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Background: The phase 3 GIMEMA-MMY-3006 trial, which compared bortezomib, thalidomide, and dexamethasone (VTD) combination therapy with thalidomide and dexamethasone (TD) as induction therapy before and consolidation therapy after double autologous haematopoietic stem-cell transplantation (HSCT) for newly diagnosed multiple myeloma, showed the superiority of the triplet regimen over the doublet in terms of increased complete response rate and improved progression-free survival. We report the results from the final analysis of the study. Methods: In this randomised, open-label, phase 3 study, patients aged 18–65 years with previously untreated symptomatic multiple myeloma and a Karnofsky Performance Status of 60% or higher were enrolled at 73 centres in Italy. Patients were randomised (1:1) by a web-based system to receive three 21-day cycles of thalidomide (100 mg daily orally for the first 14 days and 200 mg daily thereafter) plus dexamethasone (total 320 mg per cycle; 40 mg on days 1–2, 4–5, 8–9, and 11–12 in the VTD regimen, and 40 mg on days 1–4 and 9–12 in the TD regimen), either alone (TD group) or with bortezomib (1·3 mg/m2 intravenously on days 1, 4, 8, and 11; VTD group). After double autologous HSCT, patients received two 35-day cycles of either the VTD or TD regimen, according to random assignment, as consolidation therapy. The primary outcome was the rate of complete response and near complete response after induction (already reported). In this updated analysis we assessed long-term progression-free survival and overall survival (secondary endpoints of the study) with an extended 10-year median follow-up, and analysed the variables influencing survival. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, NCT01134484. Findings: Between May 10, 2006, and April 30, 2008, 480 patients were enrolled and randomly assigned to receive VTD (241 patients) or TD (239 patients). Six patients withdrew consent before start of treatment. 236 (99 [42%] women) in the VTD group and 238 (102 [43%] women) in the TD group were included in the intention-to-treat analysis. The data cutoff date for this analysis was May 31, 2018. Median follow-up for surviving patients was 124·1 months (IQR 117·2–131·7). The 10-year progression-free survival estimate for patients in the VTD group was 34% (95% CI 28–41) compared with 17% (13–23) for the TD group (hazard ratio [HR] 0·62 [95% CI 0·50–0·77]; p

100 Ph+ CML patients submitted to BMT were studied cytogenetically before grafting and serially after transplantation. The 12 European institutions participating in the study, including transplant units and laboratories of cytogenetics, collected a total of 520 studies. The Ph chromosome was observed after BMT in 22 patients who did not enter relapse during the observation time (10–1400 days — median 420 d) following initial detection of the chromosome. This abnormality was observed in 1 to 30% of the cells analyzed. In 10 patients, abnormal cells were detected only within the first 90 d after BMT, in 5 patients both before and after 90 d and in 7 patients only after 90 d. 44% of these 22 patients had a moderate-to-severe cGVHD. Future studies are needed in order to better evaluate the real incidence of persistent disease and the correlations with the GVHD.

Leukemic cells spontaneously secrete cytokines involved in the proliferation of the clone; in this study we evaluated the effects of all-trans retinoic acid (ATRA) on the in vitro autocrine production of cytokines by acute myeloid leukemia cells. Thirty acute nonlymphoid leukemia cases (ANLL) (10 APL and 20 ANLL of other cytotypes than APL) were studied; the in vitro secretions of IL-1 alpha, IL-3, IL-4, IL-6, IL-10, G-CSF, GM-CSF, TNF-alpha were tested with and without ATRA addition. After 5 d exposure to ATRA 10(-6) M APL-treated samples showed a significant reduction of IL-6 (p = 0.008) and GM-CSF (p = 0.03) and a significant increase of IL-1 alpha (p = 0.01) production, if compared to untreated APL samples. No difference was seen in IL-3, IL-10 and IL-4 productions; G-CSF production resulted absent in all but 3 APL cases, in which addition of ATRA determined increase in the production. Interestingly, the 3 G-CSF-producing cases did not obtain clinical remission with ATRA; GM-CSF and IL-6 were spontaneously produced by all the cases, and 7 of 10 APL patients subsequently obtained complete remission after induction. TNF-alpha was produced only in 1 case. No statistical difference was seen in all the productions obtained from other than promyelocytic acute leukemic cells, both with and without ATRA addition. However, it is noteworthy that the production of IL-6 was more than twice as high in ANLL non-APL than in APL cases. In conclusion, these data could thus suggest possible complementary mechanisms of the exhaustion of the leukemic clone upon treatment with ATRA.

Fludarabine (FLU) is a fluorinated purine analogue with a promising antineoplastic activity in lymphoproliferative disorders. In this study, we evaluated the efficacy of FLU in 12 previously treated (primary refractory and refractory relapse) patients with Waldenstrom's macroglobulinemia. All patients were treated at a dosage of 25 mg/m2 per day for 5 consecutive days for a total of six courses. Of the 12 patients, 5 (41%) achieved partial response (PR), and the remaining 7 showed no benefit from the treatment. An increased response rate was obtained in the 4 primary refractory patients in which 2 PR were documented. Treatment was well-tolerated and there were no Fludarabine-related fatalities. With a mean follow-up of 10 months, only 1 PR patient has relapsed. Fludarabine is an interesting new salvage agent effective against recurrent/resistant Waldenstrom's macroglobulinemia and should be evaluated in further studies in untreated patients with Fludarabine in monochemotherapy or in combination with other active modalities.

Imatinib mesylate (STI571), a specific Bcr-Abl inhibitor, has shown a potent antileukemic activity in clinical studies of chronic myeloid leukemia (CML) patients. Early prediction of response to imatinib cannot be anticipated. We used a standardized quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) for BCR-ABL transcripts on 191 out of 200 late-chronic phase CML patients enrolled in a phase II clinical trial with imatinib 400 mg/day. Bone marrow samples were collected before treatment, after 12, 20 and at the end of study treatment (52 weeks) while peripheral blood samples were obtained after 2, 3, 6, 10, 14, 20 and 52 weeks of therapy. The amount of BCR-ABL transcript was expressed as the ratio of BCR-ABL to beta2-microglobulin (beta2M). We show that, following initiation of imatinib, the early BCR-ABL level trends in both bone marrow and peripheral blood samples made it possible to predict the subsequent cytogenetic outcome and response. We propose this method as the method of choice for monitoring patients on imatinib therapy. QRT-PCR studies may be able to identify degrees of molecular response that predict both complete cytogenetic response and long term stability, as well as patterns of response that provide an early indication of relapse and imatinib resistance.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Background Among the third-generation chemotherapy regimens specifically adapted in the last decade for elderly aggressive non-Hodgkin’s lymphoma (NHL) patients, we designed an 8-week cyclophosphamide, mitoxantrone, vincristine, etoposide, bleomycin and prednisone (VNCOP-B) plus granulocyte colony-stimulating factor (G-CSF) regimen which, in a national multicenter trial, induced good complete response (CR) and relapse-free survival rates with only moderate toxic effects. Here we report a prospective, multicenter, randomized trial comparing the efficacy and toxicity of 8- and 12-week regimens of VNCOP-B plus G-CSF. Patients and methods From February 1996 to June 2001, 306 consecutive previously untreated stage II–IV aggressive NHL patients ≥60 years of age were enrolled from 12 Italian cooperative institutions. Of the 297 evaluable patients, 149 and 148 received 8- and 12-week regimens, respectively, of VNCOP-B. Results The CR rates were 63% and 56% in the 8- and 12-week groups; at a median of 32 months (range 3–62 months), relapse-free survival rates were 59% and 55%, respectively. Hematological and non-hematological toxicities were similar in both treatment groups. Conclusions Our data show that extending induction treatment with the VNCOP-B plus G-CSF regimen from 8 to 12 weeks does not raise the CR rate or provide a more durable remission.

Clinical trial results indicate that lenalidomide, an immunomodulatory drug, is a promising treatment in relapsed/refractory non-Hodgkin lymphoma (NHL). This retrospective multicenter study was conducted in patients with relapsed/refractory NHL treated with lenalidomide monotherapy through a Named Patient Program in Italy. Principal endpoints were overall response rate (ORR), safety and overall survival (OS). The ORR in 64 evaluable patients was 42.2\% and was similar among patients receiving 10, 15 or 25 mg/day lenalidomide. Response rates in patients with mantle cell, diffuse large B-cell and follicular lymphoma were 45.5\%, 42.1\% and 20\%, respectively. Among patients who responded to most recent prior therapy, ORR was 50.0\% versus 36.8\% in patients with refractory NHL. Mean duration of response in patients receiving any lenalidomide dose was 10.5 months; 1-year progression-free survival and OS were 50.3\% and 82.6\%, respectively. These findings suggest that lenalidomide is effective and safe for heavily pretreated patients with NHL in the clinical setting.

Summary Background Up to 40% of elderly patients with untreated diffuse large B-cell lymphoma (DLBCL) given a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone every 21 days (R-CHOP21) relapse or develop refractory disease. Lenalidomide has high activity in relapsed or refractory aggressive B-cell lymphomas. In phase 2 of the REAL07 trial, we aimed to establish the safety and efficacy of the combination of lenalidomide and R-CHOP21 in elderly patients with untreated DLBCL. Methods REAL07 was an open-label, multicentre trial that was done in 13 centres in Italy and one in Germany. Eligible patients were aged 60–80 years; had newly diagnosed, untreated, CD20-positive, Ann Arbor stage II–IV DLBCL or grade 3b follicular lymphoma; had an Eastern Cooperative Oncology Group performance status of 0–2; had an International Prognostic Index (IPI) risk of low-intermediate, intermediate-high, or high; and were fit according to comprehensive geriatric assessment. Participants were to receive 15 mg oral lenalidomide on days 1–14 of six 21-day cycles, and standard doses of R-CHOP21 chemotherapy (375 mg/m 2 intravenous rituximab, 750 mg/m 2 intravenous cyclophosphamide, 50 mg/m 2 intravenous doxorubicin, and 1·4 mg/m 2 intravenous vincristine on day 1, and 40 mg/m 2 oral prednisone on days 1–5). The primary endpoint was frequency of overall response (complete response [CR] and partial response [PR]), which was assessed by 18 F-fluorodeoxyglucose ( 18 F-FDG) PET at the end of the treatment. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00907348. Findings 49 patients were included in phase 2: nine had been enrolled into phase 1 between Oct 23, 2008, and June 4, 2009, and had received the maximum tolerated dose of 15 mg lenalidomide; and 40 were enrolled into phase 2 between April 28, 2010, and June 3, 2011. 45 patients (92%, 95% CI 81–97) achieved a response (42 [86%] CR; three [6%] PR). Three patients (6%) did not respond and one (2%) died for reasons unrelated to treatment or disease. 277 (94%) of 294 planned cycles of lenalidomide and R-CHOP21 were completed. Grade 3–4 neutropenia was reported in 87 cycles (31%), grade 3–4 leukopenia in 77 (28%), and grade 3–4 thrombocytopenia in 35 (13%). No grade 4 non-haematological adverse events were reported. No patients died during the study as a result of toxic effects. Interpretation Lenalidomide with R-CHOP21 is effective and safe in elderly patients with untreated DLBCL. Funding Fondazione Italiana Linfomi and Celgene.

