ALBERT

Description: Abstract 3124 Background. Outcome of aggressive HIV-associated NHL (HIV-NHL) with adverse prognostic features is not satisfactory with standard therapy. High dose therapy (HDT) and autologous stem cell transplantation (ASCT) has been demonstrated safe and active in HIV-NHL in the salvage setting. Aims. To define the role of HDT and ASCT in the upfront treatment of HIV-NHL at high risk, in terms of efficacy and toxicity. We report the interim results of a multicenter study including HDT and ASCT as consolidation after first-line treatment of HIV-NHL at high-risk. Patients and methods. Inclusion criteria: untreated aggressive HIV-NHL (including DLBCL, plasmablastic, anaplastic lymphoma), aa-IPI 2–3, age 18–60, CD4+ count 〉50/mcl and availability of effective HAART. Burkitt and CNS lymphoma are excluded. Patients (pts) receive R-CHOP-21 (CHOP-21 or CHOP-14 for CD20 negative lymphoma) for 6 cycles and, if responsive, undergo stem cells mobilization and collection after Cyclophosphamide 4gr/ms + G-CSF followed by HDT with BEAM and ASCT. Involved-field radiotherapy on previous bulky or residual disease is recommended. Pts receive HAART during the entire treatment program. Overall survival (OS) at 2 years is the primary endpoint. Results. Since January 2007 to July 2012, 23 pts were registered and 20 entered the study. Median age was 47.5 years (range, 27–62). Fifteen pts (75%) had DLBCL, 4 (20%) plasmablastic and 1 (5%) anaplastic lymphoma. ECOG PS was 〉1 in 11 pts (55%); Ann-Arbor stage III/IV, 5(25%)/15(75%); B symptoms, 12 (60%); LDH 〉 n.v., 18 (90%); aaIPI 2/3, 10 (50%)/10(50%). Fourteen pts (70%) had a prior history of HIV-positivity and 13 (65%) were on HAART at NHL diagnosis; thirteen (65%) had detectable HIV-viremia. Median CD4+ count was 283/mcl (58–571). Nine pts (45%) had HCV infection. Seventeen pts received (R)-CHOP-21 and 3 pts with plasmablastic histology received CHOP-14. One pt is still on treatment and 19/20 are evaluable for (R)-CHOP response. One had toxic death (due to hepatic failure), 2 had prolonged cytopenia (1 with severe hepatic toxicity) that lead to withdrawal of therapy and 16 completed (R)-CHOP therapy: 11 pts had complete remission (CR), 4 partial remission (PR) and 1 disease progression (PD) (ORR 79%,CR 58%, PR 21%, according to intention to treat). One pt is ongoing and 14 collected CD34+ cells after Cyclophosphamide + G-CSF (median CD34+ cells 7.35 × 10e6/Kg, range 2.65–10.0). Two pts had early disease progression after collection, 1 did not receive HDT because of cardiac ejection fraction 〈 50% at evaluation before BEAM and 1 is ongoing. So far, 10 pts received ASCT according to the protocol. Moreover, three pts received radiotherapy (2 on previous bulky and 1 on testicular disease). HDT-related toxicities included 2 grade II and 5 grade III GI and 1 grade III hepatic toxicity. Prior to engraftment 1 VZV infection, 1 Clostridium difficile colitis, 1 sigmoiditis and 5 episodes of FUO were registered. There were no transplant-related deaths. One case of CMV reactivation and no opportunistic infections were registered during observation. All transplanted pts are alive and relapse-free after a median of 43 ms (range, 4–58) after transplant (Figure 1). In the entire series, with a median f-up of 29.5 ms (range, 4–65), the 2-years PFS and OS of the entire series from study entry were 73% (+10.3%) and 76% (+10.6%), respectively (Figure 2). Conclusions. This is the first prospective trial addressing the role of HDT and ASCT in first line treatment of HIV-LNH. The procedure was well tolerated and the clinical results highly encouraging. Interim evaluation of OS in this very high risk series of pts is satisfactory and accrual is ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: Rituximab is active as single agent in relapsed CD20-positive NHL. Second line chemotherapeutic regimens in NHL are mainly based on cisplatin (DDP) and are most frequently employed as cytoreductive regimens for high dose chemotherapy and for peripheral blood CD34-positive (PBSC) harvesting. On the basis of preclinical and clinical studies, the substitution of DDP with oxaliplatin (OXDDP) in regimens like DHAP can result in a lower toxicity and higher activity. We studied the tolerability and efficacy of R-DHAOX regimen in the treatment of relapsed and refractory NHL. From November 2002 until June 2004, 33 pts affected by relapsed/refractory NHL were treated with R-DHAOX regimen (Rituximab 375 mg/m2, e.v. day 1, Desametazone 40 mg e.v. days 1–4, Oxaliplatin 130 mg/m2 e.v. day 2 and Cytarabine 2000 mg/m2 e.v. twice a day, day 3). The median age was 51 yrs (range 19–74). Twenty-one pts were in first relapse, 7 pts 〉1 relapse and 5 pts refractory to the first line. The results are summarized in table 1. These data suggested that the R-DHAOx regimen is a novel combination in salvage therapy for relapsed/refractory NHL. It has clinically significant activity with an acceptable toxicity profile, even in HIV-positive pts. R-DHAOX permits adequate stem cell harvest with G-CSF mobilisation and in our experience no delay in engraftment occurred. Supported by ISS and AIRC grants. Table 1 °30 pts evaluable; ^5 HIV-positive pts, 1 HCV-positive pts, 22 elegible for ASCT M/F 21/12 PS 1 (0–2) Median age 51 (19–74) HIV-positive 10 HCV-positive 1 Histology Lymphocytic 2 Follicular 8 (24%) Mantle 2 Diffuse large cell 18 (55%) Burkitt 1 Large cell CD30+ 2 Stage II 1 III 6 (18%) IV 26 (79%) s-IPI 0–1 10 (30%) 〉1 23 (70%) # previous lines 1 (1–2) Dose reduction 65 (55%) Toxicity Neutropenia G3/G4 22 (66%) Anemia G3/G4 8 (24%) Thrombocytopenia G3/G4 21 (64%) Mucositis G3/G4 2 Neurotoxicity G3/G4 1 Response° CR 8 (27%) PR 13 (43%) SD 1 (3%) PRO 8 (27%) Day of PBSC peak +14 (12–18) CFU-GM x10 4 /kg 93 (10.9–230) CD34+ 10 6 /kg 7 (0.65–20) # apheresis 1 (1–2) High dose chemotherapy^ 15 (68%)

