ALBERT

Description: We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, acquisition of clonogenic activity and increased expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in immunodeficient mice similar to Lin-CD34+ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34−cells. Imatinib mesilate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin− CD34− cells in vitro. Moreover, leukemic CD34− cells survived to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Description: The anti-CD20 monoclonal antibody Rituximab is now widely used in B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL) patients. Its administration is associated with a profound reduction of the number of normal and malignant CD20+ B cells, so that the procedures used so far for NHL and CLL diagnosis, detection of minimal residual disease (MRD) and follow-up (F-UP) should be revised. We have retrospectively evaluated our consecutive 4-year record of 647 NHL and CLL patients. Our aims were: 1) to compare sensitivity and concordance between 4-colour flow cytometry, qualitative and quantitative PCR and FISH. 2) to compare concordance between bone marrow (BM) and peripheral blood (PB). Sensitivity of the procedures (evaluated by serial dilution of NHL cell lines in healthy donors’ BM and PB) were found to be 10−5, 10−6, 10−4/5 and 10−3 for 4-colour flow cytometry, qualitative PCR, quantitative PCR and FISH, respectively. A total of 955 paired BM and PB samples were investigated. Most frequent diagnoses were follicular NHL (29%), diffuse large B-cell NHL (22%), marginal zone NHL (11%), CLL (10%), and mantle cell NHL (8%). In 236/955 cases (25%), 4-color flow cytometry identified a monoclonal or a phenotypically aberrant cell population. Concordance between BM and PB was 87% (p

Description: Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in a variety of different strains of immunodeficient mice has led to preclinical models extensively used to investigate AML and ALL stem cells, biology, and drug sensitivity. We investigated the engraftment kinetics of two AML cell lines (HL-60 and KG-1), two ALL cell lines (MOLT-16 and 697) and AML primary cells from an AML M4 patient (QD1-EIO, described in Fusetti et al, Cancer Res 2000) in 3 different strains of NOD/LtSz-Prkdcscid (NOD/SCID, NS)-related immunodeficient mice. NS, NS/beta2 null (NSB) and NOD/SCID/IL-2Rgamma null (NSG) mice were injected ip with 10x106 AML or ALL cells. Mice were observed daily and sacrificed when leukemia-related symptoms were evident. Overall, leukemia-related symptoms were observed in 71, 84 and 86% of NS, NSB and NSG mice, respectively (n=42, p

Description: The aim of this prospective multicenter trial was to evaluate the efficacy of the nucleoside analogue 2-CdA in combination with Rituximab and to identify possible genetic factors that could influence the clinical response of the combination. From December 2003 to November 2006, 29 pretreated/newly diagnosed pts affected by WM were enrolled in the study. Pts characteristics included: sex (M/F) 9/19, median age 64 (range 36–75 yrs), a median IgM level before treatment of 2567mg/dL; 16 pts were newly diagnosed. The combination therapy was Rituximab at standard schedule (375 mg/mq) on day 1 followed by 2-CdA 0.1 mg/kg (sc injection) for 5 consecutive days. Each cycle was administered monthly for a total of 4 cycles. Clinical responses were evaluated two months after the end of treatment, according to Response Criteria from the 3rd International Workshop on WM. Expression analysis of the genes involved in the activity of 2-CdA (deoxycytidine kinase, deoxyguanosine kinase, 5′-nucleotidase, ribonucleotide reductase 1 and 2, human equilibrative nucleotide transporter and hCNT1) was performed on bone marrow cells, at baseline in 22 pts. The therapy was well tolerated, except in three patients who discontinued Rituximab due to cardiac toxicity during the first or second infusion. The treatment was delayed in 3 pts because of haematologic toxicity (G3 neutropenia) and in 2 of them the 2-CdA dose was reduced after 2 cycles. The only non-haematological complications observed were late respiratory infections which occurred in 3 pts. At a median follow-up of 21 (13–41) months we observed 17 (59%) CR/PR, 7 (24%) MR, 1 (3%) SD and 4 (14%) PD/NR. Pharmacogenomic analysis showed a statistically significant lower expression of the hCNT1 gene in pts who failed the treatment and who achieved a MR or SD than in those who achieved a CR or a PR (p=0.014) suggesting a possible relationship between reduced hCNT1 levels and a diminished clinical activity of 2-CdA. No significant difference was detected in the expression of the other genes analyzed. Our results suggest that the combination of 2-CDA and Rituximab is safe and active in WM pts; the use of pharmacogenomic analysis might contribute to a better selection of the patients who are more likely to respond to such a combination treatment.