Salvage therapy of elderly patients with advanced, relapsed and refractory multiple myeloma (MM) is often limited by poor marrow reserve and multi-organ impairment. In particular, renal failure occurs in up to 50% of such patients, an this can potentially limit the therapeutic options. Both thalidomide and bortezomib have proven effective in these patients, with an acceptable toxicity, while, in clinical practice, lenalidomide is generally not considered a first-choice drug for MM patients with renal failure as early reports showed an increased hematological toxicity unless appropriate dose reduction is applied. Aim of this study was a retrospective evaluation of the efficacy of the combination Lenalidomide +Dexamethasone in a population of elderly MM patients treated in 5 Italian Centers. The study included 20 consecutive MM patients (9 M, 11 F, median age 76.5 years) with relapsed (N= 6) or refractory (N=13) MM and moderate to severe renal failure, defined as creatinine clearance (Cr Cl) < 50ml/min. Four patients were undergoing hemodyalisis at study entry. 85 % of the patients had been previously treated with bortezomib-containing regimens. Lenalidomide dose was adjusted according to renal function and patients clinical conditions Median treatment duration was 16 months (1-22), therapy was interrupted after 1 21-day cycle in 2 patients. Grade III-IV neutropenia was observed in 7 patients (35%) ; grade III-IV non hematological toxicity was recorded in 3 cases (28%). A > partial response was observed in 8 patients (40%), 1 of whom obtained a VGPR; 4 additional patients achieved a minor response. Median response duration was 16 months (range 2-19+ months). A complete and partial renal response was obtained in 4 and 3 patients, respectively, all of them were responsive to Lenalidomide-dexamethasone According to our data, LEN+DEX has shown efficacy and acceptable toxicity in this population of elderly patients with advanced MM and renal failure

Methods of detecting minimal residual disease (MRD) in chronic myeloid leukemia (CML) include chromosome analysis, Southern blotting, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) techniques. We report a novel method to detect intracellular messenger RNA (mRNA) by combining the techniques of reverse transcription (RT) and PCR performed directly inside the cells, without extraction of the nucleic acid. We applied this method, which we call “in-cell RT-PCR”, to detect hybrid BCR/ABL transcript within single cells. After cellular permeabilization and fixation of single cells in suspension, the neoplastic mRNA was reverse transcribed into cDNA, and the cDNA was amplified by PCR with fluorescent primers, specific for bcr/abl. Flow cytometry was used to detect cells positive for the amplified DNA within the cell cytoplasm. After transferring the amplified cells onto slides by cytospin, the positive cells for BCR/ABL cDNA were observed by fluorescent microscopy. The technique was capable of detecting low abundancy signals and distinguishing different levels of gene expression. The amplification products were found in the cells and supernatants. The distribution was critically affected by the protease digestion condition. The specificity of amplification was confirmed by a nested RT-PCR of BCR/ABL performed on extracted mRNA from the same sample, and by reamplification of supernatants. We have used the technique to study 10 Ph+ CML patients and three normal subjects as controls. Four patients were 100% Ph+ at diagnosis time and RT-PCR+ at cytogenetic and molecular analysis, respectively. In-cell RT- PCR showed that the residual non-neoplastic cells could be observed in all cases. In two patients undergoing interferon-alpha (IFN-alpha) therapy and in four bone-marrow transplanted patients, the in-cell RT- PCR was used to compare the level of Ph+ positivity detected by cytogenetic analysis with the number of cells expressing BCR/ABL transcript. In this manner, we could estimate the MRD. Our preliminary application of the technique suggests that it is capable of accurately identifying cells transcribing bcr/abl, and that it may have significant clinical applications in the detection of MRD.

The degree of matching of HLA genes between the selected donor and recipient is an important aspect of the selection of unrelated donors for allogeneic bone marrow transplantation (UBMT). The most sensitive methods currently used are serological typing of HLA class I genes, mixed lymphocyte culture (MLC), IEF and molecular genotyping of HLA class II genes by direct sequencing of PCR products. Serological typing of class I antigenes (A, B and C) fails to detect minor differences demonstrated by direct sequencing of DNA polymorphic regions. Molecular genotyping of HLA class I genes by DNA analysis is costly and work-intensive. To improve compatibility between donor and recipient, we have set up a new rapid and non-radioisotopic application of the 'fingerprinting PCR' technique for the analysis of the polymorphic second exon of the HLA class I A, B and C genes. This technique is based on the formation of specific patterns (PCR fingerprints) of homoduplexes and heteroduplexes between heterologous amplified DNA sequences. After an electrophoretic run on non-denaturing polyacrylamide gel, different HLA class I types give allele-specific banding patterns. HLA class I matching is performed, after the gel has been soaked in ethidium bromide or silver-stained, by visual comparison of patients' fingerprints with those of donors. Identity can be confirmed by mixing donor and recipient DNAs in an amplification cross-match. To assess the technique, 10 normal samples, 22 related allogeneic bone marrow transplanted pairs and 10 unrelated HLA-A and HLA-B serologically matched patient-donor pairs were analysed for HLA class I polymorphic regions. In all the related pairs and in 1/10 unrelated pairs, matched donor-recipient patterns were identified. This new application of PCR fingerprinting may confirm the HLA class I serological selection of unrelated marrow donors.

Abstract 805FN2 Background. Achievement of CR is generally associated with improved clinical outcomes for patients (pts) with MM and represents a primary endpoint of current clinical trials. The GIMEMA Italian Myeloma Network designed a phase 3 study to demonstrate that the triplet VTD regimen was superior over a doublet such as thalidomide-dexamethasone (TD) as induction therapy prior to double ASCT for newly diagnosed MM. On an intention-to-treat basis, the rate of complete or near complete response (CR/nCR) was 31% for the 236 pts on VTD induction therapy, while it was 11% (p Methods. For this purpose, in a molecular substudy to the main clinical study we assessed the ability of gene expression profile (GEP) to predict attainment of CR/nCR in 122 pts enrolled in the VTD arm of the study. Their characteristics at baseline, including cytogenetic abnormalities, were comparable with those of the whole population of 236 pts. Highly purified CD138+ plasma cells were obtained at diagnosis from each of these pts and were profiled for gene expression using the Affymetrix U133 Plus2.0 platform. In order to build a low-dimensional signature with optimal performance, genomic data were analyzed with an original algorithm that exploits quadratic discriminant analysis with a bottom-up approach that builds N-gene signatures starting from two-dimensional signatures. Gene models were applied to test datasets to predict achievement of either CR/nCR or less than nCR, and classification performances were validated by a leave-one-out crossvalidation procedure. Results. Thirty four pts out of the 122 (28%) who were included in the present analysis achieved a CR/nCR, while the remaining 88 patients failed this objective. The molecular approach described above allowed to identify several gene signatures among which we choose a 163-gene signature that provided a predictive capability of 79% sensitivity, 87% specificity, 71% positive predictive value (PPV) and 92% negative predictive value (NPV). These expression values were used in an unsupervised hierarchical clustering to stratify the population of 122 profilated pts into 3 well defined subgroups. Seventy nine pts were included in subgroup A, while the remaining 43 pts were included in either subgroup B (n=22) or subgroup C (n=21). Notably, 19 out the 34 CR/nCR pts (56%) clustered in subgroup B, whereas the remaining 15 pts were randomly distributed within subgroup A. Analysis of demographic and disease characteristics of the pts belonging to the 3 major subgroups, revealed that in subgroup B the frequencies of pts carrying del(13q) (78%) or del(17p) (22%) or with an IgA isotype (54%) were significantly higher in comparison with the corresponding values found in subgroup A (47%, 4%, and 10%, respectively) and subgroup C (38%, 10%, and 5%, respectively). In order to obtain a more feasible set of genes predictive of CR/nCR, several smaller signatures originating from the 163-gene signature were further analyzed by means of the same algorithm described above. The best predictive capability was obtained with a 41-gene signature that provided 88% sensitivity, 97% specificity, 91% PPV and 95% NPV. A GeneGo ® network analysis of genes included in the signatures showed that the most relevant network nodes included tumour suppressor genes (FBXW7 and MAD), genes involved in inflammatory response (TREM1 and TLR4) and genes involved in B cell development (IKZF1, IL10 and NFAM1). Genes included in the signatures do not gather in specific chromosomes, thus confirming the absence of bias on selection of signatures genes, potentially due to prevalence of MM typical chromosomal aberrations. Conclusions. GEP analysis of a subgroup of pts who received VTD induction therapy allowed to provide a 41-gene signature that was able to predict attainment of CR/nCR and, conversely, failure to achieve at least nCR in 91% and 95% of cases, respectively. These favorable results might represent a first step towards the possible application of a tailored therapy based on the single patient's genetic background. Supported by: Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants (M.C.) BolognAIL. Disclosures: Bringhen: Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Merck Sharp & Dhome: Membership on an entity's Board of Directors or advisory committees. Offidani:Janssen: Honoraria; Celgene: Honoraria.

