5/11 vs 0/33 (p=0.0004). Preventing relapse with DLI in MRD+ patients. Of the 30 MRD positive patients 16 were given no DLI because the donor was not available or unsuitable for second donation at the time of MRD positivity: 14 patients relapsed (87%) and 3 (19%) survive. Fourteen patients were given DLI, one relapsed (7%) and 12 survive (86%). The difference of hematologic relapse in MRD positive patients receiving DLI (8%) or not receiving DLI (57%) is highly significant (p=0.0001). This particularly true for ALL patients receiving DLI (n=10) (1 relapse and 8 survivors) as compared to ALL patients not receiving DLI (n=9) (9 relapses and 1 survivor). Conclusions. This study shows that (a) MRD positivity is frequent after allogeneic HSCT, (b) MRD positivity predicts hematologic relapse and (c) treatment of MRD with DLI protects against hematologic relapse, and improves survival. This study also confirms our previous data on the efficacy of DLI in acute lymphoblastic leukemia, when the tumour burden is low.

Background. It has been demonstrated that about 70% of patients with CML in chronic phase (CP) at diagnosis co-expressed p210 and p190 BCR/ABL transcripts, although at a much lower level (Blood1996;87:5213–17). In previous studies, the co-expression of p210 and p190 BCR-ABL transcripts at diagnosis was considered as indicative of higher tumor burden. However, the clinical relevance of p190 BCR-ABL mRNA monitoring in CML pts under Imatinib on bone marrow (BM) samples is not known. Materials and Methods. BM samples were obtained from 83 pts with CP-CML treated with Imatinib at a daily oral dose ranging between 300–500mg. These included 192 samples from 43 pts with late CP-CML (post-IFN failure) and 140 samples from 40 pts with early CP-CML who received Imatinib as first line therapy. Median follow-up was 18 (3–58) and 39 (12–58) months for early and late CP-CML, respectively. As part of a diagnostic work-up, BM samples from each patient were assessed for expression of both p210 and p190 BCR/ABL levels by real-time quantitative reverse transcription PCR (QRT-PCR) using a TAQ-Man system (ABI Prism 7700 Perkin Elmer) for BCR-ABL and ABL genes. The median number of BM assessment was 3 (2–6) for early CP-CML and 4 (2–10) for late CP-CML. A major molecular response (MMR) was defined as BCR-ABL/ABL ratios less than 0.05%. A specific nested RT-PCR screening was assessed for detection of p210 (b2a2, b3a2) and p190 (e1a2) BCR-ABL transcripts to confirm the negative data of p210 and p190 in QRT-PCR. Results. A MMR was obtained in 20 pts (50%) and 20 pts (46%) in early and late CP-CML respectively. However, early CP-CML pts showed a significantly greater reduction in p210 BCR-ABL levels compared to late CP-CML after 12 months of Imatinib therapy (p=0.006), indicating a different kinetic of molecular response. Co-expression of p210 and p190 BCR-ABL transcripts at diagnosis was 73% for early CP-CML, whereas it was not available for late CP-CML. To test whether the persistence of p190 BCR-ABL transcript was predictive of MMR, we divided CML pts in 2 groups, those with 0 or 1 p190 BCR-ABL positive samples (group 1) and those with 2 or more positive samples (group 2) during the follow-up. We found that CP-CML pts of the group 2 showed a significant lower probability to obtain MMR molecular response compared to pts of group 1 both for late and early CML patients respectively [17/24 (71%) vs 5/19 (26%) with p=0.0039)], [15/21 (71%) vs 6/18 (33%) with p=0.017)]. This correlation holds also for complete cytogenetic response (data not shown). Conclusions. In this study, approximately 50% of pts reached a MMR; half of them had undetectable values of p210 BCR-ABL transcripts. However, in a proportion of pts with complete cytogenetic response and low level of p210 BCR-ABL transcript, the expression of p190 is still detectable. The persistence of p190 signal despite the 2–3log fall in p210 BCR-ABL levels, may be of prognostic significance and may disclose unfolded concepts of biological relevance.

