ALBERT

Description: Background: Telomeres are linear DNA structures located at the ends of chromosomes which undergo progressive shortening in somatic tissues with high replicative rate, including hematopoietic cells. Due to this peculiarity, telomere length (TL) has been used as a marker of cell senescence and of previous replicative stress. Following hematopoietic stem cell transplant (SCT) a variable shortening of TL has observed, this variability has been in part ascribed to differences in post-graft replicative stress. In a recent report (Ricca I et al, Leukemia 2005), we have analysed TL on peripheral blood progenitor cell (PBPC) harvested after 2 tightly-spaced hd-chemotherapy courses, i.e. hd-Cyclophosphamide (CY) and hd-Ara-C and found that TL of PBPC was markedly shortened at the second hd-course. Aim of this study was to investigate how TL of grafted cells may influence telomere status of post-SCT hematopoietic cells in both the autologous and allogeneic setting. Patients and Methods: TL was monitored in 20 patients undergoing autograft with PBPC collected after hd-CY (10 patients) or hd-Ara-C (10 patients) and in 10 patients undergoing allograft with PBPC mobilized from healthy donors. Overall, patient median age was 48 years (range 19–64). The amount of CD34+ cells grafted was 7 × 106 CD34+/Kg (range 2,03–22,6). TL was assessed both on the graft and on Bone Marrow (BM) samples taken at a median time of 14 months (range 12–17) after SCT. All patients were in continuous complete remission at the time of TL analisis. TL was evaluated by Southern blot, as previously described (Rocci A et al, Exp Hematol 2007). Results: As expected, TL was found markedly shortened in post-Ara-C, with median TL of 8051 bp (range 5209–11024 bp) and 7066 bp (range 4800–8906 bp) in PBPC post-CY and post-Ara-C, respectively (p

Description: BACKGROUND. Germinal Center (GC) experience is a basic prognostic feature in B-CLL. Patients with VH-mutated GC-experienced CLL have a good prognosis while those with VH-unmutated GC-inexperienced CLL have a poor prognosis. In a recent study we demonstrated that telomere length (TL) of lymphoproliferative disorders strongly correlates with GC, pre-GC or post-GC origin (Ladetto M et al, Blood 2004). Aims of this study were to further define the relationship between TL and VH mutational status in B-CLL and correlate both these parameters with clinical outcome. PATIENTS AND METHODS. 109 B-CLL patients have been analyzed for telomere restriction fragments (TRF) length and are under evaluation for VH mutational status. All samples were taken at diagnosis or during the "watch and wait" phase. Male were 68, females 41. Median age was 62 years (range 34–87). Fifty-three patients were in stage A, 30 patients were in stage B and 16 were stage C according to Binet staging system. Our patient population has been monitored for a median time of 53 months (range 1–290). Sixty-three patients have been already treated for their disease while 46 have not required treatment, so far. TRF length was evaluated by Southern blot and VH mutational status by direct sequencing, as previously described (Ladetto M et al, Blood 2004). The standard cut-off of 2% deviation from any germ line VH sequence was employed to define VH mutational status. Survival analyses were performed using the Kaplan-Meier method. RESULTS. Overall, median TRF length was 5898bp (range 1737–14837bp). There was no correlation between TRF length and patient age, sex or stage. A cut-off of 4500bp discriminated two subgroups of patients characterized by different clinical outcome in terms of time to first treatment (TTFT) and time to disease progression (TTP) following first line treatment. Patients with TL 〈 4500bp had a median TTFT of 16 months and a median TTP of 14 months while patients with TL 〉 4500bp had a median TTFT of 36 months and a median TTP of 50 months (p

Description: This is an update of the GITMO-IIL trial comparing R-HDS and CHOP-R in high-risk FL 1 47%. R-HDS has been already described (Ladetto et al ASH 2005). The CHOP-R arm consisted of CHOP and Rituximab delivered sequentially as already published (Rambaldi et al Blood 2002). Cross-over was allowed for pts failing CHOP-R. Centralized minimal residual disease (MRD) analysis with the bcl-2/IgH was planned on BM cells. Analysis was “intention to treat”. Toxic deaths were 4 (2 in each arm); in addition 1 gastric cancer and 2 MDS-ANLL occurred in the R-HDS arm and 1 head and neck cancer in the CHOP-R arm. CR rates were 59% with CHOP-R and 85% with R-HDS (p

