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Summary We explored activity and safety of a dose-dense/dose-intense adriamycin, bleomycin, vinblastine and dacarbazine regimen (ABVDDD-DI) in 82 patients with advanced Hodgkin Lymphoma. Patients entered a two-stage Bryant-Day Phase II study to receive six cycles of ABVDDD-DI without consolidation radiotherapy. Cycles were supported with granulocyte colony-stimulating factor and delivered every 21 d; drugs were administered on days 1 and 11 at the same doses of standard ABVD except for doxorubicin (35 mg/m2; first four cycles only). Co-primary endpoints were complete response (CR) rate and severe acute cardiopulmonary toxicity; secondary endpoints were event-free (EFS) and disease-free survival (DFS). All patients received the four doxorubicin-intensified courses and 96% concluded all six cycles (82·3% within the intended 18 weeks). This translated into a 66·9% increase of received dose-intensity for doxorubicin and 31·8% for the other agents over standard ABVD. The CR rate was 95·1% (78/82) and 87·8% (72/82) achieved a metabolic CR after two cycles. Cardiopulmonary toxicity never exceeded grade 2 and affected 14·6% of patients. Most frequent toxicities were grade 4 neutropenia (10%) and anaemia (9%), grade 3 infection (17%) and grade 2 mucocutaneous changes (30%). Five-year EFS and DFS was 88·3% and 93·7%, respectively. ABVDDD-DI regimen was well-tolerated and ensured substantial CR and EFS rates without radiotherapy.

Specific trials on adult Burkitt lymphoma (BL) and 'unclassifiable' lymphomas with features intermediate between BL and diffuse large B-cell lymphoma (BL/DLBCL) are advocated which include substantial numbers of older patients, to improve treatment feasibility, while countering risks of systemic and central nervous system (CNS) recurrences. We prospectively evaluated a modified CODOX-M/IVAC (CODOX-M: cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate; IVAC: ifosfamide, etoposide and high-dose cytarabine) regimen by the addition of rituximab (R) and liposome-encapsulated cytarabine (D) to increase antitumour activity and halve the number of intrathecal treatments. Thirty adults (40% >60years) with BL (n=15) and BL/DLBCL (n=15) were accrued. Primary endpoints were progression-free survival (PFS), CNS recurrence, and liposomal cytarabine-associated toxicity. Eighty percent of patients received the whole treatment programme, the remaining cases received at least three full courses. Application of the RD-CODOX-M/IVAC regimen resulted in remarkable 4-year PFS (78%) and complete remission (CR) rates (93%). However, PFS was significantly lower in patients older than 60years as compared to younger ones (49%vs 93%, P=0·03; median, 36months), despite high actual dose-intensity, CR rate and tolerability. Reduced-intensity intratechal prophylaxis through liposomal cytarabine was effective because the CNS failure rate was low (3·4%) and without severe neurological toxicities. The RD-CODOX-M/IVAC strategy is feasible and highly effective, but improving outcomes in elderly patients remains a priority.

Non-Hodgkin lymphomas arising from mature post-thymic T cells (T-NHL) encompass biologically heterogeneous malignancies with a dismal prognosis (1, 2). Patients with histotypes such as peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), kinase-negative anaplastic large cell lymphoma (ALK-negative ALCL), angioimmunoblastic T-cell lymphoma, and Sezary syndrome (SS) experience early disease recurrence with 5-yr overall survival rates rarely exceeding 40%, also because of the lack of effective salvage strategies (1–3). While the biologic basis of T-NHL aggressiveness is under extensive research, investigation into novel active agents remains a critical need, especially in the setting of stem cell transplantation ineligibility/failure and end-stage disease (2, 4). Fotemustine is a third-generation chloroethylnitrosourea displaying a significant clinical efficacy in highly chemoresistant cancers such as disseminated melanoma and primary or metastatic brain tumors (5–9). While second-generation nitrosoureas have been incorporated into standard and high-dose salvage regimens for NHL, including T-cell subtypes (10–13), the activity of fotemustine in lymphoma has been poorly explored, despite its lower hepatic and renal toxicity, as compared to congener agents, and several more favorable pharmacodynamic properties (14, 15). First, the presence of a phosphoalanine carrier group renders fotemustine more lipophilic because its octanol/water partition coefficient results within the optimal lipophilicity interval, differently from both carmustine and lomustine (16, 17). Second, preclinical studies on rat and human tissues have shown that intracellular penetration of fotemustine is superior to that of carmustine (18, 19). Third, studies have documented that fotemustine is cleared 2–5 times more slowly than carmustine from tumor tissues (17–19). The higher lipophilic properties, the more efficient tissue distribution, and slower elimination of fotemustine from tumor tissues, as compared to other nitrosoureas, might provide a preclinical rationale for testing this drug in chemorefractory lymphomas. Because normal and malignant T cells express a low level of O6-alkylguanine-DNA alkyltransferase (AGT) and of O6-methylguanine-DNA methyltransferase (MGMT) (20, 21), two DNA repair enzymes implicated in cellular resistance to nitrosureas, and specifically to fotemustine (22–25), we speculated that alkylator-refractory T-NHL could represent a proper setting to explore the potential clinical activity of this newer nitrosourea derivative. Here, we report the first evidence ever that fotemustine is able to induce substantial clinical responses in patients with post-thymic T-cell malignancies.