Description: In CLL/SLL pts the combination of purine analogue and rituximab represents a well known effective therapy. Human concentrative nucleotide transporter (hCNT1) and human equilibrative nucleotide transporter (hENT1) are proteins involved in uptake of nucleoside analogues into the tumour cells. The aim of this study was to investigate R-2CDA as first line therapy and in pre-treated pts with active CLL and SLL, and to identify, by pharmacogenomic approaches, genetic factors that may predict clinical response to such treatment. 45 pts with active CLL (27 pts) or SLL (18 pts) were treated. The median age was 59 years (31–76). Patients received 4 cycles of rituximab 375 mg/m2 on day 1 and 2-CDA 0.1 mg/kg (subcutaneously) on days 2 to 6. The treatment was repeated every 4 weeks. A CT scan of the abdomen was performed at baseline and at the end of treatment in all pts; CT scan before treatment was abnormal in 42 pts. Minimal residual disease was evaluated by 6-colour flow-cytometry and PCR methods. Treatment outcome was evaluated according to NCI-WG updating guidelines: to confirm a CR the marrow should be free of clonal B-CLL cells by flow-cytometry and a CTscan should be negative. At baseline we investigated, on bone marrow for SLL and on peripheral blood for CLL, the expression of human concentrative nucleotide transporter (hCNT1) and human equilibrative nucleotide transporter (hENT1) using ABI PRISM 7000 Real Time RT-PCR platform in 24 pts. 42 pts (26 untreated) were fully evaluable for response.LDH and beta2microglobulins were abnormal in 14% and 30% of pts respectively. The percentage of neoplastic cells in the bone marrow was more than 70% in 17% of pts. The overall response rate was 88% (26% CR and 62% PR), with 21% of untreated pts and 5% of pre-treated pts achieving a CR. Severe neutropenia (grade 4) developed in 7% of pts. 4 pts developed pneumonia, 1 with neutropenia. 1 pt had reactivation of herpes zoster virus and 1 pt experienced febrile neutropenia of unknown origin. The median TTP was 41 months. There was not statistically significant difference in terms of duration of response between untreated and pre-treated pts (TTP: 44 vs 35 months, p=0.1814). Pts achieving a CR had a longer response duration than pts with PR (p=0.0047). Low serum lactate dehydrogenase levels, a lower (〈 70%) neoplastic marrow infiltration at baseline and a normal CT scan at the end of therapy (independent of response) predicted a longer response duration (p=0.0145, p=0.0164 and p=0.008 respectevely). The pharmacogenomic analysis showed a difference (p=0.8345) in terms of hCNT1 expression levels between patients achieving a CR and pts with PR or NR. Pts in CR had higher levels of hCNT1 expression and the increase of 1 unit of the expression level of hCNT1 is associated with a reduced risk of PD by 33%. The refractory pts had lower levels of hCNT1 than non refractory pts but the difference was not statistically significant. There was no statistically significant difference in terms of hCNT1 expression between CLL and SLL pts. The combination of 2-CDA and rituximab induces a high response rate, including CR in pre-treated pts. The treatment is well tolerated with acceptable toxicity also in pts over 70 years. The achievement of a CR is important to obtain durable response. The correlation between the levels of expression of hCNT1 and the response to therapy needs to be confirmed in larger studies.

