ALBERT

Description: Abstract 1658 Poster Board I-684 Introduction NHL patients with refractory disease or relapsing after autologous or allogeneic stem cell transplant (SCT) have very poor prognosis with currently available salvage chemotherapy. Sorafenib (Nexavar, BAY43-9006, Bayer) is an oral multikinase inhibitor approved by FDA for the treatment of renal cell carcinoma and hepatocellular carcinoma. Sorafenib exerts a broad range of antiproliferative, antiangiogenic, and proapoptotic effects against a variety of nonhematological tumors through the inhibition of the RAF/MEK/ERK pathway, the receptor tyrosine kinases c-kit, Flt3, RET, as well as the proangiogenic vascular endothelial growth factor receptors (VEGFRs), and platelet-derived growth factor receptor-β (PDGFR-β). Several lines of evidence suggest that Sorafenib might have a significant clinical impact in the treatment of malignant lymphomas by overcoming the cytoprotective effects of Bcl-XL, ERK, and Mcl-1 and eventually targeting additional signalling pathways relevant to lymphomagenesis. Our preclinical data demonstrating a marked cytotoxic activity of Sorafenib against NHL cell lines in vitro and in vivo in xenograft models, established the rationale for this currently ongoing phase II study aimed to determine safety and activity of Sorafenib in relapsed/refractory NHL. Methods Between March 2008 and May 2009, 21 patients (16 males and 5 females; median age, 65 years; range, 29-74 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 11), follicular lymphoma (FL, n =4), mantle cell lymphoma (MCL, n =2), lymphoplasmacitoid lymphoma (LPL, n =1), chronic lymphocytic leukemia (CLL, n =2), and peripheral T-cell lymphoma (PTCL, n =1) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this phase II trial. Prior to study entry, patients received a median of 4 (range 2 - 7) lines of treatment, including autologous SCT in 15 (71%) and an additional allogeneic SCT in 5 (24%) cases. At study entry, 7 (33%) patients had relapsed and 14 (67%) refractory disease. Eligibility criteria included absence of any available treatment options of proven efficacy, at least one target lesion ≥2 cm, ECOG performance status of 0-1, and adequate bone marrow, liver and renal functions. Sorafenib (400 mg BID, per os) was administered continuously until disease progression or appearance of clinical significant toxicity probably related to study drug. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. Results To date, 21 patients received a median of 3 months (range, 1 – 11) of Sorafenib therapy. All patients are evaluable for toxicity and response, and 1 patient is still on therapy. Overall, therapy was well tolerated without significant adverse events. The most common drug-related non-hematological toxicities were grade 1-2 mucositis (14%), diarrhea (24%), hand-foot syndrome (24%), anorexia (29%), and fatigue (29%). Grade 3-4 hand-foot syndrome occurred in 19% of patients. Hematological toxicities included grade 1-2 neutropenia (10%) and thrombocytopenia (24%). Grade 3-4 neutropenia and thrombocytopenia were observed in 14%, and 24% of patients, respectively. Best response to Sorafenib included 1 (5%) complete remission (CR) occurring in the patient with LPL, and 1 (5%) partial remission (PR) in a patient with cutaneous DLBCL, for an overall response rate (ORR) of 10%. In both patients, response duration was 6 months. In addition, 9 (42%) patients achieved stable disease (SD) for a median of 3 months (range, 2 – 10), with 3 (14%) patients achieving SD for ≥6 months, while 10 (48%) patients progressed. Upon Sorafenib treatment, an extensive necrosis involving the central area of the tumor associated with a nearly complete disappearance of tumor vascularization was documented by computed tomography and contrast-enhanced ultrasound in two DLBCL and one FL patients bearing latero-cervical or abdominal lymphoid masses. Conclusions Sorafenib as a single agent was well tolerated. Despite limited clinical activity (10% ORR), disease stabilization was experienced by 42% of patients. The potent antiangiogenic activity of Sorafenib in NHL patients bearing highly vascularized lymphoid masses suggests that further research should focus on combinations of Sorafenib with molecularly targeted agents eventually exerting antivascular activities. Disclosures Off Label Use: The multikinase inhibitor Sorafenib has been used in a phase II trial in patients with relapsed/refractory NHL.