This Phase II study assessed feasibility and efficacy of a biweekly R-COMP-14 regimen (rituximab, cyclophosphamide, non-pegylated liposome-encapsulated doxorubicin, vincristine and prednisone) in untreated elderly patients with poor-risk diffuse large B-cell lymphoma (DLBCL) and moderate to high ‘life threat’ impact NIA/NCI cardiac comorbidity. A total of 208 courses were delivered, with close cardiac monitoring, to 41 patients (median age: 73 years, range: 62–82; 37% >75 years) at a median interval of 15·6 (range, 13–29) days; 67% completed all six scheduled courses. Response rate was 73%, with 68% complete responses (CR); 4-year disease-free survival (DFS) and time to treatment failure (TTF) were 72% and 49%, respectively. Failures were due to early death (n = 3), therapy discontinuations (no-response n = 2; toxicity n = 6), relapse (n = 6) and death in CR (n = 3). Incidence of cardiac grade 3–5 adverse events was 7/41 (17%; 95% confidence interval: 8–31%). Time to progression and overall survival at 4-years were 77% and 67%, respectively. The Age-adjusted Charlson Comorbidity Index (aaCCI) correlated with failures (P = 0·007) with patients scoring ≤7 having a longer TTF (66% vs. 29%; P = 0·009). R-COMP-14 is feasible and ensures a substantial DFS to poor-risk DLBCL patients who would have been denied anthracycline-based treatment due to cardiac morbidity. The aaCCI predicted both treatment discontinuation rate and TTF.

To determine the efficacy and safety of the combination of gemcitabine plus oxaliplatin, with and without rituximab, in patients with relapsed/refractory B-cell lymphoma unsuitable for high dose therapy.Patients were prospectively enrolled in two subsequent trials, GEMOX [gemcitabine (1200 mg/m(2), days 1 and 8) and oxaliplatin (120 mg/m(2), day 2), three-weekly] and R-GEMOX [rituximab (375 mg/m(2), day 1), gemcitabine (1200 mg/m(2), day 1) and oxaliplatin (120 mg/m(2), day 2), bi-weekly], up to six courses.Sixty-two patients were enrolled: GEMOX [n = 30; median age, 66 years (range, 46-85); previous chemotherapyor =2, 70%; PS ECOGor = 2, 57%]; R-GEMOX [n = 32; median age, 65 years (range 32-79); previous chemotherapyor =2, 75%; PS ECOGor = 2, 47%]. Overall and complete response rates were 57 and 30% (95% CI, 15-49) for GEMOX and 78 and 50% (95% CI, 32-68) in R-GEMOX, respectively. Grade 3/4 neutropenia occurred in 57 and 47% of cycles and grade 3/4 thrombocytopenia in 26 and 17% of courses for GEMOX and R-GEMOX, respectively. At 42 months, the failure-free survival (FFS) was 7% (95% CI, 0-16) for GEMOX and 28% (95% CI, 9-47) for R-GEMOX (P = 0.014), with overall survivals of 7 (95% CI, 0-16) and 37% (95% CI, 20-55), respectively (P = 0.016).Both regimes showed good tolerability and appealing response rates. FFS was more prolonged in R-GEMOX, but patients continuously relapsed without a clear plateau on survival curves.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the emergence of a GPI-defective clonal hematopoiesis. Its clinical features are hemolytic anemia, cytopenia, and thrombosis. Circulating monocytes and granulocytes are largely GPI-defective in PNH patients. This study aims to investigate the granulocyte functional properties in PNH. We analyzed bacterial-dependent intracellular ingestion and the consequent activation of oxidative burst in GPI-defective granulocytes from four neutropenic PNH patients. Our data show a significant increase in the ability of GPI-defective granulocytes to ingest opsonized bacteria. In addition, an impaired respiratory burst effectiveness in response to two independent bacterial stimuli, the N-formyl-MetLeuPhe (fMLP) synthetic bacterial peptide and E. coli, was revealed. The occurrence of neutropenia and the severe impairment of oxidative burst, occurring in chronic granulomatosis disease, were unable to significantly affect phagocytosis. Thus, additional mechanisms, able to differentially affect ingestion ability and respiratory burst effectiveness, have to be hypothesized. The reduced burst effectiveness of GPI-defective granulocytes was maintained after treatment with phorbol 12-myristate 13-acetate, a pharmacological stimulus able to extensively recruit and to trigger intracellular protein kinase C (PKC). Moreover, blocking of PKC has been observed to severely affect granulocyte respiratory burst with a mild effect on the phagocytosis. These data suggest a role for a modulation of intracellular PKC in the pathogenesis of the impaired granulocyte oxidative burst. (c) 2006 Wiley-Liss, Inc.