Description: Introduction: NLABRs are frequently observed in cancer free-subjects. We recently observed that NLABR-positive clones can persist up to 60 days (Ladetto et al, J Clin Oncol 2003). However the long-term kinetics and potential pre-neoplastic role of NLABR-carrying cells are unknown. To define the natural history of NLABR-positive clones, long term monitoring of cancer-free subjects carrying these lesions has been performed. Methods: 118 subjects undergoing periodical blood examinations for warfarin therapy were screened for the bcl-2/IgH translocation. PCR-positive subjects underwent subsequent monitoring at least once every three months. NLABR-positive clones were monitored using both nested and real time-PCR according to previously published approaches (Ladetto et al Exp Hematol 2001). Sequence homology of NLABRs has always been confirmed by direct sequencing of nested PCR products. Results: 15 NLABR-positive subjects were identified out of 118 (12.7%) subjects. NLABR-positive subjects were monitored for a median time of 13 months (mos) (range 3–30 mos) for a total number of 60 timepoints. In eight subjects (53%), NLABRs detected at study initiation were not detected again in follow-up samples. These eight subjects have been monitored for median period of 12 mos (range 3–28 mos). Follow-up samples in this group were usually PCR-negative, although transient PCR-positivity due to unrelated NLABRs were noticed in two samples. In seven subjects (47%), the same NLABR observed at study initiation was detected one or more times at follow-up. In four subjects, NLABRs detected at diagnosis were amplified in every available follow-up sample (three to seven samples were available for each subject). In three, NLABRs detected at diagnosis were amplified only in a fraction of follow-up samples while the remaining were PCR-negative. Overall, persistent NLABRs were followed on these subjects for a median time of 15 months (range 3–30). The median burden of persistent NLABRs assessed by real-time PCR was 33 rearrangements (rg)/106 diploid genomes (dg) (range

Description: Abstract 3434 Poster Board III-322 Introduction Fludarabine alone or in combination is considered the standard treatment for Chronic Lymphocytic Leukemia (CLL). A high response rate is usually observed following fludarabine, particularly if delivered together with rituximab. In spite of the high therapeutic efficacy, most patients subsequently relapse. For these patients there are no defined salvage treatments. Moreover, fludarabine-containing regimens may have some restrictions due to non-negligible side effects. In particular, there are some concerns in employing fludarabine first-line in patients with autoimmune hemolytic anemia (AHA) or with renal function impairment. In addition, fludarabine has been reported to compromise the capacity of progenitor cell mobilization. Thus, novel effective treatment approaches are needed, for the management of patients failing after fludarabine-based regimens and for those unfit to receive fludarabine first-line. A very effective salvage regimen for refractory/relapsed lymphoproliferative disorders is the DHAP combination. DHAP has been widely used in the rescue of both low-grade and high-grade lymphoma. However, its efficacy in CLL has not been verified yet. The main concern with DHAP is the well known renal toxicity of cisplatin. However, the use of the less toxic analog Oxaliplatin, might circumvent this problem. Indeed, recent reports have shown that the inclusion of oxaliplatin into the original DHAP regimen (Ox-DHA) markedly improves the tolerability and widens regimen applicability. Aim: To evaluate retrospectively feasibility and efficacy of the DHAP and Ox-DHA regimens in CLL and in other non-follicular low-grade lymphomas, in particular in Waldenström Macroglobulinemia (WM). Patients and Methods Between 2002 and 2008, 84 low-grade lymphoma patients received DHAP or Ox-DHA; their median age was 60 yrs. (range: 24-84), 58 were male; 70 patients had CLL with advanced stage (65 patients with Binet stage B and C) and 14 had WM. Thirty-eight patients were treated at first relapse, 27 at second or subsequent relapse, whereas 19 received the DHAP or Ox-DHA schedule as first-line treatment, in place of Fludarabine, due to: i. AHA (3 patients), ii. concomitant second malignancy (4 cases), iii. need of initial debulking, before a high-dose program with autograft. The original DHAP schedule requires hospitalization for three to five days; it includes: Cisplatin 100 mg/sqm on day 1, Cytarabine 2 g/sqm/b.i.d. every 12 