Description: We have investigated signaling mediated by GPIb-IX-V, GPVI and α2β1 during platelet adhesion to collagen type I either in the presence or absence of von Willebrand factor (VWF) A1A2A3 domain under flow conditions (wall shear rate 600 or 3000 s−1). We used platelets labeled with FLUO3-AM and real-time videomicroscopy with high-speed image acquisition to analyze concurrently adhesion, translocation and calcium transients in single platelets interacting with the surface. All experiments were performed in the presence of integrillin (100 μg/ml) to block the possible involvement of αIIbβ3. Platelet adhesion and activation at 600 s−1 were similar in the absence or presence of VWF A1A2A3 domain. Calcium signaling consisted of both short lasting peaks (release from intracellular stores) and long sustained waves (transmembrane flux). In our experimental condition blockade of α2β1 or GPVI with monoclonal antibodies reduced platelet adhesion by 50% and the proportion of adherent platelets that became activated by 20–30%. Concomitant inhibition of both receptors decreased platelet adhesion by 〉90%. Thus, both α2β1 and GPVI participate in the initial stage of platelet adhesion to collagen type I. At the higher shear rate of 3000 s−1, platelet adhesion and activation was 40–50% less than at 600 s−1 in the absence of VWF A1A2A3 domain; addition of the latter enhanced adhesion and activation by 12 to 14-fold, and 〉80% of the platelets exhibited stable adhesion during the 30 s observation period. Intracellular Ca++ peaks in adherent platelets reached 2–3 μM. Blockade of GP Ib-IX-V inhibited both platelet adhesion and activation by at least 90%. Blockade of α2β1 markedly reduced the number of adhering platelets, which were mostly (〉90%) translocating on the surface and exhibited only short lasting Ca++ transients. Blockade of GPVI greatly decreased the number of adherent platelets, most of which, however, were firmly attached to the surface (