The prognostic value of the site of DNA rearrangement within the M-BCR on chromosome 22 or of the type of transcript has been debated in the last years. The majority of the studies do not support the hypothesis of a predictive value of such molecular parameters. Results coming from a multicentric, prospective trial, based on alpha-IFN therapy, seem to indicate a better karyotypic response in 3' rearranged patients. The possibility of evoking a cytotoxic immune response directed towards peptides originating from each of the different BCR/ABL junctions constitute an important challenge for the future.

The radiosensitivity of lymphomas as well as the local targeted delivery of high doses of radiation make radioimmunotherapy (RIT) an attractive therapeutic option. RIT is indeed an underestimated tool. In follicular lymphoma (FL), RIT represents one of the most effective single agents. The two most commonly used radioimmunoconjugates are 90Y-ibritumomab tiuxetan (Zevalin®) and 131I-tositumomab, both based on murine anti-Cd20 antibodies. RIT has demonstrated efficacy in relapsed/refractory FL and was approved for this indication [1]. Subsequently, many trials have evaluated the role of RIT consolidation after initial tumor debulking with chemotherapy or immunochemotherapy [2] and several phase II studies supported these results [3–5]. We now report updated long-term efficacy and toxicity results of a multicenter nonrandomized phase II trial of fludarabine and mitoxantrone plus RIT (fludarabine, mitoxantrone, zevalin trial), demonstrating that this combination was safe and very effective in untreated FL patients.

The cytotoxic effects and the induction of programmed cell death (apoptosis) by Fludarabine (FLU), 2-chlorodeoxyadenosine (2-CdA), and deoxycoformycin (DCF) with/without alpha-interferon (alpha-IFN) were evaluated in vitro against freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells. Cytotoxicity was evaluated according to the soluble tetrazolium/formazan assay. Regarding the cytotoxicity, FLU, 2-CdA, and DCF showed a mean antitumor activity of 45% +/- 3.39 (mean +/- S.D.), 55% +/- 4.72, and 20% +/- 3.16, respectively. alpha-IFN alone showed a mean cytotoxic activity of 10% +/- 2.72. The cytotoxicity of these purine analogues in combination with alpha-IFN was 52% +/- 2.97, 75% +/- 3.41, and 26% +/- 7.09, respectively. We observed a statistically significant increase of cytotoxicity compared to controls in FLU alone (P < 0.05), 2-CdA (P < 0.05), and their combination with alpha-IFN (P < 0.05). Apoptosis was evaluated by electrophoresis gel of DNA oligonucleosomal fragments and by a cytofluorimetric method. Only FLU and 2-CdA activated the apoptosis and DCF showed a minor apoptotic pathway amount. These apoptosis data were confirmed by both gel electrophoresis of DNA and by propidium iodide cytofluorimetric method. FLU and 2-CdA show activity in B-CLL cells by direct cytotoxic action and the induction of cell death by apoptosis; in the future, it would be interesting to utilize these in vitro assays in monitoring chemosensitivity and predicting response for the clinical use.

Summary. By applying direct immunofluorescence with a dual-staining technique we were able to demonstrate that 20/37 (54%) patients with acute lymphoblastic leukaemia (ALL) expressed both lymphoid and myeloid antigens on the same leukaemic cells. CD13 and CD33 myeloid antigens were detected in 18/20 and in 15/20 cases respectively, and both in 13. Molecular studies confirmed that the four patients with T-cell phenotype had molecular rearrangement of T-cell receptor (TcR) β chain, and 26 ALL patients with ‘B-cell’phenotype showed JH rearrangement. Two ALL patients without (ALL/My-) and three with myeloid antigens (ALL/My+) also demonstrated bcr/abl rearrangement. Both groups of patients had similar presenting features such as age, sex, Hb level, white blood cells, platelet counts and cytogenetic features. Complete response was achieved in 16/17 (94%) ALL/My- patients and in 15/18 (83%) ALL/My+ patients (two deaths occurred during induction) with a mean duration of 17 and 16 months respectively and with similar survival and event-free survival curves. Myeloid antigen expression in adult ALL patients may occur more frequently than previously reported. The presence of myeloid antigen does not identify, in our series, a higher-risk subgroup of patients, although lack of any statistical evidence of prognostic significance needs to be confirmed in a larger case study.

Abstract 5123 Vertebral compression fractures occur in approximately 60% of MM patients and can cause pain, persistent disability and dismail quality of life. Appropriate therapy of MM or radiotherapy can lead to improvement of symptoms in a significant percentage of patients, but these positive effects can take time to be perceived. Vertebral agumentation techniques have been recently proposed as suitable options to relieve bone pain from vertebral compression fractures in patients with benign osteoporosis or neoplastic diseases such as MM. Aim of this study was to analyze the clinical course and outcome of 40 consecutive MM patients (23M, 17F, median age = 67.6yrs) treated in the Centers referring to GER, who underwent percutaneous vertebroplasty from 2006 to 2010. Seventeen patients (43%) were newly diagnosed while 23 patients were relapsed or refractory after 1–3 lines of therapy. All the patients were treated because of severe pain, the extent of vertebral fractures was assessed by nuclear magnetic resonance imaging. Sixty-nine procedures were performed at C2-L5 levels, 51% of the patients were treated at a single level, a maximum of three levels were treated in 6 patients, 13 procedures (32%) were performed at L1 level. Thirty seven patients (92%) experienced reduction of pain, with 55% showing complete disapperance of symptoms prior to any further treatment, 3 patients reported no or little improvement. Responses were durable, after a median follow-up of 14 months no further collapse of the treated vertebrae was observed. After vertebroplasty, first line or salvage therapy was administered to 35 patients, 10 newly diagnosed patients were scheduled to receive autologous stem cell transplant, and peripheral blood stem cell collection was not affected by the procedure. In conclusion, percutaneous vertebroplasty appears to be useful in MM patients with painful vertebral fractures as it allows rapid and durable achievement of pain control, without interfering with further therapeutic programs Work supported in part by RiminiAIL Disclosures: No relevant conflicts of interest to declare.

Abstract 112 BCR-ABL kinase domain (KD) mutation analysis may be an useful tool for physicians and is being performed in a growing number of laboratories. Recommendations aimed to rationalize the use of mutation testing in chronic myeloid leukemia (CML) have recently (Blood 2011) been compiled by a panel of experts appointed by European LeukemiaNet (ELN) – including specific recommendations as to when mutation analysis should be performed. They came from the expert opinion of the panel members whenever published data were insufficient or contradictory. In order to provide further data to validate or refine these recommendations, we have analyzed the GIMEMA CML WP database recording the results of mutation analyses performed in CML pts (n=1301) receiving imatinib and/or 2nd generation TKIs between January 2004 and July 2011. At dagnosis, mutation analysis was recognized to be useful in the few pts who present in accelerated phase or blast crisis (BC), while it was not recommended in chronic phase (CP) pts. Interrogating our database, we could retrieve 58 mutation analyses in newly diagnosed pts in CP and 12 in newly diagnosed pts in BC. Imatinib-resistant mutations were detected in 0 and 2 pts, respectively. In pts receiving 1st-line imatinib, mutation analysis was recommended both in case of failure and in case of suboptimal response. We have analyzed 399 chronic phase (CP) CML pts receiving first-line imatinib because they were found to meet one of the criteria for failure or suboptimal response. Overall, 45/166 (27.1%) failures were found to be positive for one or more BCR-ABL KD mutations. In particular, mutations were detected in 3/16 (18.8%) pts with less than CHR at 3 months, 1/9 (11.1%) pts with no CyR at 6 months, 4/24 (16.7%) pts with less than PCyR at 12 months, 6/36 (16.7%) pts with less than CCyR at 18 months, 15/49 (30.6%) pts who lost CCyR and 16/32 (50%) pts who lost CHR. More interestingly, only 11/233 (4.7%) suboptimal responders we analyzed were positive for mutations. Among ‘cytogenetic' suboptimal responders, mutations were detected in 1/15 (6.7%) pts with no CyR at 3 months, 1/20 (5.0%) pts with less than PCyR at 6 months, 5/51 (8.2%) pts with less than CCyR at 12 months. Among ‘molecular' suboptimal responders, mutations were detected in 0/52 pts with less than MMR (but having achieved CCyR) at 18 months and in 4/95 (4.2%) pts who lost MMR (but not CCyR). Which rise in Bcr-Abl transcript level should trigger a mutation analysis was the most difficult issue to provide recommendations upon, given the lack of convincing and reproducible data in the literature. It was finally agreed to recommend mutation analysis only in case of MMR loss. In 159 of the CP pts we have analyzed, mutation analysis was specifically requested because of a transcript increase at a single RQ-PCR assessment: 29 pts had less than 1-log increase and 41 pts had a 1-log increase or more – but with no loss of MMR. None of these pts was found to have mutations. Another 36 pts had less than 1-log increase and 53 had a 1-log increase or more, leading to loss of MMR. Mutations were detected in 1 (2.8%) and 3 (5.7%) pts, respectively. In pts receiving dasatinib or nilotinib as 2nd-line agents, mutation analysis was recommended at baseline and then in case of failure according to the provisional definitions proposed by Baccarani et al (J Clin Oncol 2009). Nineteen among the pts we analyzed met these criteria; overall, mutations were detected in 11 (57.8%), including 5/7 pts with no CyR at 3 months, 6/9 pts with minimal CyR at 6 months, 1/4 pts with less than PCyR at 12 months. In addition, newly acquired mutations were detected in 93/131 (71%) pts who lost a previously achieved HR or CyR. We also tested 19 pts who met the provisional definitions for suboptimal response to dasatinib or nilotinib 2nd-line. Mutations were detected in 4/19 pts (21%), including 2/5 pts with minor CyR at 3 months, 1/7 pts with PCyR at 6 months, 1/7 pts with less than MMR at 12 months. Our data indicate that: a) pts harbouring mutations can more frequently be found among cytogenetic suboptimal responders than among molecular suboptimal responders; b) any Bcr-Abl transcript increase that is not associated with MMR loss shouldn't indeed trigger a mutation analysis; c) although definitions of response to dasatinib or nilotinib 2nd-line are still provisional and might soon be refined, not only failures but also suboptimal responses are frequently associated with mutations. Supported by AIL, AIRC, PRIN and FIRB. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Martinelli:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

We retrospectively analyzed the data of 337 patients with cytogenetically normal (CN) acute myeloid leukemia (AML), aged ≤ 65 years (training set). A prognostic index score (PIS) was calculated by totaling the score derived from the regression coefficients of each clinical variable, significantly associated with prognosis by multivariate analysis. The variables that were independent prognostic factors for event-free survival (EFS) and overall survival (OS) in the training set were: age ≥ 50 years, secondary AML and white blood cell count (WBC) ≥ 20 × 10(9)/L. The patients of the training set were stratified into three groups: low-, intermediate- and high-risk. The median EFS was 25, 12 and 7 months in the low-, intermediate- and high-risk groups (p < 0.0001), respectively. The median OS was not reached in the low-risk group and was 19 and 10 months in the intermediate- and high-risk groups (p < 0.0001). This PIS was validated in a series of 193 patients with CN-AML. The median EFS was 66, 16, and 3 months (p < 0.0001) and the median OS was 66, 16, and 5 months in the three risk groups, respectively (p < 0.0001). This PIS may be useful for clinical decision-making in CN-AML and may be prospectively integrated with the newest biological markers which at present are not routinely assessed and need prognostic validation.