Paroxysmal nocturnal haemoglobin- uria (PNH) is a haematopoieis disorder character- ized by the expansion of a stem cell bearing a somatic mutation in the phosphatidylinositol gly- can-A (PIG-A) gene, which is involved in the bio- synthesis of the glycosylphosphatidylinositol (GPI) anchor. A number of data suggest the inability of the PIG-A mutation to account alone for the clonal dominance of the GPI-defective clone and for the development of PNH. In this context, additional immune-mediated mechanisms have been hypoth- esized. We focused on the analysis of T lympho- cytes in three PNH patients bearing a mixed GPI and GPI - T cell population and showing a marked cytopenia. To analyze the biological mechanisms underlying the control of T cell homeostasis in PNH, we addressed the study of CD40-dependent pathways, suggested to be of crucial relevance for the control of autoreactive T cell clones. Our data revealed significant, functional alterations in GPI and GPI - T cell compartments. In the GPI - T cells, severe defects in T cell receptor-dependent prolif- eration, interferon- production, CD25, CD54, and human leukocyte antigen-DR surface expres- sion were observed. By contrast, GPI T lympho- cytes showed a significant increase of all these pa- rameters, and the analysis of CD40-dependent pathways revealed a functional persistence of CD154 expression on the CD48CD4 lympho- cytes. The alterations of the GPI T cell subset could be involved in the biological mechanisms underlying PNH pathogenesis. J. Leukoc. Biol. 78: 000-000; 2005.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, acquired hematopoietic disorder characterized by a phosphatidylinositol (PI) glycan-A gene mutation, which impairs the synthesis of the glycosyl-PI (GPI) anchor, thus causing the absence of all GPI-linked proteins on the membrane of the clonal-defective cells. The presence of a consistent GPI-defective monocyte compartment is a common feature in PNH patients. To investigate the functional behavior of this population, we analyzed its in vitro differentiation ability toward functional dendritic cells (DCs). Our data indicate that GPI-defective monocytes from PNH patients are unable to undergo full DC differentiation in vitro after granulocyte macrophage-colony stimulating factor and recombinant interleukin (IL)-4 treatment. In this context, the GPI-defective DC population shows mannose receptor expression, high levels of the CD86 molecule, and impaired CD1a up-regulation. The analysis of lipopolysaccharide and CD40-dependent, functional pathways in these DCs revealed a strong decrease in tumor necrosis factor α and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.

DMSO-associated encephalopathy during autologous peripheral stem cell infusion: a predisposing role of preconditioning exposure to CNS-penetrating agents?

Specific trials on adult Burkitt lymphoma (BL) and 'unclassifiable' lymphomas with features intermediate between BL and diffuse large B-cell lymphoma (BL/DLBCL) are advocated which include substantial numbers of older patients, to improve treatment feasibility, while countering risks of systemic and central nervous system (CNS) recurrences. We prospectively evaluated a modified CODOX-M/IVAC (CODOX-M: cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate; IVAC: ifosfamide, etoposide and high-dose cytarabine) regimen by the addition of rituximab (R) and liposome-encapsulated cytarabine (D) to increase antitumour activity and halve the number of intrathecal treatments. Thirty adults (40%60years) with BL (n=15) and BL/DLBCL (n=15) were accrued. Primary endpoints were progression-free survival (PFS), CNS recurrence, and liposomal cytarabine-associated toxicity. Eighty percent of patients received the whole treatment programme, the remaining cases received at least three full courses. Application of the RD-CODOX-M/IVAC regimen resulted in remarkable 4-year PFS (78%) and complete remission (CR) rates (93%). However, PFS was significantly lower in patients older than 60years as compared to younger ones (49%vs 93%, P=0·03; median, 36months), despite high actual dose-intensity, CR rate and tolerability. Reduced-intensity intratechal prophylaxis through liposomal cytarabine was effective because the CNS failure rate was low (3·4%) and without severe neurological toxicities. The RD-CODOX-M/IVAC strategy is feasible and highly effective, but improving outcomes in elderly patients remains a priority.

Patients with recurring T-cell non-Hodgkin lymphoma (T-NHL) are incurable and candidate for investigational agents. Here, we report on five patients with T-NHL refractory to multiple chemotherapy lines, including in all cases alkylators and gemcitabine, who received the third-generation chloroethylnitrosourea fotemustine at a dose of 120 mg/m(2) every 21 d, up to eight courses. Median actual dose intensity was 79%; toxicity was manageable and mainly hematological. One complete remission, one partial remission, two protracted disease stabilization, and one transient, minor response were achieved. Time to progression ranged from 48 to 240+ d. This is the first evidence ever reporting the activity of fotemustine in end-stage T-NHL. Formal studies with this agent are warranted in T-cell malignancies.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is unique among the members of the TRAF family, as it lacks the N-terminal RING/zinc-finger domain. Also the function of TRAF1 is not clearly established, with many papers reporting contradictory results. Here we show that TRAF1 interacts with BAFF receptor, a member of the TNF receptor family, and positively regulates activation of the alternative NF-kappaB pathway. Ectopic expression of TRAF1 causes degradation of TRAF3, stabilization of NIK, and processing of p100 to produce the mature form p52. In addition, we show that knocking-down expression of TRAF1 in the Hodgkin's disease derived cell line L1236, interfere with p100 processing and with p52 mediate gene transcription. Collectively these results support a role for TRAF1 as a positive regulator of the NF-kappaB alternative pathway.

Patients with Hodgkin lymphoma (HL) failing salvage stem cell transplantation are candidates to investigational strategies. Lenalidomide represents an attractive option as it targets several signaling pathways, which regulate survival of HL cells and their microenvironmental interactions. We report the occurrence of Grades 2 and 3 tumor flare reactions in the first three patients entered a lenalidomide-based compassionate program for treatment-refractory HL. Flares occurred in concomitance of the scheduled week-off lenalidomide and upon withdrawal of symptomatic steroid treatment, and were associated with changes in B-cell regulatory cytokines and the concurrent expansion of polyclonal B-cells. Flares mimicked tumor progression but were effectively managed with anti-inflammatory treatment and followed by a clinical response, suggesting that they may mirror the pleiotropic actions of lenalidomide on HL microenvironment.