Description: Patients undergoing allogeneic cell transplantation (HCT), who develop chronic Graft versus Host Disease (GVHD), can show a cutaneous involvement similar to that of patients with systemic sclerosis (SSc). Human Cytomegalovirus (hCMV) has been shown to be implicated in the pathogenesis of SSc, since a subset of anti-hCMV antibodies directed against the late viral protein UL94, cross-react with the cell surface tetraspanin NAG-2, and induce the vascular and fibrotic damage observed in the disease. HCMV infection and/or its reactivation have also been associated with the increased risk to develop GVHD and with the worsening of clinical manifestations. Immunosuppressive treatment for GVHD can be intuitively correlated to the viral reactivation, however, the pathogenetic link between hCMV infection and chronic GVHD is still lacking. In this study we wanted first to evaluate the presence of anti-hCMV antibodies directed against the viral protein UL94 in the plasma of HCT patients and second to study their possible role in the pathogenesis of chronic GVHD. Eighteen patients undergone allogeneic HCT for hematological malignancies (16 with nonmyeloablative conditioning regimen and 2 with myeloablative), between 2003 and 2006, with a median follow-up of 7.35 months (range 1.2–55.7), were retrospectively studied. Five of them were anti-hCMV antibodies negative but their donors were anti-hCMV IgG positive. Twelve patients experienced hCMV reactivation evaluated by pp65 and PCR and they needed either Gancyclovir or Valgancyclovir or Foscavir as preemptive therapy. Interestingly, almost all patients (11/12) with hCMV reactivation developed chronic GVHD, and six of the eleven had acute GVHD. All GVHD patients showed skin involvement, and some of them had evidence of diffuse skin fibrosis remarkably similar to that of SSc. None of the patients nor of the donors were suffering from autoimmune diseases. Five of 18 are still alive and in complete remission, included the 2 patients with a skin involvement similar to diffuse SSc. One patient with diffuse skin involvement was lost to the follow-up. Thirteen patients died: 8 for progression of disease, 4 because of steroid resistant GVHD, 1 for sepsis and multi-organ failure. Patients who died before day 100 were not suitable for chronic GVHD evaluation. Patients who developed chronic GVHD showed the presence of anti-hCMV derived late viral protein UL94. Remarkably, the 3 patients with chronic GVHD who developed a diffuse SSc-like skin involvement, had antibodies directed against NAG-2, a cell surface tetraspanin that forms complexes with integrins, already reported to play a pivotal role in the pathogenesis of SSc. Such antibodies bound endothelial cells and fibroblasts and were able to induce endothelial cell apoptosis and fibroblast proliferation, as already shown in patients with SSc (Methods as previously published by Lunardi C. et al. on Nat Med. 2000 Oct; 6(10):1183–6 and PLoS Med. 2006 Jan; 3(1 e 2): 94–108). These findings sustain the involvement of hCMV infection in the pathogenesis of chronic skin GVHD through a mechanism of molecular mimicry between the viral late protein UL94 and the cell surface molecule NAG-2. Moreover, these data provide a previously unknown pathogenetic mechanism by which hCMV infection contributes to the development of chronic skin GVHD resembling SSc. Perspective studies with a larger number of patients are needed to further support these results. Figure Figure

Description: Here we present the clinical results and the outcomes of 143 consecutive newly-diagnosed follicular lymphoma (FL) patients (pts) treated with 3 different modalities of treatment in our institution from 1994 until 2007. During this period, from 1994 to 2002, 55 pts received Chlorambucil and Prednisone (Chl+PDN), subsequently 31 pts Rituximab with Chlorambucil (R-Chl) and 57 pts a 4-weekly standard-dose of Rituximab alone (R). Chlorambucil was given in an induction phase at 10mg/day for 6 consecutive weeks followed by a longer maintenance phase in the first group: 2-week pulses of 10 mg daily with 2-week intervals for a total of 12-months of treatment versus four 2-week pulses with monthly-Rituximab. In the single agent R group 36 pts received maintenance with at least 4-bimonthly administrations. The 3 groups were comparable for demographic and prognostic factors: median age at diagnosis was 55 yrs, over 60% of pts were in advanced stages and mainly asyptomatic (about 90%) and over 50% of pts in each group were low-risk FLIPI. A third of pts in both Rituximab-based treatment groups had bone marrow involvement and bulky disease. In terms of ORR 78% of pts treated with Chl+PDN, 97% with R-Chl and 72% with R single agent, obtained a clinical response, with a percentage of complete response of 58%, 90% and 47% respectively. No significant incidence of adverse events were reported and none discontinued the therapy because of toxicity. One case of myelodysplatic syndrome was described in a relapsed patient of the Chl-PDN group. With a median follow up of 72, 56 and 27 months in the 3 groups evaluated, the CR rate was maintained in 29%, 74% and 37% of pts respectively with a median duration of response of 36, 40 and 24 months. Rituximab as first-line in FL pts seems safe, feasible and able to induce a high rate of clinical response similar to those achieved with standard chemotherapy alone. On the other hand, the immunochemotherapy seems to confirm its superiority in terms of response rate and duration of response.