Abstract 2756 Background: Nilotinib is a potent and selective BCR-ABL inhibitor. The phase 3 ENESTnd trial demonstrated superior efficacy nilotinib vs. imatinib, with higher and faster molecular responses. After 24 months, the rates of progression to accelerated-blastic phase (ABP) were 0.7% and 1.1% with nilotinib 300mg and 400mg BID, respectively, significantly lower compared to imatinib (4.2%). Nilotinib has been approved for the frontline treatment of Ph+ CML. With imatinib 400mg (IRIS trial), the rate of any event and of progression to ABP were higher during the first 3–4 years. Consequently, a confirmation of the durability of responses to nilotinib beyond 3 years is extremely relevant. Aims: To evaluate the long term outcome of patients treated with nilotinib 400mg BID as frontline therapy. Methods: A multicentre phase 2 trial was conducted by the GIMEMA CML WP (ClinicalTrials.gov.NCT00481052). Median 48-month follow-up data for all patients will be presented. Definitions: MR3.0 (Major Molecular Response) as a BCR-ABL/ABL ratio Results: 73 patients enrolled: median age 51 years; 45% low, 41% intermediate and 14% high Sokal risk. The cumulative incidence of CCgR at 12 months was 100%. CCgR at each milestone: 78%, 96%, 96%, 95%, 92% at 3, 6, 12, 18 and 24 months, respectively. The overall estimated probability of MR3.0 was 99%, while the rates of MR3.0 at 3, 6, 12, 18 and 24 months were 52%, 66%, 85%, 81% and 82%, respectively. Two out of 73 patients never achieved a MR3.0, 1 who progressed to AP/BP (see below) and 1 in stable and confirmed CCgR at 36 months. Three pts had a confirmed loss of MR3.0 due to low adherence (all 3 still on nilotinib). The overall estimated probability of MR4.0 was 79%, while the rates of MR4.0 at 12, 24 and 36 months were 12%, 27% and 25%, respectively. One third (21/73 pts) showed a stable MR4.0 (defined based on 3 consecutive MR4.0 samples 4 months apart). Only one patient progressed at 6 months to ABP and subsequently died (high Sokal risk, T315I mutation). Adverse events were mostly grade 1 or 2 and manageable with appropriate dose adaptations. During the first 12 months, the mean daily dose was 600–800mg in 74% of patients. The nilotinib last daily dose was as follows: 800mg in 46 (63%) patients, 600mg in 3 (4%) patients and 400mg in 18 (25%), 6 permanent discontinuations. Detail of discontinuation: 1 patient progressed to ABP; 3 patients had recurrent episodes of amylase and/or lipase increase (no pancreatitis); 1 patient had atrial fibrillation (unrelated to study drug) and 1 patient died after 32 months of mental deterioration and starvation (unrelated to study drug). Two patients are currently on imatinib second-line and 2 on dasatinib third-line. With a median follow-up of 39 months, the estimated probability of overall survival, progression-free survival and failure-free survival was 97%, the estimated probability of event-free survival was 91%. Conclusions: The rate of failures was very low during the first 3 years. Responses remain stable. The high rates of responses achieved during the first 12–18 months are being translated into optimal outcome for most of patients. Acknowledgments: European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures: Gugliotta: Novartis: Honoraria; Bristol-Myers-Squibb: Honoraria. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Cuneo:Roche: Consultancy, Speakers Bureau. Soverini:Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Summary. The prognostic value of the location of the breakpoint on chromosome 22 in patients with Ph 1+ chronic myeloid leukaemia (CML) is still controversial. We analysed both DNA rearrangement and transcript type in a new continuous series of CML patients. By Southern blotting analysis, we found that, out of 72 patients, 43 had a 5’rearrangement and 29 a 3’one, of the 43 5′-rearranged patients, 35 carried an a2b2 transcript and eight an a2b3 one, while, of the 29 patients rearranged in the 3’part of the M-BCR area, 26 had an a2b3 transcript, one had an a2b2 transcript and two carried both types of transcript. Thus, mRNA studies allow to detect an a2b3 transcript in 17·7% of 5’rearranged patients. However, no correlation was observed between type of transcript and survival, as after DNA studies.

An 8;14 chromosome translocation with breakpoints at q11 and q32, respectively, is described as the sole abnormality in bone marrow cells of two adult patients with common acute lymphoblastic leukemia (ALL). The Southern blot analysis revealed a rearrangement after BamHI and HindIII digestion and hybridization with a JH probe, thus demonstrating the involvement of the gene coding for the heavy chains of the immunoglobulins (IgH). Therefore, a pathogenetic mechanism similar to that observed in Burkitt's lymphoma and its variants, or in other lymphomas with t(11;14) or t(14;18), may be hypothesized. In all these cases IgH is juxtaposed to an oncogene (c-MYC, BCL-1, and BCL-2, respectively). A similar structure, with oncogene type potential, could be present on 8q11. The patients underwent a complete remission after induction therapy.

We studied 36 DNA samples of 18 patients affected with chronic myeloid leukemia (CML) for the presence of mutations in the first exon of the BCR gene was divided into four regions amplified by polymerase chain reaction (PCR). By single strand conformation polymorphism analysis (SSCP) and direct sequencing of amplified fragments, we found different banding profiles in 9 out of 18 patients in the PCR fragment spanning nucleotide 506-826. In one patient, sequence analysis revealed the presence of a point mutation at nucleotide 669 (A-T; Gln-Leu). No difference was found between DNA samples collected during the chronic phase and the blastic transformation. No different mobility shifts of single stranded PCR products were found in the other amplified fragments. The activation of BCR-ABL involves direct interaction between BCR first exon sequences and the tyrosine kinase regulatory domains of ABL. In the first BCR exon, and around the mutated sequences two SH-2-binding sites, are retained. These domains are essential for BCR-ABL-mediated transformation. Our results demonstrate the presence of point mutation in this regulatory region, which may suggest a role for the altered BCR sequence in activation of the BCR-ABL oncogene.

Abstract 3413 Introduction Imatinib mesylate is highly effective in inducing rapid hematologic and cytogenetic responses in the vast majority of chronic myeloid leukemia (CML) patients. Yet, discontinuation of treatment is associated with disease relapse probably due to the persistence of resistant leukemic stem cells representing a reservoir of the disease. On this regard it has been reported that BCR-ABL positive progenitor cells can still be detected in patients in complete cytogenetic response (CCyR) after short term of imatinib treatment (Bhatia R, et al. Blood. 2003;101:4701-4707) but also after a stable long lasting CCyR (Bocchia M, et al. Leukemia. 2008;22:426-428). Compared to imatinib, the second generation Tyrosin Kinase Inhibitor (TKI) nilotinib appears to eradicate more rapidly the bulk of CML cells, inducing high rate of CCyR and major molecular response (MMolR) after a very short period of treatment (78% CCyR and 52% MMolR at 3 months) (Rosti G, et al. Blood. 2009;114(24):4933-8). Despite nilotinib is a more potent TKI, it didn't appear to be more effective in eliminating in vitro CML progenitors than imatinib (Konig H, et al Leukemia. 2008;22:748-755). Up to date no data evaluating the persistence of Ph+ stem cells in early chronic phase CML patients during first line treatment with nilotinib have been reported. Patients, materials and methods We investigated the presence of residual CD34+/Ph+ cells in 24 CML patients in CCyR during first line nilotinib treatment. Patients were enrolled in 2 clinical trials (GIMEMA CML0307 and CAMN107A2303) and evaluation of residual leukemic stem cells was performed during a routine bone marrow aspirate after receiving specific patients informed consent. Bone marrow purified CD34+ cells were evaluated for BCR-ABL fusion gene by fluorescent in situ hybridization (FISH) analysis. A minimum of 100 interphase nuclei of purified CD34+ cells was considered optimal for FISH analysis. Results All 24 patients have been treated exclusively with nilotinib since diagnosis (17/24 at 400mg bid; 5/24 at 300mg bid; 2/24 at 400mg/day). At the time of analysis all 24 patients were in CCyR for a median time of 27 months (range 6–29) after being treated for a median time of 30 months (range 9–30) with this second generation TKI. Regarding molecular response 20/24 (83%) were in MMolR while only 1/24 (4%) was in CMolR. Harvest, purification and subsequent FISH analysis of bone marrow CD34+ cells was optimal in 15/24 (63%) patients, suboptimal in 5/24 (21%) patients (less than 100 interphase nuclei analyzed) and not adequate in 4/24 (16%) patients (less than 50 interphase nuclei). With respect to leukemic stem cells, residual CD34+/Ph+ cells were found only in 1/20 (5%) evaluable patients. Of note, in this patient 140 CD34+ interphase nuclei were analyzed and only 1 was found bcr-abl positive (0.7%). Conclusion Our study shows for the first time that in patients in CCyR during front line Nilotinib treatment residual CD34+/Ph+ stem cells are very rarely detected. These results quite differ from what was previously found in imatinib treated patients (Bocchia M, et al. Leukemia. 2008;22:426-428). In fact in the present series, only 1/20 (5%) patients treated with nilotinib in CCyR for a median time of 27 months showed residual CD34+/Ph+ cells, while in our prior study residual leukemic CD34+ cells were still detectable in 14/31 (45%) imatinib treated patients in stable CCyR (median of 39 months). Despite the limited number of patients studied, this novel evidence may support the better short term clinical results observed with nilotinib as first line treatment in CML. Disclosures: Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