Background: Given the consistent antitumor activity and favorable toxicity profile in heavily pretreated patients, BDM represents a suitable platform for combination with target-based agents in the setting of recurrent HL. The anti-CD30 antibody-monomethyl auristatin E (MMAE) conjugate brentuximab vedotin (BV) appears a most valuable candidate by coupling an impressive clinical activity with the lack of serious overlapping toxicities with BDM. While the combination of BDM and BV is being evaluated in Phase II studies, no information is available as to possible mechanisms through which BDM may regulate the antitumor efficacy of BV towards HL cells. Methods: We evaluated the effects of acute and extended exposure to BDM on CD30 expression and sensitivity to the cytotoxic activity BV in the HL cell line L1236. Cells were cultured in the presence of BDM at its IC50 for different time points of acute exposure. Through continuous exposure to increasing concentrations (25.0, 50.0, 75.0, 100 micromol/L) of BDM, we then performed a serial in vitro selection for BDM-resistant (R) L1236 cell clones and determined their CD30 expression by flow cytometry, qRT-PCR, and Western analysis. Results: Acute exposure to BDM (48 to 72 hrs) of L1236 cells led to a sizeable upregulation of CD30 as shown by flow cytometry. This effect was unsustained since CD30 intensity returned to baseline levels upon culturing in BDM-free medium for one week. Expression of other surface antigens, i.e. HLA-DR, was unaffected by BDM while acute exposure to doxorubicin and other cytotoxic drugs did not modify the CD30 expression. We established four L1236-derived cell clones (R25, R50, R75 and R100) able to proliferate across the different concentrations of BDM with growth/viability curves superimposable to native cells. Clonal identity among clones and with parental cells was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. All R-clones displayed an up to 900% increase in CD30 median fluorescence intensity, relative to native L1236 cells (Figure 1A). The sustained CD30 upregulation was confirmed by qRT-PCR and Western blotting since R-clones expressed up to 10-fold higher levels of CD30-specific mRNA and CD30-specific 120 kDa components as compared to parental cells. To rule out a general upregulation of surface molecules as a result of the chronic exposure to BDM we evaluated the expression of other surface antigens. As opposed to CD30, HLA-DR and PDL-1 relative transcript levels and cell surface expression were significantly reduced in R-cells. Intriguingly, BDM-induced CD30 upregulation was specific to L1236 cells since extended exposure to BDM did not modify expression levels of CD30 in other lymphoma cell types (SUDHL1, JEKO). BDM-induced CD30 overexpression significantly enhanced the sensitivity of HL cells to the cytotoxic effects of BV. MTS assay showed the IC50 of R100 cells to BV shifted to 0.21 ± 0.06 mcg/mL (48 hrs) and 0.19 ± 0.05 mcg/mL (72 hrs) vs. 3.16 ± 0.75 mcg/mL (48 hrs) and 3.87 ± 0.68 mcg/mL (72 hrs) of parental cells, a 15- and 20-fold increase, respectively (p Conclusions: Whileacute exposure to BDM induced a transient upregulation of CD30, extended exposure to the agent resulted in the selection of L1236 subclones with a stable overexpression of surface CD30 and reduced levels of HLA-DR and PDL-1. Upregulation of CD30 was associated with a significantly enhanced sensitivity to cytotoxic effects of BV. Translated into a clinical context these findings suggest that HL patients progressing upon BDM treatment may turn exquisitely sensitive to BV and prompt the exploration of a sequential treatment with BDM and BV in late and earlier disease phases. Results also provide a mechanistic insight as to the efficacy of the BDM-BV combination in HL, highlighting non-overlapping resistance pathways between BDM and MMAE. BDM-induced selection of CD30hi, HLA-DRlow/PDL-1low tumor cell clones might be relevant to the design of novel treatment strategies for HL based on the combination of BDM with BV and anti-PD1/PDL-1 antibodies. Figure 1. Expression of CD30 and HLA-DR in parental (L1236) and BDM-resistant (R) HL cells Figure 1. Expression of CD30 and HLA-DR in parental (L1236) and BDM-resistant (R) HL cells Figure 2. Sensitivity to BV of parental (L1236) and BDM-resistant (R100) HL cells Figure 2. Sensitivity to BV of parental (L1236) and BDM-resistant (R100) HL cells Disclosures Pinto: Takeda: Research Funding; Takeda, Celgene, Roche, TEVA: Honoraria.