hrs. on day 2, Dexamethasone 40 mg days 1-4. Ox-DHA can be delivered in the outpatient setting, compared to DHAP, there are two main modifications: Oxaliplatin 100 mg/sqm in two-hr i.v. infusion on day 1, and Cytarabine 2 g/sqm two doses delivered in two consecutive days (day 2 and day 3). Rituximab (375 mg/sqm) was added in 12 DHAP and 28 Ox-DHA courses. Patients aged over 70 yrs had variable dose reductions (25% to 50%). Results Ox-DHA had hematological toxicity analogous to that commonly observed with the original DHAP schedule; 11 patients required short hospitalization for severe infectious complications; 7 patients developed fever of unknown origin, 4 showed reversible peripheral neurotoxicity. The program was discontinued in four patients due to disease progression (3 patients) and AHA (1 case). There were no severe liver or renal toxicities. No toxic deaths were recorded. The overall response (OR) rate was 90%; in details, complete remission (CR), or very good partial remission (VGPR), was achieved in 41% of patients receiving DHAP without rituximab and 50% of those receiving DHAP supplemented with rituximab. In the Ox-DHA group, 50% of patients treated without rituximab and 75% of those treated with rituximab reached CR or VGPR. Among patients treated at diagnosis, overall 47% reached CR or VGPR and 42% reached partial remission (PR). The OR rate was unexpectedly high at 91% in patients treated for refractory/relapsed disease, with 66% of them achieving CR/VGPR (74% and 55% for patients at 1st and ≥ 2nd relapse, respectively) and 35% PR. Among 14 WM patients, 1 obtained a CR, 5 a VGPR, 6 a PR and 2 had a stable disease. Conclusions Dexamethasone, Cytarabine and Cisplatin or Oxaliplatin schedule is a well tolerated regimen, highly effective both front-line and in the rescue for relapsed/refractory disease in CLL and WM patients. Future clinical trials will define the real efficacy of the DHAP or Ox-DHA regimen in the management of CLL and WM patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1975 Introduction: Telomeres are reliable indicators of previous cell proliferation and cell ageing. Moreover, a marked though variable loss of telomere length (TL) has been observed in several hematological malignancies. In particular, some recent studies have reported a marked TL reduction in patients with Ph-negative Chronic Myeloproliferative Neoplasms (Ph-neg-CMNs) (Ferraris AM et al, Br J Haematol 2005; Bernard L et al, Leukemia 2009). This supports the possible influence of TL in the development of CMNs. Moreover, TL might be of prognostic relevance in these disorders. The present study reports the analysis of TL in a large series of patients with Ph-neg-CMNs, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF), and in a few cases of Secondary Erythrocytosis (SE), as well. Aims of the study were: i. to verify the rate of TL reduction in Ph-neg-CMNs; ii. to compare TL in ET, PV and MF; iii. to investigate the role of TL as a marker of proliferation, evaluating differences in TL compared to healthy subjects and subjects with Secondary Erythrocytosis (SE); iv. to verify telomere dynamics according to the treatment received. Methods: Peripheral blood (PB) samples were obtained from 239 Ph-neg-CMNs patients (median age 68 yrs, range 10–91): 78 had PV, 100 had ET and 61 MF. Most patients were analyzed for JAK2 mutations: among 72 evaluated PV patients, a JAK2V617F mutation was found in 66 (92%) and an exon 12 mutation was identified in 2 patients; a JAK2 V617F mutation was detected in 50 (50%) ET patients and in 44 (73%) MF patients. Samples were obtained either at diagnosis or during follow-up. More than a half of CMNs patients received before TL analysis at least 1 year of cytoreduction (129 patients); 90% received Hydroxyurea (HU) and 26% other cytoreductive drugs with or without HU. As control, PB samples from 202 healthy age-matched subjects and from 14 SE subjects were analyzed. TL was assessed by Southern blot analysis, according to standard procedures (TeloTAGGG Telomere Length Assay Kit, Roche Diagnostic, Mannheim, Germany). JAK2V617F mutation analysis was performed by ASO-PCR and digestion with BSAXI (Guerini et al, Leukemia 2008). Results: PV, ET and MF patients showed individual progressive TL shortening correlated with age as observed in the healthy population. However, CMNs patients had TL significantly shortened (5,890 bp, ± 1,305) compared to healthy age-matched individuals (median: 7,330 bp, ± 1574) (p