Description: Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband’s plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband’s plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband’s plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.

Description: Platelet adhesion to von Willebrand factor (VWF) activates αIIbβ3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates αIIbβ3 activation directly or through other signaling proteins physically associated with it (eg, FcR γ-chain), possibly with the contribution of other agonist receptors and of VWF signaling through αIIbβ3. To resolve this question, human and GP Ibα transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain and the adapter molecule, ADAP, and triggered intracellular Ca2+ oscillations and αIIbβ3 activation. Inhibition of Ca2+ oscillations with BAPTA-AM prevented αIIbβ3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented αIIbβ3 activation but not Ca2+ oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibα transgenic platelets lacking FcR γ-chain. These data establish that GP Ib-IX-V itself can signal to activate αIIbβ3, through sequential actions of Src kinases, Ca2+ oscillations, and PI 3-kinase/PKC. (Blood. 2004;103:3403-3411)

Description: After the introduction of highly active antiretroviral therapy (HAART), intensive treatment, including high-dose therapy (HDT) and peripheral blood stem cell transplantation (PBSCT), has become feasible in HIV-positive patients with Hodgkin (HL) and non-Hodgkin (NHL) lymphoma. Herein, we report the long-term results, on an intention-to-treat basis, of a prospective study on HDT and PBSCT in 50 HIV-positive HAART-responding patients with refractory/relapsed lymphoma. After debulking therapy, 2 patients had early toxic deaths, 10 had chemoresistant disease, 6 failed stem cell mobilization, 1 refused collection, and 4 progressed soon after PBSC harvest. Twenty-seven actually received transplant. Twenty-one patients are alive and disease-free after a median follow-up of 44 months (OS, 74.6%; PFS, 75.9%). Only lymphoma response significantly affected OS after transplantation. In multivariate analyses both lymphoma stage and low CD4 count negatively influenced the possibility to receive transplant. Median OS of all 50 eligible patients was 33 months (OS, 49.8%; PFS, 48.9%). Low CD4 count, marrow involvement, and poor performance status independently affected survival. PBSCT is a highly effective salvage treatment for chemosensitive AIDS-related lymphoma. It seems rational to explore its use earlier during the course of lymphoma to increase the proportion of patients who can actually receive transplant.

Description: We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca++ concentration ([Ca++]i) that are associated with sequential stages of integrin αIIbβ3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibα (GPIbα) and the VWF A1 domain led to Ca++ release from intracellular stores (type α/β peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca++]ioscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbα. Once adhesion was established through the integrin αIIbβ3, new [Ca++]i oscillations (type γ) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbα-VWF bonds leads to αIIbβ3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied αIIbβ3, with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbα and αIIbβ3 and lead to platelet adhesion and aggregation on immobilized VWF.

Description: Key Points The production of NO by platelets and its possible role are controversial. We visualize NO formed by single platelets adhering to collagen under flow conditions and show that it depends on Ca++ and modulates adhesion.