8564 Background: Patients (pts) with peripheral T/NK cell lymphomas (PTCL) and intermediate-high/high IPI risk have a 5-yr overall survival < 20%. Current chemotherapy is unsatisfactory while benefit of upfront autologous transplantation (ASCT) is limited by high pre-transplant progression rates and pts advanced age. We evaluated efficacy and stem cells (SCs)-mobilizing activity of a biweekly regimen of gemcitabine (G), ifosfamide (Ifo) and oxaliplatin (Ox) (GIFOX), as an upfront strategy ensuring fast cytoreduction and early ASCT access or an effective alternative to CHOP-like programs in transplant-inelegible pts. Methods: Six biweekly courses of GIFOX [G 1000 mg/m2 D1, Ox 130 mg/m2 D2, Ifo 5 g/m2 D2 as 24h infusion (fractionated over days 2-4 in pts>65 yrs), G-CSF DD 7-11] were planned for all pts, with SCs mobilization at course 3 in ASCT-eligible pts. Simon's minimax two-stage design was adopted with the primary and secondary endpoints of response rate (RR) and progression-free survival (PFS), respectively. Results: Thirty-four pts (median age 63 yrs, r 42-80) [PTCL, nos (n=16), AITL (n=7), extranodal NK/T-cell (n=5), SS (n=6)], with IPI score intermediate-high (62%) or high (38%) were accrued [stage IV: 71%; BM involvement: 38%; E-site >1: 47%; hi LDH: 71%; ECOG>1: 38%; B-symptoms: 44%]. A total of 172 courses was delivered (median 6, r 2-6). Only 5 pts received + cells harvest: 4.36x106/kg) and 8 (7CRs,1PR) underwent ASCT, 6 to 13 weeks after the 6th course. Estimated 5-yr PFS was 48% (95%CI: 28-65); median PFS for non-transplanted pts was 15 mo.s. Estimated 4-yr disease-free survival was 58%. Relevant toxicities were G4 thrombocytopenia (13%), G4 anemia (23%), G3/G4 infection (29%/6%), G3 encephalopathy (6%). Conclusions: Response and survival rates of GIFOX in high-risk PTCL compared more than favorably to CHOP-based regimens. Effective cytoreduction and prompt access to ASCT were ensured, together with safe delivery of a full induction program to transplant-ineligible pts.

8066 Background: The role of consolidation RT on MBD after upfront chemotheraphy for advanced HL is debated, also given the supradditive iatrogenic risk. We present the results achieved in the subset of patients (pts) with MBD (max width > 1/3 of thoracic diameter) accrued in a phase II study of an intensified ABVD program without RT. Methods: The current analysis derives from the final evaluation of our trial for advanced HL (stage IIXB-IV) conducted from 06/2004 to 03/2010 (Russo et al, ASH 2009 abst 715). Pts were scheduled to 6 cycles of a ‘time-densified’ ABVD (3-week intercycle, drugs on days 1 and 11) with the first 4 cycles being also ‘dose-intensified’: doxorubicin (ADM) 35 mg/m2, days 1 and 11 and G-CSF on days 6-8 and 17-19. Results: Of 82 accrued pts, 39 had BMD at presentation. Median age was 29yrs (r 16-58); male 46%; stage IIB 48%, III 8%, IV 43%; B-sympt 87%, E-disease 53%; IP Score ≥3 51%. All pts completed the intensified program. Median actual dose intensities for ADM, bleomycin, vinblastine and dacarbazine were 23.12, 6.69, 3.96 and 245 mg/week, respectively; the increase over conventional ABVD was 85% for ADM and averaged 32% for the other agents. PET2 negativity was achieved in 36/39 (92%; 95% CI 79-98), complete responses (CR) in 37/39 [94%; 95% CI 82-99]. At a median f.u. of 54 mo.s (r 20-91) all pts are alive with an event-free survival of 89% (95% CI, 80-98). Events were: 1, DLCO,FEF25-75) were seen. Conclusions: Intensified ABVD can achieve PET2 negativity in a very high proportion of pts with MBD and ensure a long-term disease-free status even without RT. While results need confirmation on a randomized basis, the low mediastinal failure rate seems in line with recent suggestions that RT could be omitted in MBD when CR is achieved upon intensified chemotheraphy.