Description: INTRODUCTION High-dose chemotherapy (HDT) with autologous stem cell transplantation is an effective treatment option for both non-Hodgkin’s Lymphoma (NHL) and Hodgkin’s Lymphoma (HL). Its clinical applicability has been considerably widened by peripheral blood progenitor cells (PBPC). Indeed, HDT with autograft is now the most frequently employed treatment for patients

Description: Introduction. Prophylaxis with factor concentrates reduces bleeding events and improves quality of life for adults and children with severe hemophilia. However, the optimal dosing and infusion frequency is not yet established. Integration of PK data into decision making is gaining support, in particular at the transition between conventional and EHL products. Here we report about 29 PK data of patients affected by hemophilia treated at our centre since childhood. Improved quality of life was our first aim, supposed that decreasing frequency of infusions or increasing the target through factor level allows a more active life without increased risk of bleeding. Patients' characteristics and methods. 18 patients (62%) were ≤ 18 years of age at PK time. 16 were affected by severe hemophilia A, 5 by moderate hemophilia A, 6 by severe hemophilia B and 2 by moderate hemophilia B. At PK time, 28 patients were on prophylaxis and 1 was on demand with recombinant factor IX. Median age at onset of prophylaxis was 9 years (range 3 months-38 years). Genetic assessment was available in 24 patients. Of these, 37.5% and 62.5% were carriers of null and not null mutations respectively. 4 patients were undergone to PK with standard products (1 Octocog alfa, 1 Simoctocog alfa, 1 Octocog alfa-Kovaltry®, 1 Turoctocog alfa) in order to define timing and dosage of successive infusions, while 25 patients switched to EHL factors (15 Efmoroctocog alfa, 2 Ionoctocog alfa, 7 Albutrepenonacog alfa, 1 Eftrenonacog alfa). In 15 patients a population-based PK (popPK) according to WAPPS-Hemo program was also performed. The annualized bleeding rate (ABR) was counted from patient's home bleeding records for one year before PK until now. Results. According to PK data, 21 patients (75%) decreased infusion frequency (100% hemophilia B and 67% hemophilia A patients). The remaining 7 hemophilia A patients maintained the same timing in order to increase the through factor level. Notably, 1 hemophilia B patient switched from on demand treatment to prophylaxis with EHL product due to the more acceptable schedule. 66% of null mutation patients and 73% of not null mutation patients decreased timing. Of 28 patients available at follow-up, 32%, 50% and 18% decreased, increased and maintained the same annual average factor consumption/kg, respectively. All patients had a good adherence after switch. In particular, the on demand patient started a regular prophylaxis with optimal compliance. ABR displayed a reduction with a median of 0 (range 0-5) after PK analysis compared to 1 (range 0-12) before the switch. Full PK vs popPK data obtained using at least two individual PK sampling points were almost similar. Conclusions. Our results remark the necessity of PK study especially in children due to the inter-individual variability independent of genetic assessment. Regarding factor IX, PK allowed us to propose timing even longer than that recommended by prescribing indications resulting in a better personalized prophylaxis. Moreover, our study demonstrates that a full PK analysis is feasible also in children. However, given similar results, popPK could be more feasible in most patients. Regarding consumption, the reduction of only 32% of patients reflects our aim to maintain a high safety profile in an active pediatric population. Nevertheless, the mean annualized consumption was just 0.6-fold increased in the remaining patients. This approach led us to further reduce ABR and in some cases to obtain a persistent no-bleeding status even with a full active life. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: array-CGH is significantly impacting on cancer cytogenetic. We used this technique to perform pan-genomic screening in 15 patients with MM and 4 with de novo PCL. Patients and methods. Bone marrow samples were employed. If tumor contamination was below 20% plasma cells were purified with Myltenyi columns. Array-CGH was performed as follows: genomic DNAs, from both the tumor and normal reference cells, labeled with different fluorescent dyes were cohybridized to 1 Mb resolution arrays containing 2600 Bacteria Artificial Chromosome (BAC) clones (Spectral Genomics Inc, Houston, TX, USA) according to manufacturer’s recommendations. Variations in