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

Abstract 2273 Background: While Imatinib (IM) has revolutionized treatment for chronic myeloid leukemia (CML), demonstrating outstanding survival figures, currently no data exist on mid to long term impact of disease burden and therapy from the patients’ perspective. Aim: The main objective of this study is to identify specific limitations of quality of life (QoL) in CML survivors who are undergoing first line treatment with IM in comparison with controls from the general population. Patient-reported symptom prevalence was also investigated. Patients and methods: Patients were recruited in 26 centers, randomly selected to geographically represent the whole study country. Patients selection criteria included: being in treatment with IM for at least three years and being in complete cytogenetic response at the time of study entry. All patients were invited by their treating physicians in the hospital to participate and all consenting patients were requested to complete a Health Survey Packet at home. Pre-paid reply envelopes were also provided with the request to send back completed Surveys to an independent National coordinating Data Center. Generic QoL was assessed with the SF-36 that consists of 36 items covering eight generic health status/QoL domains: physical functioning (PF), role limitations due to physical health (RP), bodily pain (BP), general health perceptions (GH), vitality (VT), social functioning (SF), role limitations due to emotional problems (RE), and mental health (MH). All scales ranging between 0 and 100 with higher scores representing better outcomes. A previously devised patient-reported CML Symptom Checklist was used to investigate 9 symptoms of possible major concern in these patients. Mean SF-36 scores were compared to available national general population reference values and all analyses were adjusted for age and gender. Statistical comparisons were all adjusted for multiple testing. Differences in mean scores were expressed in Cohen effect sizes (ES; with 0.2, 0.5, and 0.8 indicating small, medium, and large ES, respectively) and clinical significance. Results: Between March and December 2009, 448 patients were recruited in a large national-based survivorship project. Patients’ compliance was optimal with 94% of patients (N=421) returning a valid Health Survey Packet to the National coordinating Data center. At study participation, mean age of patients was 56 years (59% male and 41% female) and median time of IM therapy was 5 years. Seventy-seven percent of patients were receiving standard dose of 400 mg and 43% had at least one comorbidity. Age and gender adjusted comparisons with general population norms revealed worse outcomes for the following scales: RP (P75 years) suggested an almost uniform pattern in all scales with worse outcomes between CML patients and population controls among the youngest groups. GH was significantly worse in younger patients (55-64) (P=.03; ES=0.4) and no differences were found in the older age groups compared with population norms. Prevalence of reported symptoms (with any level of concern) was: fatigue (82%); problems with muscular cramps (78%); problems with musculoskeletal pain (72%); problems with edema (70%); skin problems (47%); diarrhea (43%); headache (39%); abdominal discomfort and nausea (28%). Conclusion: This study suggests that while still being on treatment with IM for years, CML patients might expect to have a QoL profile broadly similar to that of general population in many areas. However, role limitations (i.e., in work or other regular daily activities) due to physical health seem the major constraint faced by these patients; there is also an indication that younger patients might be those experiencing major limitations. Additional analyses will be undertaken to ascertain the impact of symptoms and other laboratory and clinical data on specific QoL domains. Such unique patient-reported data supplements conventional information on clinical efficacy of IM and may support both clinicians and patients in making more informed treatment decisions in this area. Disclosures: Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol M. Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

Abstract 359 Background: Nilotinib is a potent and selective inhibitor of BCR-ABL. In the phase 3 ENESTnd trial, nilotinib demonstrated superior efficacy to imatinib with higher and faster molecular responses. With a median follow-up of 18.5 months (ASCO/EHA 2010), the rates of progression to accelerated or blast phase (AP/BC) were 0.7% and 0.4% with nilotinib 300 mg and 400 mg BID, respectively, and significantly lower in comparison to imatinib (4.2% P = .006 and .003, respectively). Based on the results of the ENESTnd trial, nilotinib has been approved (FDA) for the frontline treatment of Ph+ CML. With imatinib 400 mg (IRIS trial), the rate of any event and the rate of progression to AP/BC were higher during the first 3 years on treatment (15.6% and 6.1%, respectively). Consequently, a confirmation of the durability of nilotinib responses at 3 years is extremely important. Aims: To evaluate responses (either cytogenetic and molecular) and to investigate outcomes of patients treated for 3-years with nilotinib 400 mg BID as frontline therapy. Outcomes evaluated include Overall Survival (OS), Progression-Free Survival (PFS), Failure-Free Survival (FFS) and Event-Free Survival (EFS). Method: A multicentre phase 2 trial (nilotinib 400 mg BID) was conducted by the GIMEMA CML Working Party (ClinicalTrials.gov.NCT00481052). The median follow-up is currently 30 months (3 years by November 2010). Definitions: Major Molecular Response (MMR): BCR-ABL/ABL ratio < 0,1% IS; Complete Molecular Response (CMR): undetectable transcript levels and nested PCR negative; failures: no CHR at 3 months, no CgR at 6 months, no PCgR at 1 year, no CCgR at 18 months, loss CHR or CCgR, progression and death (according to the revised European LeukemiaNet recommendations); events: failures and treatment discontinuation for any reason. All the analysis has been made according to the intention-to-treat principle. Result: 73 patients have been enrolled; median age 51 years (range 18–83); 45% low, 41% intermediate and 14% high Sokal risk. The cumulative CCgR rate (primary endpoint) at 12 months was 100%. CCgR at each milestone: 78% at 3 months, 96% at 6, 12 and 18 months, 92% at 24 months. The cumulative rate of MMR was 96%, while the rates of MMR at 3, 6, 12, 18 and 24 months were 52%, 66%, 85%, 81% and 82%, respectively. The cumulative rate of CMR was 41%, while the rates of CMR at 12 and 24 months were 7% and 12%, respectively. None of the patients who achieved a MMR progressed to AP/BC. Only one patient progressed at 6 months to AP/BC: a 63 years old female with a high Sokal risk disease in CCgR at 3 months, who developed a T315I mutation. During the first 12 months, the mean daily dose was 600–800 mg, 400–599 mg, and less than 400 mg in 74%, 18% and 8% of patients, respectively. The nilotinib last daily dose was as follows: 800 mg in 48 (71%) patients, 400 mg in 19 (28%) patients and 200 mg in 1 (1%) patient. Adverse events (AEs) were mostly grade 1 or 2 and manageable with appropriate dose adaptations. Two patients (3%) showed a prolongation of the QTcF above 450 msec (none above 50 msec). Four events lead to permanent discontinuation of nilotinib: 3 patients discontinued after 9, 15 and 27 months on treatment for recurrent episodes of amylase and/or lipase increase (no pancreatitis) and 1 patient after 25 months due to atrial fibrillation, unrelated to study drug. Three of them are currently on imatinib second-line and 1 on dasatinib third-line. Overall, 5 events have been recorded so far (1 progression to AB/BC and 4 permanent discontinuation of nilotinib due to AEs). At 30 months the OS, PFS and FFS are 99% and the EFS is 92%. Conclusion: The rate of failures was very low during the first 3 years. Responses remain stable. The very high rates of responses achieved during the first 12 months on treatment are being translated into optimal outcome for most of the patients. Acknowledgments: European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures: Rosti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Gugliotta:Novartis: Honoraria. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; pfizer: Consultancy. Baccarani:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Research Funding; Wyeth: Consultancy, Research Funding.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P < .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P < .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.

A simple prognostic classification of Ph+ chronic myeloid leukemia (CML) was proposed in 1984 by an international study Group [23] and is now widely used for clinical purposes. That study was retrospective, and was based on disease features at diagnosis. To test prospectively and to check the value of the classification, and to investigate if additional information on the early course of leukemia can help in refining the prognosis, 505 Ph+, nonblastic and nontransplanted patients first registered between 1984 and 1986 were followed up to June 1990. It was found that the prognostic formulation predicted survival in that series exactly the same way as in the original series (median survival: > 60 months for low-risk patients, 46 months for intermediate-risk patients, and 32 months for high-risk ones). It was also found that several objective or subjective assessments of disease course during the first 8 months after diagnosis were significantly related to survival length within any risk group. This study provides full confirmation of Sokal's international prognostic classification, and shows that the definition of prognosis can be improved some months after diagnosis by taking into account the course of the disease and the response to therapy. These conclusions apply to patients receiving conventional treatment. The course of Ph+ chronic myeloid leukemia (CML) is rigidly programmed to progress to an acute phase, which is called blastic metamorphosis (BM) and which can develop either suddenly (blastic crisis) or by a slow progression (accelerated phase) [7,24,29]. In all recent series, 2-year survival ranged between 65% and 80%, median survival was slightly shorter than 4 years, and the proportion of patients who were still alive after 10 years was less than 10% [24,29]. However, a number of prognostic variables were identified [2,8,9,12,15,16,19,23, 28] and a prognostic formulation that was claborated by an international study [23] was shown to provide a reliable estimate of survival length and is currently used for that purpose. The value of that formulation, and the formulation itself, require periodical controls and revisions. For that purpose, the Italian Cooperative Study Group on CML registered and followed up all CML patients who were first seen between 1984 and 1986. A very preliminary analysis of that cohort of patients has already been performed, but it was limited to the first 2 years after diagnosis [10]. This report extends prior findings, confirming the validity of the formula up to the 5th year after diagnosis, and shows that the definition of prognosis can be significantly improved by taking into account dynamic clements which reflect the course of the disease and the response to conventional treatment during the first 8 months after diagnosis.