Abstract 267 Introduction: The tumor cells of HL, the Hodgkin and Reed-Sternberg (H-RS) cells, derive from germinal center B-cells with a deranged B-cell transcription program due to epigenetic silencing and acquired genetic lesions. Tissue H-RS cells are surrounded by a preponderant infiltrate of mixed non-malignant reactive cells, including B-lymphocytes, which provide essential signals for their survival and proliferation. Small percentages of B-cells, clonally related to H-RS cells, were found in the blood of HL patients (pts), suggesting they may represent putative HL-initiating cells (Jones, 2009). Since the serum free light chain (sFLC) assay detects and quantifies monoclonal and polyclonal B-cell populations expanding in lymphohemopoietic tissues, we exploited the sFLC testing to further explore the biologic significance of B-lymphocytes in HL. Patients and Methods: Frozen (-80°C) serum samples from 119 untreated cHL pts (48% males), with normal renal function and serum immunochemistry, were assayed by immunonephelometry (Freelite, The Binding Site, Ltd., UK). After quantization of free κ and λ concentrations (normal ranges, κ: 3.3-19.4 mg/L; λ: 5.71-26.3 mg/L), sFLC κ/λratio was calculated (reference range 0.26-1.65). The median age was 31 years (r, 15–70), with 22% aged ≥ 45 years. Histology was nodular sclerosis in 85 pts (71.4%) and 72 (60.5%) were in stage I–II. According to GHSG criteria, 16% had early favorable, 29% early unfavorable and 55% advanced disease. The International Prognostic Score (IPS) was of 0-2 and ≥ 3 in 66.4% and 33.6% of pts, respectively. Following ABVD (4-6 courses ± RT), the median Event-free survival (EFS) was 78 mo.s [95% CI, 68-88] for the entire population and 75 mo.s [95% CI, 60-90] and 64 mo.s [95% CI, 50-77] for pts in early and advanced stage, respectively. Results: Elevated κor λsFLC concentrations were found in 47% (median 29.55 mg/L; r, 19.44 - 64.70) and 29.4% (median 32.70 mg/L; r, 26.60 - 79.77) of pts, respectively. In 30 pts (25.2%) levels of polyclonal κ and λ sFLC were concurrently elevated. The sFLC κ/λratio was abnormal in only 7.5% of pts (clonal λ: 8/119; clonal λ: 1/119). The presence of high sFLC levels correlated with lymphopenia (< 0.6 × 109/L; p= 0.04), leukocytosis (WBC > 15 × 109/L; p=0.03), ESR (> 50; p=0.04) and unfavorable IPS (≥ 3; p=0.03), but not with EBV status and risk factors such as stage, B symptoms, bulky and extra nodal disease, LDH and albumin. The association with leukocytosis may in part result from inhibition of spontaneous neutrophils apoptosis exerted by FLC (Cohen, 2003). Most interestingly, while we found no significant association with response rate, baseline elevation in sFLC predicted for EFS in 51 evaluable pts with early stage disease. Patients were divided into tertiles and best break point value for predicting EFS coincided with the upper limit of the highest tertile for both κ (>25.53 mg/L) or λsFLC (>26.66 mg/L) (Figure 1). The pts in the top tertile had the worst outcome compared with the 2 lower tertiles (κ, p=0.015; λ, p=0.002). Outcomes for the 2 lower tertiles were comparable. Data remained significant after stratification for the IPS. In contrast, baseline sFLC levels, κ or λ, were not predictive for EFS in pts with advanced disease. Interestingly, sFLC levels in a control group of 30 pts in continuous CR from 2 years, were within the normal ranges. Conclusions: We have shown that about 50% of cHL pts displays elevated levels of sFLC mirroring the presence of a consistent polyclonal B-cell expansion at diagnosis. The role of reactive B-cells in HL is poorly understood. While some data indicate that high intratumoral B-cell counts may predict for a better outcome, the activity of rituximab in cHL, regardless of CD20 expression on H-RS cells, was suggested to result by depletion from the HL microenvironment of normal B lymphocytes required for tumor cell growth (Younes, 2003). Our study support that elevated sFLC levels may reflect an increased polyclonal B cell activity in cHL microenvironment which appears to negatively influence the outcome of pts in early stage disease. This effect is lost in advanced disease, suggesting that rituximab might result more active for pts in early than advanced stages. That putative HL-initiating small B-cells may emerge from the expanded polyclonal B-cell population present from the early phases of disease development, is an intriguing possibility. Disclosures: Marchei: Radim, Italy: Employment. Amoroso:The Binding Site, Ltd: Consultancy.

Abstract 715 We previously reported interim results (Blood-ASH Annual Meeting Abstracts-2007 110: abstract 2324) of a prospective study evaluating dose-dense and dose-intense variants of ABVD regimen. Seventy pts with a newly diagnosis of cHL were enrolled from 06/04 to 03/08. Pts had intermediate-(INT) or advanced-stage (ADV) according to GHLSG criteria (Tab.1). INT (n=24) were treated with dd ABVD, and ADV (n=46) with dd-di ABVD. Briefly, the strategy concepts of treatment were: 6 cycles of chemotherapy; the inter-cycle period shortened from 28 to 21 days; drugs delivered at day 1 and 11 of each cycle; In the dd-di-ABVD, adriamycin was escalated from 50 to 70 mg/m2 in cycles 1-4; primary G-CSF was given; the therapy was driven by interim-FDG-PET; radiotherapy was planned with very stringent criteria. TOXICITY: On a total of 838 courses of chemo we had 18 events which needed red cell transfusion; 5 of severe thrombocytopenia completely reversed within one week; 25 of severe neutropenia ( DOSE INTENSITY STUDY: Median duration of chemotherapy (planned 18-wk) was 19.7-wk (range 17.6-21.7). Planned and delivered RDIs of drugs were significantly higher as compared with the most used regimens (Tab.2). RESPONSE: Early-CR (PETneg > 2 cycles) was obtained in 65/70 pts (95%). No statistical differences was noted between INT and ADV subsets. At the end of 6thcycle 69/70 pts (98,6%) were in CR. Three out of 69 complete responders (4.3%) had a biopsy-proven relapse: a 33-yr old man ( IVEA), a 27-yr old girl (IIB) and 29-yr old girl ( IVXEB). Relapsed occurred at 3, 10 and 14 months from the end of Tx, respectively. SURVIVAL: Data with a minimum follow up of 12-mo from the end of Tx were available in all 70 pts. Fig.1 shows the EFS rates of 24 INT-stage (95.8%), and 46 ADV-stage (91.3%) pts, respectively. A comparative analysis (Fig.2) between this series of 70 pts treated with dd ABVD or dd-di ABVD and the last 70 historical INT-stage (n=25) and ADV-stage (N=45) pts treated with baseline ABVD shows a statistically significant increment in EFS rate in pts receiving intensified ABVDs (93.0% vs 73.2% p=0.0041). CONCLUSIONS: The final results of this study shows that the activity of intensified ABVD is significantly higher than baseline ABVD in terms of response (CR) and survival (EFS) rates, still maintaining a low-toxicity profile. Based on these results a randomised comparison of intensified versus baseline ABVD seems justified. Disclosures: No relevant conflicts of interest to declare.