Description: Abstract 3210 Poster Board III-147 BACKGROUND High-dose chemotherapy followed by autologous Peripheral Blood Stem Cell (PBSC) transplantation represents an effective option in relapsing/refractory malignant lymphoma as well as in selected solid tumors. Collection of a sufficient amount of stem cells (CD34+ ≥ 2.0×106/kg) by apheresis is mandatory for the procedure. However many factors could influence the mobilization of PBSC and about 30% of patients eligible for this therapeutic option fail stem cell mobilization. Peripheral CD34+ counts of 20/μL is conventionally considered the cut-off for a successful collection. METHODS With the aim to identify those factors affecting PBSC mobilization, we retrospectively reviewed data about our experience at European Institute of Oncology from 01/2005 to 05/2009. By evaluating the number of CD34+cells after mobilization, patients were considered as good mobilizers (peripheral CD34+ ≥ 20/μL, group A), relative poor mobilizers (peripheral CD34+ counts 2.0×106 CD34+cells/kg. According the Two-sided Fisher's exact test, more than 3 previous chemotherapy lines before mobilization (p

Description: Abstract 3754 Poster Board III-690 Introduction Follicular lymphoma (FL), one of the most common B-cell non-Hodgkin's lymphoma (NHL), is an indolent disease characterized by a continuous relapse pattern. In the last years the treatment have changed with the introduction of Rituximab, that in combination with chemotherapy prolongs the progression-free survival (PFS) and the overall survival (OS) of FL pts. We report here the results of a regimen containing Rituximab and Chlorambucil (Chl) in untreaed FL pts. Methods Since December 2001 48 pts (22 male and 26 female) with naive FL were treated in first-line with R-Chl. The schedule consisted of an induction phase with four weekly infusion of Rituximab at standard dose and 6 consecutive weeks of Chl at 6mg/mq/daily. Pts were restaged and in absence of disease progression received four cycles of consolidation with a monthly Rituximab infusion and 14 days of Chl each month. Results Median age at diagnosis was 56 years (range 29-79). Ann Arbor stage was I-II in 13 pts and III-IV in 35 pts (73%); 15 pts had bulky disease and 14 pts presented an extranodal localization. B symptoms were present in 8 pts. Histological grading was available in 42 cases, and was 1 in 10 pts, 2 in 26 pts and 3 in 6 pts. FLIPI was evaluable in 46 pts, and was 0-1 in 25 pts (52%), 2 in 12 pts (25%), and 〉2 in 9 pts (18%). After the induction phase overall response rate (ORR) was 98% with 14 pts in complete remission (CR). After the consolidation phase 39 pts (81%) obtained a CR and eight (17%) a partial response (PR); the remaining patient had a stable disease and underwent high-dose chemotherapy and autologous transplantation. All pts but one concluded the planned treatment. The mean daily dose of Chl administered during the induction phase was 10 mg, while 8 mg in the prolonged treatment. Twenty-eight pts (58%) experienced a neutropenia during treatment, and Chl dose was reduced in 22 pts (46%); however, only 11 pts (23%) had a G3-4 neutropenia. The Chl was stopped only in a patient because of a persistent G3 neutropenia after the first consolidation cycle. After a median observation time of 42.5 months (range 7-94) nine pts relapsed (19%), with a median time to relapse of 20 months (range 7-66). Two pts died, one of disease progression and one of a pre-existing ovarian cancer. Conclusions Our results described a safe and feasible combination of immuno-chemotherapy in untreated FL pts. With respect to traditional