Summary Background Follicular lymphoma is the most common form of lymphoma in Europe and the USA. In this prospective, single-arm, open-labelled, multicentre non-randomised phase II trial (FLUMIZ [FLUdarabine, MItoxantrone, Zevalin] trial) we aimed to assess the efficacy and safety of fludarabine and mitoxantrone plus radioimmunotherapy in untreated patients with follicular non-Hodgkin lymphoma (NHL). Methods Patients with stage III or IV untreated indolent follicular NHL were enrolled between June 1, 2004, and April 15, 2006, at 13 Italian institutions, and were treated with oral fludarabine (40 mg/m 2 on days 1 to 3) and intravenous mitoxantrone (10 mg/m 2 on day 1) every 28 days for six cycles. Patients who had at least a partial response (PR) with normal platelet counts (>100×10 9 /L) and granulocyte counts (1·5×10 9 /L), and bone-marrow infiltration less than 25% 4–6 weeks after completion of the sixth cycle of chemotherapy were deemed eligible for consolidation treatment 6–10 weeks after the sixth cycle with one course of yttrium-90 ( 90 Y)-labelled ibritumomab tiuxetan (Zevalin), which consisted of an initial infusion of intravenous rituximab (250 mg/m 2 ) on day 1 followed by a second 250 mg/m 2 infusion on day 7, 8, or 9. The second infusion was followed by a weight-based dose of 90 Y-ibritumomab tiuxetan, administered as a slow intravenous push over 10 min. Primary endpoints were complete response (CR) and haematological toxic effects and secondary endpoints were overall survival and progression-free survival. Responses were classified according to the International Workshop for Response Criteria for non-Hodgkin's lymphomas. Analysis was per protocol. This trial is registered as a European Standard Controlled Trial on the EudraCT website http://oss-sper-clin.agenziafarmaco.it, number 2004-002211-92. Findings 61 patients were enrolled in the trial and received six cycles of fludarabine and mitoxantrone, after which an overall response was noted in 98% (60 of 61) of patients (43 of 61 patients had CR and 17 of 61 patients had PR). 57 patients (43 with CR and 14 with PR) were deemed eligible for subsequent 90 Y-ibritumomab tiuxetan. Of the 14 patients who had PR after the initial treatment, 12 obtained CR after 90 Y-ibritumomab tiuxetan. By the end of the entire treatment regimen 55 of 57 patients achieved CR. With a median follow-up of 30 months (range 21–48), 3-year progression-free survival was estimated to be 76% (95% CI 72·3–82·4) and 3-year overall survival 100%. 36 of 57 patients had grade 3 or 4 haematological toxic effects, and blood transfusions were given to 21 of 57 patients. Interpretation This trial has provided evidence for the feasibility, tolerability, and efficacy of fludarabine and mitoxantrone plus 90 Y-ibritumomab tiuxetan in untreated patients with follicular NHL. Funding Italian Association for Leukaemias, Lymphomas, and Myeloma, Bologna, Italy.

Over the last few years, Thalidomide-Dexamethsone (TD) has been one of the most commonly used induction regimens for the treatment of newly diagnosed multiple myeloma (MM). In a phase III study conducted by the Italian Myeloma Network GIMEMA, TD was compared with Velcade-Thalidomide-Dexamethasone (VTD) as induction therapy in preparation for, and as consolidation after, melphalan (200 mg/m2)-based double autologous stem-cell transplantation (ASCT) in pts aged ≤65 years with symptomatic MM. Up-front VTD comprised Velcade, 1.3 mg/m2 on d 1, 4, 8, and 11; Dexamethasone, 40 mg on each day of and after Velcade administration; Thalidomide, 200 mg/d through d 1 to 63. Pts randomized to TD received Thalidomide as in VTD and Dexamethasone, 40 mg/d on d 1–4 and 9–12 of every cycle. Primary study end point was the rate of complete response (CR)/near CR (nCR) following three 21-d cycles of induction therapy. The study was started in May 2006 and was closed to accrual in April 2008, after a total of 480 pts were enrolled. Of these, 399 pts (199 randomized to VTD and 200 to TD) could be evaluated for primary study end point and toxicity of induction regimens (secondary end point). All analyses were intent to treat. In comparison with TD, VTD effected significantly higher rates of response (≥partial response: 78.5% vs 92%, P

Objectives: Infections are the major cause of morbidity and mortality in patients with acute myeloid leukaemia (AML). They primarily occur during the first course of induction chemotherapy and may increase the risk of leukaemia relapse, due to a significant delay in consolidation therapy. The intensification of induction chemotherapy and the use of non-conventional drugs such as fludarabine are considered responsible for the increased risk of infections. Methods: In this study, we retrospectively analysed the infections occurred in 224 newly diagnosed AML patients ≤65 yr, consecutively treated between 1997 and 2002 with an induction regimen including fludarabine, arabinosyl cytosine and idarubicin, with or without etoposide (FLAI/FLAIE), in the context of three multicentric prospective trials (AML97, AML99, AML02). Results: During the induction phase, 146 (65%) patients experienced fever of undetermined origin (FUO), 30 (13%) and 47 (21%) patients had Gram-negative and positive bacteremias, respectively, and 10 (4%) patients developed a probable/proven invasive fungal infection (IFI). The fatality rate for Gram-negative, Gram-positive bacteremias and probable/proven IFI was 10%, 8% and 60% respectively. During consolidation, 75 (35%) patients had FUO, 43 (20%) and 40 (19%) patients had Gram-negative and positive bacteremias, respectively, and 5 (2%) patients developed a probable/proven IFI. The fatality rate for Gram-negative, Gram-positive bacteremias and probable/proven IFI was 14%, 5% and 80% respectively. Interestingly, the overall incidence of microbiologically documented infections during induction was 38% and the incidence of probable/proven IFIs during the induction/consolidation programme was 7%. No infections caused by viruses or opportunistic pathogens were observed neither during induction, nor during consolidation. Conclusions: These data, although retrospectively collected, suggest that fludarabine-based chemotherapy is not associated with an increased incidence of infections, in particular IFIs, compared to conventional regimens commonly used for AML induction.

This randomized phase III clinical trial explored the efficacy of DaunoXome (DNX) versus Daunorubicin (DNR) in acute myeloid leukaemia (AML) patients aged >60 years. Three hundred and one AML patients were randomized to receive DNR (45 mg/m(2) days 1-3) or DNX (80 mg/m(2) days 1-3) plus cytarabine (AraC; 100 mg/m(2) days 1-7). Patients in complete remission (CR) received a course of the same drugs as consolidation and then were randomized for maintenance with AraC+ all trans retinoic acid or no further treatment. Among 153 patients in the DNR arm, 78 (51.0%) achieved CR, 55 (35.9%) were resistant and 20 (13.1%) died during induction. Among 148 patients in the DNX arm, 73 (49.3%) achieved CR, 47 (31.8%) were resistant and 28 (18.9%) died during induction. Univariate analysis showed no difference as to induction results. After CR, DNX showed a higher incidence of early deaths (12.5% vs. 2.6% at 6 months, P = 0.053) but a lower incidence of relapse beyond 6 months (59% vs. 78% at 24 months, P = 0.064), with a cross in overall survival (OS) and disease-free survival (DFS) curves and a later advantage for DNX arm after 12 months from diagnosis. DNX seems to improve OS and DFS in the long-term follow-up, because of a reduction in late relapses.

Imatinib (IM) 400 mg daily is the standard treatment for chronic myeloid leukemia in early chronic phase (ECP): the results of the IRIS trial have shown a 72 months overall survival of 95%; EFS and PFS were 83% and 93%, respectively; the cumulative rate of complete cytogenetic response (CCgR) for the IM 400 mg arm was 25% at 3 months (at 6, 12, 18 and 60 months it was 51%, 69%, 76% and 87%, respectively). Nilotinib, a second generation TKI, has a higher binding affinity and selectivity for Abl with respect to IM, being 20 to 50 times more active in IM-sensitive cell lines and is highly effective in IM resistant patients, across every disease phase. To investigate the therapeutic efficacy and the safety of nilotinib 400 mg BID in untreated, ECP, Ph-pos CML patients, the italian GIMEMA CML Working Party is conducting an open-label, single stage, multicentric, phase II study trial (ClinicalTrials.gov. NCT00481052); all patients provided written informed consent. The primary endpoint is the CCgR rate at 1 year; the kinetic of molecular response is studied by Q-PCR baseline and after 1, 2, 3, 6, 9 and 12 months from treatment start. PATIENTS Seventy-three patients have been enrolled from 20 Centres between June, 2007 and February, 2008. The median age was 51 years (range 18–83), 45% low, 41% intermediate and 14% high Sokal risk. Median follow-up is currently 210 days (range 68–362). RESULTS All 73 patients and 48/73 (66%) completed 3 and 6 months on treatment, respectively. Response at 3 and 6 months (ITT): the CHR rate was 100% and 98%, the CCgR rate 78% and 96%, respectively. A MMR, defined as a BCR-ABL:ABL ratio < 0.1% according to the International Scale, was achieved by 3% of all treated patients after 1 month on treatment, but this proportion rapidly increased to 22% after 2 months, 59% after 3 months and 74% after 6 months. One patient progressed at 6 months to accelerated-blastic phase with the T315I mutation. NILOTINIB DOSE AND COMPLIANCE No dose escalation was permitted in case of resistance; the median daily average dose was close to the intended dose, 789 mg (range 261 – 800); 34/73 patients (47%) interrupted nilotinib at least once, with a median duration of dose interruption of 15 days (range 2–98). The dose of nilotinib at the last visit was 400 mg BID for 52 patients (71%), 400 mg daily for 20 patients (27%) and 200 mg daily for 1 patient (1%). ADVERSE EVENTS: AEs (grade III/IV) were manageable with appropriate dose adaptations: hematologic toxicity was recorded so far in 4 pts (5% - only 1 event grade IV neutropenia); the most frequent biochemical laboratory abnormalities (grade III) were total bilirubin increase (15%), GOT/GPT increase (11%) and lipase increase (4%). Only 1 episode of grade IV lipase increase was recorded. It is noteworthy, considering the 48 cases with at least 6 months of follow-up, that the incidence of any grade II and III nonhematologic adverse event, decreased from 50% and 8% (first 3 months) to 23% and 6% (second trimester), respectively. ECG monitoring: in 16 patients (22%), transient and not clinically relevant ECG abnormalities have been recorded; 2 more patients (3%) revealed a transient and uneventful QTc prolongation (>450 but CONCLUSIONS: The results that have been achieved in these unselected patients and within a multicentric trial, strongly support the notion that in ECP Ph-pos CML patients both cytogenetic and molecular responses to nilotinib are substantially faster than the responses to IM. Acknowledgments: Work supported by European LeukemiaNet, COFIN, University of Bologna and BolognAIL.