Abstract 3716 Poster Board III-652 Introduction Intrathecal (IT) chemotherapy is an integral component of treatment for Burkitt lymphoma (BL), together with intensive systemic chemotherapy including blood brain barrier crossing agents such as high dose cytarabine (Ara-C) and methotrexate (MTX). However, the optimal IT treatment is yet to be established. Since cytotoxic concentrations of conventional IT agents (Ara-C and MTX), are maintained in the cerebrospinal fluid (CSF) for only a few hours, repeated lumbar punctures are necessary which may turn cumbersome for patients (pts) and pose technical difficulties in some instances. The availability of a sustained-release formulation of Ara-C (liposome-encapsulated Ara-C; Depocyte®) may offer the opportunity of reducing the total number of IT administrations by maintaining/enhancing, the efficacy of CNS prophylaxis. In this regard, a single 50 mg IT injection of Depocyte is able to achieve cytoxic concentrations of free-Ara-C in the CSF for 10-14 days. We report the results of a prospective phase II study aimed at evaluating the safety/activity profile of IT Depocyte in pts with BL and atypical (a)BL. Patients and Methods The study was designed to assess, in untreated pts with BL and aBL, the safety and feasibility of a 50% reduction (from 8 to 4) of the number of IT injections required by substituting IT Ara-C (4 doses) and MTX (4 doses) with 4 administrations (50 mg) of IT Depocyte. IT injections were planned on days (d) 1 or 2 of each rituximab (R)-CODOX-M courses and on d 8 of each R-IVAC course. Pts with aBL received 2 additional R-CODOX courses without any further IT therapy. Primary study endpoints were safety, Depocyte-related extra-hematologic adverse events ≥G3, and CNS failure, i.e. progression/relapse at leptomeningeal and/or parenchymal sites; secondary endpoints included event- and disease-free survival (EFS, DFS). Results A total of 30 HIV negative pts (15 BL and 15 aBL) were enrolled and treated with a dose-modified Magrath regimen (Lacasce, 2004). The median age of pts (M/F: 22/8) was 53 years (r, 25-78), 10 pts (33%) were considered as a low risk (LR) category by displaying ≥ 3 of the following factors: normal LDH, WHO PS 0-1, Ann Arbor stage I-II, and ≤ 1 extra nodal sites. All remaining cases (67%) were considered as high-risk (HR). At diagnosis, 3 pts (10%) had a positive CSF for lymphoma, 6 (20%) had bone marrow involvement and 12 (40%) bulky (>10 cm) disease. Each pt received a median of 4 (r, 1-6) IT injections of Depocyte at the a median day of 0.5 (r, -1 to 1) for R-CODOX-M1 (course1), 4.0 (r, -2 to 17) for R-IVAC1 (course 2), 1 (r, -1 to 1) for R-CODOX-M2 (course 3) and 6.5 (r, -3 to 14) for R-IVAC2 (course 4). On a total of 111 applications, the following IT injection-related adverse events (NCI-CTCAE v 3.0) of G1-G2 severity were recorded (pts experiencing toxicity): headache 26.6%, nausea 6.6%, vomiting 3.3%, fever 10%, lumbar pain 10%, fatigue 26.6%, somnolence 6.6% and sinus bradycardia 3.3%. A G3 headache episode, accompanied by a transient loss in visual acuity, led to refusal of further IT Depocyte by a single pt. At 51 mo.s, the EFS was 70% with a DFS of 90% at a median observation of 24 mo.s (r, 1-49). Among the 27 complete responders, no isolated leptomeningeal relapses occurred. In particular, none of the 3 responders with CSF involvement at presentation, displayed any form of CNS progression (leptomenigeal and/or parenchymal). In contrast, a parenchymal CNS involvement, with a negative CSF, was observed, at 4.0 mo.s, as a part of the systemic progression in a single pt (3.3%) with chemorefractory aBL. This pt presented with unfavorable features (hi-LDH, stage IVB, PS 2, bulky retroperitoneal adenopathy and 4 extra nodal sites including liver, pancreas, kidney and spleen) and skipped both the first IT and systemic MTX administrations. Conclusions Within the limits of a single arm study, our results show that substitution of 8 IT injections (Ara-C/MTX) with 4 IT administrations of Depocyte within the R-CODOX-M/IVAC regimen is feasible and devoid of severe and/or life-threatening/invalidating neurotoxicity. The CNS progression/recurrence rate was at least super imposable to historical results (CNS relapse rate: 6% to 11%) achieved by the Magrath regimen including double-agent IT treatment. Based on these results, it appears that Depocyte can be safely incorporated into the Magrath regimen to provide adequate single-agent CNS prophylaxis with a reduced burden of IT applications. Disclosures: Off Label Use: Liposome-encapsulated Ara-C for CNS prophylaxis. Vitolo:Mundipharma: Lecture fees. Pinto:Mundipharma: Lecture fees.