treatments, our regimen shows a similar efficacy and a lower toxicity, that leads to an easier handling. The schedule including four monthly additional Rituximab administrations confirms the superiority of a prolonged exposure to Rituximab over the standard 4-weekly administrations. In conclusion, our data suggest that this combination may be considered as a valid first-line treatment of FL patients, especially in those not eligible for more aggressive chemotherapy regimens. Disclosures: No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) is an aggressive disease, currently incurable with standard-dose chemotherapy. Autologous cell transplantation (ASCT) is frequently considered a therapeutical approach in MCL, despite not generally being curative. In controlled studies upfront ASCT showed a significant event-free survival advantage, that might be improved by the addition of the anti-CD20 monoclonal-antibody (MoAb) (Rituximab®) in the myeloablative regimen. Based on these evidences, we prospectively analysed our patients (pts) affected by MCL, who received a continuous Rituximab administration in course on treatment. From May 2000 to the present we treated 22 pts with histological diagnosis of MCL, including 16 pts (73%) newly diagnosed. The majority were male (15/22) and the median age at diagnosis was 56 years old (range 35–66). Disease was stage III–IV: 21 pts with bone marrow involvement in 60%. Eleven pts presented extranodal involvement. The induction schedule included 2–4 CHOP-like regimen with MoAb (R-ACOD), followed by mobilization chemotherapy with cyclophosphamide, MoAb and G-CSF. Harvest was performed successfully in all pts with a median number of CD34-positive cells of 8.0 x 106/Kg (1.9–22.8 106/Kg). Subsequently the pts received 2 cycles of high-dose, cytarabine-based regimen with MoAb (R-ESHAP). Rituximab injections were given at standard dose (375 mg/mq). Before ASCT, 11 pts (50%) were in complete response (CR); in particularly 5 of them who were pretreated. Eight achieved partial response, while one was in progression. Two pts were not restaged. One patient did not undergo ASCT, because of serious pulmonary infection after high-dose cytarabine. In terms of conditioning regimen all pts received Rituximab (375 mg/mq), Melphalan (180mg/mq) in association with either Idarubicin (15mg/mq for 3 days) in 13 pts and or Novantrone (60 mg/mq for 1 day) in 8 pts. After stem cell reinfusion and G-CSF from Day +5, the median time to recovery (neutrophil counts 〉0.5x103) was 14 days (range 13–28 days) in the first regimen versus 10 days (range 8–11 days) in the more recent regimen. During aplasia the infection rate was low, as a matter of fact we observed just two episodes of pneumonia, that resolved at neutrophil recovery. No toxic death was reported. Response at 3 months after ASCT was evaluable in 19 patients (90%); one was lost at follow-up, while one has been transplanted one month ago. Seventy patients (80%) achieved complete response, maintained in 11 of them after a median time of 23 month (range 10–79) after ASCT; seven of them were in complete response before the transplant procedure. With a median time of 27 months (range 9–46 months) 7 patients progressed and 4 of these died because of lymphoma. Our study supports that ASCT can be considered a valid approach to induce high response rate. To enhance the ASCT outcomes, the addition of Rituximab can be considered a feasible approach associated with its “purging in vivo” effect. Long-term disease control might be reflected upon a plateau in the survival curve, but a randomised study and longer follow up is warranted.