A new and rare type of Bcr/Abl junction between exon C3 of the 3′ portion of the Bcr gene and Abl exon 2 has been identified in the leukemic cells of two Ph1-positive chronic myelogenous leukemia patients in chronic phase. This is the fourth type of Bcr/Abl junction so far identified in Ph1-positive hematologic malignancies and is a consequence of an unusual breakpoint position on chromosome 22 that falls approximately 20 kb downstream of the major breakpoint cluster region (bcr) of the Bcr gene. The new hybrid mRNA is 540 base pairs (bp) longer than that expressed by the K562 cell line and could codify for a Bcr/Abl protein carrying 180 additional aminoacids with respect to the larger P210 protein so far identified. The hematologic phenotype expressed by the two patients carrying this unusual type of Bcr/Abl rearrangement does not significantly differ from that commonly seen in chronic myelogenous leukemia.

Infections are the major cause of morbidity and mortality of acute myeloid leukemia (AML). Invasive Fungal Infections (IFIs) occur in at least 10 to 20% of the patients submitted to induction and consolidation treatments, are responsible for death during induction in up to the 5% of the cases and may cause a delay in consolidation and intensification therapy. Among the risk factors for IFIs it has been included the use of Fludarabine (Fluda), which can induce severe and prolonged immunosuppression. In this study we retrospectively analyzed the infections occurred in 224 newly diagnosed AML patients, aged at least 65 years, consecutively treated with an induction regimen including Fluda, Ara-C and idarubicine with or without etoposide (FLAI/FLAIE). During induction phase, 181/224 (81%) patients experienced a fever of undetermined origin (FUO), the incidence of Gram negative and positive sepsis was 16% (37/224) and 29% (65/224) respectively and 7/224 (3%) patients developed a possible/probable IFI. In 6/224 patients (3%) a proven IFI was found (4 aspergillosis and 2 candidiasis). We then collected the data of the incidence of infections during the first consolidation course (FLAI: n=70; high-dose Ara-C [HD-AC]: n=65; idarubicine and HD-AC: n=89). The overall incidence of FUO was 34% (76/224), the number of Gram negative and positive sepsis was 52/224 (23%) and 49/224 (22%), respectively and 2/224 (1%) patients developed a proven IFI (3 aspergillosis and 1 candidiasis). We subsequently evaluated the incidence of infections in the three different consolidation groups. No significant differences were observed in terms of FUO, Gram positive and negative bacteraemia/sepsis and possible, probable and proven IFIs, during consolidation with Fluda-based regimen and with HD-AC-based regimens. Interestingly, the overall incidence of IFIs during consolidation with FLAI was significantly lower than during consolidation with HD-AC-based treatment program (0% vs 9%; p=0.02). These data, even though retrospectively collected, suggest that Fluda-based chemotherapy is not followed by increased incidence of infections, in particular IFIs, in comparison with conventional non-Fluda based regimens commonly used for AML induction. In our series, Fluda-based consolidation chemotherapy caused a significantly lower incidence of IFIs compared to HD-AC-based consolidation. This may be related to the lower duration of neutropoenia in patients treated with FLAI with respect to those treated with HD-AC/HD-AC + idarubicine.

Five Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) patients with additional chromosome abnormalities at diagnosis have been followed during Imatinib therapy. In all, the Ph chromosome disappeared, while the 5 cases, additional abnormalities [dup(1); del(5), +8 (2 patients) and +14] persisted in the subsequent studies, performed over a period of 11 to 49 months, either alone or together with a karyotypically normal cell population. This finding is consistent with a secondary origin of the Ph chromosome in these patients. It is still to early to evaluate the possible prognostic value of these additional abnormalities.

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

Background: Older (age ≥60 years) patients with acute myeloid leukemia (AML) have poor outcomes and intensive induction chemotherapy is frequently unsuitable. Thus, new safe and effective therapies are urgently needed. Tosedostat, a new, orally bioavailable inhibitor of members of the M1 and M17 classes of aminopeptidases, was proven to be effective in both de novo and relapsed AML. We hypothesize that the addition of tosedostat to cytarabine may improve the response rate and remission duration over what is expected with chemotherapy or tosedostat alone. Methods: This was a phase II, prospective, multicenter study, designed according to Fleming's method. Fixing the lowest acceptable rate as 10% and the successful rate as 25%, with a significance level alpha=0.05 and a power 1-beta =0.80, the sample size was estimated in 33 patients. Thirty-three patients (median age 75 years) received Tosedostat 120 milligrams orally once daily until disease progression, coupled with intermittent low-dose cytarabine given subcutaneously at 20 milligrams twice/day for 10 days. Courses of cytarabine were repeated every 4 weeks in the absence of disease progression or unacceptable toxicity, up to 8 cycles. Global gene expression profiling (GEP, Affymetrix Human Gene 2.0 Array) was performed on purified AML blasts of 29 patients from peripheral blood or bone marrow at diagnosis before treatment initiation. Unsupervised clustering was generated using a hierarchical algorithm based on the average-linkage method. Principal component analysis (PCA) and supervised gene expression analysis was performed by using GeneSpring GX 12.0 (Agilent, USA). Results: The characteristic of enrolled patients are listed in table 1 Induction-period mortality was 12%, with 4 deaths occurring in aplasia. According to intent-to-treat, the CR rate was 48.5% (16/33 patients); 2 additional patients obtained a partial response, for an overall response rate of 54.6%. In addition, 4/33 patients remained in stable disease for a median time of 9 months (range: 4-14). Seven patients did not respond and died with progressive disease after having received a median of 2 cycles of cytarabine and 45 days of tosedostat. In responding patients, the median time to best response was 74 days (range 22-145). Responding patients (CR+PR) had a longer median overall survival than non-responders (P=0.018). Six out of 18 (33%) responding patients are still in CR after a median follow-up of 425.5 days (range 208-758); 5 additional patients are alive with stable disease. Twenty-two patients died [while in aplasia (4), in CR (1), due to resistant disease (9) or due to progressive disease (8) after a median CR duration of 192 days (87-535)]. 29 patients had GEP analysis, and a molecular signature associated with the clinical response (CR vs. no CR) was identified. By supervised analysis, 212 genes differentially expressed based on the clinical response (complete remission (CR) vs no CR) were identified (Mann-Whitney, p2). The 212 genes differentially expressed were significantly associated with six relevant biological functions and pathways: β-catenin (βcat); TNFα signaling pathway via NFκB; ERB2; STK33/SKM (serine/threonine kinase 33 expression using the SKM cell line); inflammatory response; and epithelial-mesenchymal transition pathways. Conclusions: The tosedostat and low-dose cytarabine combo produced a CR rate superior to what expected (45.4% versus 25%), and thus met the primary endpoint of study. Further, potential biomarkers were identified by GEP. Specifically, the achievement of CR could be efficiently predicted by the gene expression patterns with an overall accuracy exceeding 90%. A validation analysis is currently being conducted on additional 14 patients in order to confirm the ability of GEP to identify potential responders to TST. The study was registered at European Union Drug Regulating Authorities Clinical Trials (EudraCT) n.2012-000334-19. Acknowledgments: CTI is gratefully acknowledged for providing Tosedostat for the patients. The study was supported in part by AIL Pesaro Onlus. Table 1. Characteristic N = 33 Median age, years (range) 75 (62-85) Median WBC count, × 109/L (range) 3.05 (0.26-24.53) Blasts, % (range) 60 (20-96) Cytogenetic risk group*, n Not evaluable 3 (9%) Intermediate Karyotype 17 (52%) Unfavourable Karyotype 13 (39%) AML, n De novo 16 (48%) Secondary, n 17 (52%) Disclosures Fanin: Novartis Farma: Speakers Bureau.

Thirty untreated patients, median age 69 years (range 60 - 75 years), with diffuse large B-cell lymphoma (B-DLCL) were treated with a pegylated liposomal doxorubicin (PL-doxorubicin) modified CHOP-rituximab regimen. PL-doxorubicin 30 mg/m2, was given in combination with standard dosage of prednisone, vincristine, cyclophosphamide, rituximab (according to CHOP-R regimen) every 21 days for six courses. Cardiac toxicity was evaluated by mean of echocardiography for left ventricular ejection fraction (LVEF) evaluations and serum troponin-I levels. Overall response and complete response rates were 76% and 59%. Projected two year event free survival and overall survival are 65.5% and 68.5%. No treatment-related mortality was documented. WHO grade III-IV neutropenia and thrombocytopenia were 86% and 3%. Extra-hematological III-IV toxicity was represented, respectively, by a single case of infection, mucositis, and bleeding. LVEF evaluations and the troponin levels did not show significant changes over the course of the treatment. One patient with a previous history of atrial fibrillation experienced a single episode of arrhythmia. None of the patients developed palmar-plantar erythrodysesthesia. This regimen appears an active regimen for the treatment of elderly patients with B-DLCL. The replacement of conventional doxorubicin with PL-doxorubicin seems to be associated with a negligible incidence of extra-hematological toxicity, in particular cardiac and infectious complications.