8579 Background: Gemcitabine (G), ifosfamide (Ifo), oxaliplatin (Ox) and rituximab (R) have been accounted of cross-synergy in preclinical and early clinical studies in Hodgkin lymphoma (HL). We assessed activity, toxicity and stem cells (SCs) mobilizing capacity of a bi-weekly salvage combination with these agents in HL recurring after conventional or high dose therapy (HDT). Methods: Patients were scheduled to receive 3 R-GIFOX courses followed by SCs mobilization and HDT if elegible for autologous transplantation (ASCT) or 3 more courses if not. R-GIFOX consisted of R 375 mg/m2 D1, G 1000 mg/m2 D2, Ox 130 mg/m2 D3 and Ifo 5 g/m2 D3, as a 24-h single infusion, G-CSF 5 mcg/kg/d DD 7–11 (10 mcg/kg/d, 3rd course until SCs mobilization). Results: Twenty-one patients (median age 33 yrs, r 22–64) with relapsed (n = 16) [post-ASCT (n=6), 12 mo.s (n=3)] or primary progressive (n = 5) HL, were prospectively accrued. Ten patients (48%) had received ≥ 2 previous CHT lines and 15 (78%) had GHLSG recurring HL prognostic score ≥ 2. Eighty-three total courses were delivered (median 3, r 3–6). CTCAE v3.0 G4 thrombocytopenia occurred in 18% of courses, G4 infection in 11%. Ifosfamide was withdrawn at the 4th course in 2 patients, both aged 64 yrs, due to tachyarrhythmia and encephalopathy. Actual dose intensity of the first 3 courses was 82%, 86%, 92 % for G, Ifo and Ox, respectively. The overall response according to FDG-PET/IWC criteria after 3 courses was 86%, with 2 partial and 16 complete responses (CRs) (76%; CR=10, CRu=6). Four CRs were achieved among the 6 patients with post-ASCT relapses. Eight of 14 eligible patients had effective CD34+ cells harvest [median 4,35 × 106/kg (r 2,91–11.45)] and proceeded to subsequent ASCT. Five ’bad mobilizers’ had previously undergone radiation therapy (n=3) and radioimmunotherapy (n=2). At 42 mo.s. Failure Free Survival was 57%. At a median f.u. of 12 mo.s for CRs, Disease Free Survival was 79% in patients eligible for ASCT and 41% in those unfit treated with additional R-GIFOX. Conclusions: R-GIFOX retains an attractive therapeutic potential in recurring HL, enabling pre-ABMT cytoreduction and mobilization, and also a safe delivery of a full salvage program to patients unfit for HDT. No significant financial relationships to disclose.

Background: We evaluated the clinical activity, toxicity, and stem cells mobilizing capacity of a short-course (bi-weekly), dose intensive, salvage regimen (R-GIFOX) in patients with relapsed and refractory Hodgkin Lymphoma. The agents Gemcitabine, Ifosfamide, Oxaliplatin and Rituximab employed in this combination have been all accounted of cross-synergistic activity in both preclinical and early clinical studies in Hodgkin lymphoma. Patients and methods: Patients were scheduled to receive three courses of therapy followed by mobilization and ASCT or three more courses if ineligible for ASCT. R-GIFOX consisted of Rituximab (375 mg/m2 on day 1), Gemcitabine (1000 mg/m2 on day 2), Oxaliplatin (130 mg/m2 on day 3) and Ifosfamide (5 g/m2 on day 3), as a 24-hour single infusion. Treatment was given every two weeks with G-CSF support (5 mcg/kg/day or 10 mcg/kg/day at the end of the third course for stem cells mobilization). Responses were evaluated by the integrated FDG-PET/IWC criteria, after the third course and at the end of the entire program. Results: Fifteen patients (median age 36 years, range 24–64 years) with relapsed (n = 12) [post-ABMT (n=4), < 12 mo.s (n=6), > 12 mo.s (n=2)] or primary progressive (n = 3) Hodgkin Lymphoma, were accrued in this prospective study. Forty percent of them had received 2 or more previous treatment lines. Stage IV at recurrence was found in 9 patients (60%), B symptoms in 7 (46%). Sixty-two total courses were delivered (median 3, range, 3–6). Hematologic toxicity was tolerable, but CTCAE v3.0 G4 thrombocytopenia was found in 15% of courses and infection occurred in 9% of cycles. Ifosfamide was withdrawn in two patients; both aged 64 years, at the fourth course due to the occurrence of tachyarrhythmia and encephalopathy, respectively. Actual dose intensity of the first 3 courses was 84%, 87%, and 91% for Gemcitabine, Ifosfamide, and Oxaliplatin, respectively. All the patients completed at least 3 courses of therapy and were valuable for response. The overall response rate assessed after three courses of R-GIFOX was 87%, with 12 complete responses (CRs) (80%; CR=8, CRu=4) and 1 partial response. Interestingly 3 CRs were achieved among the four patients with post-ABMT relapses. Effective CD34+ cell mobilization was obtained in 6 out of 11 eligible patients and all of them proceeded to subsequent ASCT. The median number of collected CD34+ cells was 4,16 × 106/kg body weight (range, 3.52–10.95) In all instances a single leukapheresis was performed. Among the five ‘bad mobilizers’, two patients had previously undergone radiation therapy and one had received immunoradiotherapy with Ibritumomab Tiuxetan. Failure Free Survival was 46% at 32 mo.s and Disease Free Survival 53% at 30 mo.s. with a median follow-up of complete responders of 11 mo.s. Conclusions: The present study confirms the attractive therapeutic potential of Gemcitabine/Ifosfamide-based combination in the setting of recurrent Hodgkin Lymphoma and further suggests additional contribution from Rituximab and Oxaliplatin. R-GIFOX may represent a less toxic, cytoreductive, and stem cell mobilizing alternative to toxic cisplatin-based salvage regimes, useful for pre-ABMT cytoreduction but also for a full salvage treatment program to be safely delivered to patients unfit for high-dose procedures.