Description: Abstract 2250 Background: Autologous stem cell (SC) transplantation (ASCT) is a potentially curative treatment for several hematologic malignancies and has been demostrated feasible and effective in HIV-related lymphoma (ARL). Peripheral blood SC collection could represent a major issue in the use of ASCT in HIV infected patients (pts). Aim: To evaluate the feasibility and efficacy of SC mobilization in HIV positive (pos) pts with lymphoma and identify factors influencing harvest results. Potential “ongoing” predictors of collection were also assessed. Patients and Methods: We retrospectively analysed 98 consecutive pts with ARL, candidates to ASCT, who underwent SC mobilization at 3 Italian and 2 Spanish centers from 2000 to 2010. A collection less than 2×106 CD34+ cells/kg was defined as “mobilization failure”, between 2–5 as “suboptimal collection” and more than 5 as “good collection”. Several parameters were evaluated for correlation with outcome: age, sex, lymphoma histopathology, disease status, WBC and Plt count at start of mobilization, type of mobilizing therapy, marrow disease, previous mobilization failure, n° of previous chemotherapy (CT) lines, months from first detection of HIV positivity, CD4 count and HIV-viremia. Moreover, circulating CD34+ and WBC count on the first day of CD34+ monitoring and their ratio (SC ratio = CD34/WBC) were assessed as “ongoing” outcome predictors. Results: A total of 127 attempts of SC harvest in 98 pts were analysed. Median age was 41.5 ys (28-65). Lymphoma diagnosis was DLBCL in 42% of cases, Burkitt 10%, plasmablastic 10%, HL 31%, anaplastic 5%, follicular lymphoma 1% and PEL 1%. Disease status was complete remission in 36%, chemosensitive disease in 53% and refractory disease in 10% of cases. In 3 cases bone marrow was involved and mobilizations failed. In 18% of cases pts received mobilizing therapy after 1 previous CT line, in 67% after 2 and in 16% after 3 or more. All pts but 2 were on antiretroviral therapy. Median CD4 count was 231/mcl (50-1146) and HIV-viremia was detectable in 22%. Median time from first HIV detection was 79.5 ms (3-295). In 24% of cases G-CSF alone (10-20 mcg/Kg) was used as mobilizing treatment, while CT + G-CSF (5-10 mcg/Kg) in 76%, including single-agent Cyclophosphamide (CTX) 1.5 gr/ms (13%), CTX 〉3 gr/ms (27%), platinum containing regimens (20%), ifosfamide containing regimens (11%) and others (5%). Mobilization failure occurred in 40% of procedures, a collection between 2–5 × 10^6 CD34/Kg in 24% and 〉 5 in 35%. Finally, of 98 pts who underwent SC mobilization, 22% failed to collect enough cell to perform ASCT, 12 pts even after repeated attempts, 33% had a suboptimal and 45% a good collection (4 and 5 pts respectively after repeated mobilizations). At univariate analysis failure was significantly associated with refractory disease, Plt 〈 150.000/cmm, CTX 1.5 gr/ms as mobilizing treatment, previous mobilization failure and circulating CD34+ cell 〈 7.4/mcl on the first day of monitoring; whereas CTX 〉 3 gr/ms, CD4 count and SC ratio 〉 0.002 were associated with a reduced risk of failure. In multivariate analysis refractory disease (p 0.002 (p 3 gr/ms, WBC count and circulating CD34+ cells 〉29,7/mcl at the first day of monitoring and SC ratio 〉 0,002, whereas G-CSF alone and previous mobilization failure were negative predictive factors. Multivariate analysis confirmed CTX 〉 3 (p 29,7 (p=0.0003) and SC ratio 〉 0,002 (0.0036) as indipendent factors for good collection. Conclusions: In this series of 98 ARL and 127 SC mobilization attempts, a substantial number of pts failed SC harvest (22%) whereas 33% had a suboptimal and 45% a good collection. Lymphoma status and mobilizing treatment seems the strongest predictors for outcome, with refractory disease and low CTX dose (1.5 gr/ms) significantly associated with failure and CTX 〉 3 gr/ms predictor for good collection. A high ratio between circulating CD34+ cells and WBC on the planned day of first apheresis might represent a useful “ongoing” parameter to predict the outcome. These data might help to decide the mobilizing strategy in ARL and could provide the framework to rationally explore the use of new mobilizing agents Disclosures: No relevant conflicts of interest to declare.