Hairy cell leukemia (HCL) is a rare B-cell neoplasm highly responsive to purine analogues 2Chlorodeossiadenosine (2CdA) or Desossicoformicin or Interferons as single agents, and only a minority are refractory. Patients who obtain any response (either complete or partial) tend to have survivals as normal healthy subjects and/or will benefit from repeating the treatments in case of relapse. Conversely, the minority of patients who do not respond to one of the drugs often do not respond to the others and have a poor prognosis. We have recently observed that the majority of HCL have mutated VH genes, while a minority have unmutated VH genes. In the most common B-cell neoplasm chronic lymphocytic leukemia (CLL), VH gene status has prognostic impact and correlates with progression, treatment-response and surivival. In the process of identifying prognostic parameters of responsiveness to 2CdA, we prospectively investigated the VH and VL genes expressed by the tumor cells and response to treatment in patients receiving subcutaneous 2CdA. In newly diagnosed HCL requiring treatment, enrolled in an Italian multicenter trial (ICGHCL2004), peripheral blood mononuclear cells were obtained prior to treatment, and the expressed tumor VH and VL transcripts were identified by RT-PCR and cloning. Tumor sequences with > 98% homology to germline VH and VL genes were defined as “unmutated”. Patients received 0.1 mg/kg subcutaneous 2CdA (Litak) for 5 or 7 consecutive days and responses were evaluated by immunohistochemistry of trephine bone marrow biopsies 2 and/or 6 months after the end of treatment. Of 56 patients recruited, 22 patients were evaluable for response. Definition of response was according to consensus resolution criteria. We observed that 19/22 patients responded to subcutaneous 2CdA (15 CR, 4 PR), while 3/22 patients demonstrated refractory or progressive disease, indicating similar efficacy of subcutaneous to intravenous administration. Leukocytosis was observed in 2/3 refractory, but also in 2/21 responsive patients. In one of one patient in CR, molecular remission was also documented in the bone marrow by PCR and capillary electrophoresis. Most remarkably, the 3/3 refractory HCL shared the common feature of expressing unmutated VH and VL genes, in contrast to the responsive patients that all carried mutated VH and/or VL genes. From our series, there are indications that mutational status may relate with tumor burden (leukocytosis) and, more importantly, with response to 2CdA. Overall, the interim data suggest that HCL patients with unmutated VH genes may not benefit from single agent subcutaneous 2CdA and provide elements to build new clinical trials with combined strategies in cases of refractory/non responsive HCL where the immunogenenetic tumor profile is provided.

The kidney is frequently involved in the course of monoclonal gammopathies (MG). Renal involvement presents different clinical-morphological patterns, which can occur either at the onset or in a late phase of the hematological disease, as well as after chemotherapy. The reasons for the organ tropism of monoclonal immunoglobulins (Igs) are still unknown. Currently, it is well known that some primary structure alterations in monoclonal Igs and/or in their segments correlate to nephrotoxicity. On the other hand, it is impossible to predict the pathogenicity and the clinical manifestations induced by a specific monoclonal Ig based on its specific conformational modifications. Pathogenicity and organ tropism are probably complex phenomena, which involve specific protein factors, patient factors, target organ characteristics and monoclonal plasmacellular mass entities. However, aminoacidic sequence analysis of nephrotoxic Igs and some recent in vitro studies have allowed two different monoclonal light chain (LC) types to be distinguished. Glomerulopathic LCs (G-LCs) in the mesangium recognize their target structure and induce two distinct mesangiopathies, monoclonal Ig deposition disease (MIDD) and AL-amyloidosis (AL). Tubulopathic LCs (T-LCs) act on the proximal or on the distal tubule and cause, respectively, Fanconi syndrome (FS) and cast nephropathy. Pathogenic monoclonal Igs have the propensity to deposit in different renal parenchymal structures in extracellular sites, because of the transformation of soluble precursors in insoluble products. Evidence suggests that somatic mutations can destabilize the normal LCs globular soluble structure and this could be the major driving force for precipitation. Based on these features, MG can be classified as conformational and depositional diseases. Electronmicroscopy (EM) analysis of renal biopsies in MG patients with glomerular diseases distinguishes two morphological aspects. MIDD and a recently identified entity named proliferative glomerulonephritis (GN) with monoclonal IgG deposits are both characterized by non-organized granular electrondense deposits. AL, immunotactoid (IT) glomerulopathy and monoclonal cryoglobulinemia are, instead, characterized by organized deposits such as fibrils or microtubules. Tubular diseases in MG patients produce two different histological patterns. In FS, monoclonal Igs form crystals in the renal interstitium able to induce a local intense flogosis, while in cast nephropathy monoclonal Igs precipitate with Tamm-Horsfall protein (THP) in the proximal tubular lumen and induce tubular obstruction. The different morphological aspects are unrelated to specific clinical manifestations, while renal biopsy can diagnose different entities that can respond to different therapeutical schedules. This reveals the importance of the renal biopsy in the clinical management of the renal pathology in plasma cells dyscrasias, mainly when supported by the most advanced techniques of immunoelectronmicroscopy and polymerase chain reaction (PCR)-mediated analysis. Further elucidation of the molecular events involved in the pathogenesis of the different forms of renal damage is needed to design new and more effective therapeutical strategies. In particular, urinary proteomics seem to be promising in this setting.

Ninety ET patients diagnosed according to the PVSG criteria entered a phase II study designed to identify a treatment with PEG Interferon α-2b (PEG Intron, Schering-Plough Corp) safe and able to obtain and to maintain for a long time the Hematological Response (HR, PLTs < 500x 109/L). The patients, 30 Males and 60 Females, median age 45 years (18–72), judged at risk and in the majority of cases pre-treated with cytoreductive drugs (60%), at study entry showed a mean PLT count of 1093 ± 357 x 109/L (>1000 in 49% of cases). During the first year (part 1 of the study) the PEG Intron starting dose of 25 μg/week was gradually increased if necessary at week 13, 26 and 39 till a maximum of 100 μg/week to reach the HR. The HR at the end of the 1st year (primary endpoint) was registered in 64 patients who represented the 71% of the 90 enrolled subjects (analysis on Intent-to-Treat, ITT) and the 79% of the 81 patients still receiving PEG Intron. The PEG Intron effective dose had a mean value of 50 ± 22 μg/week resulting very low (25μg/week) in 33% of cases. The primary endpoint was reached independently by sex, age, cytoreductive pre-treatment and baseline PLT count. Nevertheless, the HR was more frequent in the patients with true ET respect those with false ET (WHO criteria, HR 93% vs. 68%). In the 64 responder patients admitted to the part 2 of the study according to the protocol, the HR at the end of the 2nd year was registered in 75% of cases on ITT and in 87% of the 55 patients still on treatment, with a PEG Intron maintenance dose gradually reduced to a mean level of 27 μg/week. No thrombotic or hemorrhagic events occurred during the study and, moreover, a decrease of splenomegaly (from 22% to 6%) and of disease related symptoms (from 42% to 2%) was registered. The unmaintained HR, in 29 valuable patients who discontinued the PEG Intron treatment after two years, had a median duration of 11 weeks (4–52). Eighteen patients discontinued PEG Intron treatment (9 during the 1st year and 9 during the 2nd year) as consequence of PEG Intron toxicity and side effects (n 7), loss of compliance (n 5) and drug unrelated reasons (n 6). Toxicity and side effects (WHO grading) were never of grade 4, in very few cases of grade 3 and in the majority of patients of grade 1 or 2 (mainly flu-like syndrome, leukopenia, hypertransaminasemia). The ECOG Performance status (PS) was of grade 0 in all but few patients who showed a transitory grade 1 PS. In conclusion, PEG Intron treatment performed at relatively low dose seems safe and able to induce and to maintain the HR for a long time in the majority of at risk ET patients.

Summary Fludarabine plus cytarabine (Ara-C) and idarubicin (FLAI) is an effective and well-tolerated induction regimen for the treatment of acute myeloid leukaemia (AML). This phase III trial compared the efficacy and toxicity of FLAI versus idarubicin plus Ara-C and etoposide (ICE) in 112 newly diagnosed AML patients

Acute myeloid leukaemias (AMLs) are a heterogeneous family of hemopoietic malignancies that share a high frequency and a high degree of Pgp-mediated multidrug resistance (MDR), one of the major causes of treatment failure. Induction treatment (FLAI), including 5 days administration of Idarubicin (IDA- 10mg/sqm/day), in combination with high-dose Arabinosyl Cytosine (HDAC-2g/sqm/day) and Fludarabine (FLUDA-25mg/sqm/day) was adopted as induction treatment of newly diagnosed patients with AML, except acute promyelocytic leukemia (APL). In our previous experience (Russo et al. Leuk. Lymphoma, 2001), FLAI regimen showed to be highly effective (CR rate 72%), also in AML MDR-pos patients, with a low extra-hematological toxicity. In this prospective randomized trial, FLAI was compared with ICE (IDA 10mg/sqm/day x 3 days + AC p.c 100mg/sqm/day x 10 days + VP16 100mg/sqm/day x 5 days) for induction of remission. Post-induction treatment program included: HDAC (3g/sqm/12h x 6 days), MEC (Mitoxantrone 12 mg/sqm/day x 4 days, Etoposide 100 mg/sqm/day x 4 days, Cytarabine 1 gr/sqm/day x 4 days) and allogeneic or autologous BMT. Over a period of 2 years, 118 patients were randomly assigned to FLAI (67) or ICE (51). The clinical and hematological characteristics of the two patient population were not different. In the FLAI group, the complete remission (CR) rate was 72% after the first course and 76% after HDAC; in the ICE group, the CR rate was 53% and 69%, respectively (P 1.0 x 109/L and platelets > 50 x 109/L was significantly better in FLAI arm than in ICE arm. In both groups, it was seen an approximately equal rate of FUO, Gram negative/Gram positive bacteremias and systemic fungal infections. Infections and haemorrhages caused death during induction (DDI) in 3% of patients treated with FLAI and in 10% of patients treated with ICE. Non-hematological toxicity of FLAI was mild and significantly lower than ICE. In particular, in the FLAI arm, only 3/67 pts developed a grade 3 or 4 gastro-intestinal toxicity, whereas in ICE arm 16/51 patients experienced this toxicity (p=0.0001). Other grade 3 or 4 toxicity (ipertransaminasemia, cutaneus toxicity, renal or cardiac failure) were seen in 1/67 pts in FLAI arm versus 7/51 pts in ICE arm (p=0.02). In conclusion, these preliminary results strongly suggest that FLAI, as single induction course, is an highly effective regimen with a limited non-haematologic toxicity. Furthermore, FLAI seems to be more effective than ICE to overcome Pgp-mediated multidrug resistance.