Ninety patients with Essential Thrombocythemia (ET) where object of a phase II prospective multicentre study designed to evaluate efficacy, safety and tolerability of a two years treatment with PEG Interferon α-2b (PEG Intron, Schering Plough). The patients, 30 M and 60 F, 18–72 years old (median 45), observed in 16 Hematological Institutions of the Gruppo Italiano Malattie Mieloproliferative Croniche (GIMMC), received the ET diagnosis according to the PVSG criteria. At PEG Intron treatment start the patients showed: previous cytoreduction 97% (IFN α 31%), platelet count >1000 x 109/L 81%, splenomegaly 22%. At the end of the first year The PEG Intron starting dose of 25 μg/week resulted increased to a mean value of 55 μg/week and the Hematological Response (HR = Plts 45 years, platelet count >1000 x109/L, Hb 50 μg/week. In conclusion, the present study shows that in ET patients a two years PEG Intron treatment, able to induce and to maintain the Hematological Response in the majority of cases, is associated to a decrease of bone marrow cellularity, granulopoiesis, erytropoiesis, MK number, size and ploidy and, moreover, with an increase of MK dystrophy and of bone marrow fibrosis. These preliminary data on bone biopsy and aspirate will be object of a planned centralized reevaluation by a Panel of Pathologists and Clinicians.

Summary. Philadelphia (Ph) chromosome-positive chronic myelogenous leukaemia (CML) was studied in a subject heterozygous for the X chromosome-linked alloenzyme system of glucose-6-phosphate dehydrogenase (G6PD). Determination of G6PD mosaicism showed homogeneous expression in granulocytes, erythrocytes and platelets. Cytogenetic studies showed the typical Ph translocation in all metaphases from bone marrow and peripheral blood myeloid cells, bcr rearrangement was detected in bone marrow and in granulocytes. B cells were stimulated with Epstein-Barr virus (EBV) in order to evaluate involvement of lymphocytes, EBV-transformed lymphoblastoid cells expressed a single G6PD pheno-type and therefore probably derived from the leukaemic stem cell. However they had a normal karyotpye and a constitutional bcr restriction pattern. Molecular analysis in this case of CML clarifies the differentiative potential of cells belonging to the leukaemic clone, by demonstrating that clonal Phnegative B cells maintain normal differentiative capacity and have a bcr gene sequence which is not rearranged.

The IEV schedule consisted of epirubicin 100 mg/m2 on day 1, etoposide 150 mg/m2 on days 1-3, and ifosfamide 2.5 g/m2 on days 1-3. Patients who proceeded to haematopoietic stem cell transplants (HDTs) received conditioning therapy with BEAM [for the Hodgkin's Lymphoma (HL) and non-Hodgkin's Lymphoma (NHL) groups], or melphalan 100 mg/m2 and mitoxantrone [for the multiple myeloma (MM) patients]. The study consisted of 65 patients with a median age of 53 years: 27 had aggressive NHL, 20 had HL, 7 had indolent NHL, and 11 had MM. Fifty-five patients received IEV for a disease that was refractory to conventional induction regimens, or that was in first or second relapse; 4 patients were treated with IEV while in complete response (CR) after chemotherapy in order to mobilise peripheral blood stem cells (PBSCs). Ninety percent of patients with HL responded to IEV, and 85% achieved CR. Both aggressive and indolent NHLs were less responsive (ORR 50 and 33%, respectively; CRR 41 and 16.5%, respectively). MM patients displayed an intermediate responsiveness (ORR 50% and CRR 30%). IEV was well tolerated in most patients. No life- threatening infections were recorded. PBSC mobilisation was successful in 37 out of 39 patients (95%) and led to the collection of a median of 16, 12, and 13.7 x 10(6) CD34+ cells/kg in patients with HL, NHL, and MM, respectively. All 37 patients underwent an autologous stem cell transplant following a 1 to 2 month interval after the end of IEV. Two patients were submitted to an allogeneic transplant. The median overall survival rate in HL, aggressive NHL, and indolent NHL is 32 (5-60), 16 (2-46), and 14 (4-42) months, respectively. Median EFS is 31 (5-60), 7 (2-46), and 7.5 (4-42) months, respectively. In conclusion, our study confirms that IEV +/- HDT is a well-tolerated and effective salvage treatment for lymphoid malignancies, and that IEV acts as an excellent stem cell mobiliser.