DNA sequence copy number for each BAC clone was assessed by relative fluorescence signal intensities, in a single hybridization, providing a locus-by-locus measure of DNA copy-number changes. Results: The assay was validated as follows: three normal DNAs were tested revealing no genetic imbalances. One normal male was tested against one normal female and sex chromosome (ch) imbalances were effectively detected. Array-CGH results for ch 13q14.3 deletions were matched with FISH results and concordance was seen in 87% of cases. The median number of lesions/patient observed in our panel was 17 (4–135) (fig 1a). Also the amount of the total genome affected by chromosomal imbalances was highly variable (median 3.9% range: 0.14%–27%) (fig 1a). The amount of involved genome did not correlate with the actual number of lesions (fig 1a). A good correlation was noticed between the amount of losses and gains in each patient (fig1b). Notably PCL do not have a more disrupted genome compared to MM patients as one might expects based on the highly malignant behavior (fig 1a). Interestingly two patients with a prolonged clinical history of MGUS prior to MM diagnosis had massive presence of losses ad gains. Of 2600 BACs 934 were never affected, 864 were targeted only in one patient (pt), 401 in two pts, 296 in 3–5 pts and only 105 were targeted in six pts or more (fig 1c). These 105 recurrent imbalances could be attributed to 9 different abnormalities. Among these we have identified five recurrent lesions (occurring in at least six patients) that have not been previously described. These are 19p13.2 (gain 9 pts, loss 1 pt), 14q12 (loss 3 pts, gain 4 pts), 16q12.1 (loss 6 pts) 11q24 (gain 6 pts), 9q23 (gain 6 pts). Conclusions: array-CGH allow effective pan-genomic screening of MM patients; the pattern of genetic disruption is highly heterogeneous with a majority of non-recurrent or uncommonly recurrent lesions; a number of highly recurrent lesions have been identified that will require assesment for prognostic impact; the overall amount of perturbed genome does not seem to correlate with more aggressive disease, and might be the reflection of alternative biologic features (f.e. a prolonged history of clonal disease). Figure Figure

Description: Introduction: Secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/AML) may occur following autologous stem cell transplantation (ASCT). The molecular pathogenesis of sMDS/AML is uncertain; moreover, no suitable indicators able to define the risk of sMDS/AML development have been identified so far. A marked though variable telomere loss has been observed in hematopoietic cells following ASCT; meanwhile, shortening of telomere length has been observed to be associated with several neoplasias, including haematological malignancies. Thus, telomere dynamics has been investigated in a series of patients who received ASCT, in order to verify whether the degree of telomere loss in hematopoietic cells might be associated with the risk of sMDS/AML development. Methods: Telomere length (TL) was retrospectively evaluated in bone marrow (BM) cells from 38 lymphoma patients (M/F=24/14; median age=51 years, range 24–68) long-term survivors following ASCT and from 51 healthy donors (M/F=31/20; median age=53 years, range 18–82). Median follow-up since ASCT was 6 years (range 1–10). All patients were in continuous complete remission and displayed normal haematological values at the time of TL assessment. Among 38 autografted patients, 7 developed sMDS/AML (3 AML, 3 sMDS, 1 persistent pancitopenia with cytogenetic abnormalities), at a median of 5 years (range 1–10) following transplant. There were no significant differences in terms of demographical and clinical features not even for the amount of CD34+ve cells reinfused (median values: 5.2 vs 6.8 × 106 CD34+ cells/kg, respectively), between patients developing sMDS/AML and the remaining autografted patients. Samples for TL analysis were obtained and stored at a median of 12 months (range 6–24) before clinical development of sMDS/AML. TL was evaluated by Southern blot analysis. Results: TL of autografted patients was found to be significantly shorter compared to that of age-matched healthy donors, consistently with previous reports (see Figure 1, squares=healthy controls, circles=autografted subjects). A further TL loss was observed in all the 7 patients who subsequently developed sMDS/AML; their TL (Figure 1, black circles) was significantly shorter compared to both healthy subjects and sMDS/AML-free autografted patients (Figure 1, grey circles) (p