Description: Recent trials have shown that anti-CD20 monoclonal antibody Rituximab may be effectively employed in association with high-dose (hd) chemotherapy and peripheral blood progenitor cell (PBPC) autograft in the management of high-risk B-cell lymphoma. Addition of Rituximab has a dual effect: increased tumour cytoreduction and in vivo purging prior to PBPC harvesting. We here report the results of a prospective, multicenter trial evaluating Rituximab-supplemented hd-sequential chemotherapy (R-HDS) as frontline treatment in patients with high-risk Diffuse Large B-Cell Lymphoma (DLBCL). So far, 6 Italian Centres associated to GITIL have participated to the study. Eligibility criteria included: i. biopsy-proven DLBCL, with CD20+ phenotype; ii. no previous cytotoxic treatments; iii. age between 16–60 yrs.; iv. advanced stage disease with 2–3 aaIPI score. The R-HDS regimen includes an initial debulkying (3 APO courses) and then the sequential administration, at 15–20 day intervals, of: i. cyclophosphamide (CY) 7gr/sqm (day 1) + Rituximab 375mg/sqm (day +2 and +10), followed by PBPC harvest; ii. Ara-C 2gr/sqm b.i.d. for 6 days, reinfusion of 1–3x106 autologous CD34+ve cells/kg (day 7) and then Rituximab 375mg/sqm (day +8 and day +18); iii. etoposide 2.4gr/sqm day +1 + Cisplatin 100mg/sqm day +2; iv. a final myeloablative regimen (Mitoxantrone 60mg/sqm + L-Pam 180mg/sqm), with PBPC autograft (≥5x106 CD34+ve cells/kg) + Rituximab 375 mg/sqm (day +30 and +37); v. involved-field radiotherapy on areas of previous bulky lesions or residual lesions, within 2–3 mos. following autograft. Presently, 91 patients (median age: 48 yrs.) have been enrolled and are evaluable. They all had 2 (58) or 3 (33) aaIPI score; in addition, 63 (69%) presented with disease-related symptoms, 52 (57%) had extranodal disease, and 27 (30%) had BM involvement. There were 4 early toxic deaths (three due to sepsis following CY, Ara-C and autograft, respectively, and one due to leucoencephalopathy from JC-virus infection 2 mos. after autograft); one more toxic death due to pneumonia occurred at 10 mos. after R-HDS, for an overall TRM of 5.5%. In addition, 21 patients had CMV or VZV reactivation that resolved after antiviral therapy. Overall 73 patients (80 %) reached CR. So far, at a median follow-up of 24 mos., 76 patients (83.5%) are alive and 68 (75%) are in continuous CR (CCR), with 4.3-yr OS and and EFS projections of 80% and 74%, respectively. There was a trend towards a better outcome in aaIPI 2 vs. 3, although the difference was not statistically significative. Among 27 patients with BM+, 17 (63%) are presently in CCR, at a median follow-up of 25 mos. In conclusion, R-HDS was feasible at the multicenter level although the occurrence of severe infectious complications should not be underestimated. Nevertheless, both CR rate and survival curve projections compare favorably with the poor outcome usually observed in aaIPI 2–3 patients managed with conventional chemotherapy. The results here presented urge a comparative analysis between conventional vs. intensified Rituximab-supplemented chemoimmunotherapy in younger patients with high-risk DLBCL.

Description: Background. Recent reports have shown that Rituximab added to conventional chemotherapy may significantly improve the prognosis of CD20-positive Diffuse Large B-cell Lymphoma (DLB-CL). However, patients with unfavorable clinical presentation still have a poor outcome. Other studies have documented an increased anti-lymphoma activity upon addition of Rituximab to intensified treatments with autologous peripheral blood stem cell (PBSC) transplantation. Based on these premises, a prospective multicenter study has been performed on the use of a Rituximab-supplemented high-dose sequential (R-HDS) chemotherapy schedule with PBPC autografting in patients with unfavorable DLB-CL, defined as score 2 and 3 (intermediate-high or high) according to the age-adjusted International Prognostic Index (aaIPI). Methods. The R-HDS regimen included: (i) an initial debulkying with 3 APO courses; (ii) a high-dose (hd) phase consisting in the sequential administration, at 15–20 day intervals, of hd- cyclophosphamide (7gr/sqm, with two Rituximab doses at 375 mg/sqm), hd-Ara-C (2gr/sqm b.i.d. for 6 days with Rituximab), and hd etoposide + Cisplatin; (iii) a final myeloablative phase (hd-Mitoxantrone + L-Pam) with PBSC autografting and 2 more doses of Rituximab. Involved-field radiotherapy was scheduled on areas of previous bulky disease or residual lesions. Six Centers affiliated to the GITIL group (Gruppo Italiano Terapie Innovative nei Linfomi) participated in the multicenter study. Patient enrollment started in November 1999 and was closed in September 2004. Results. Overall, 112 previously untreated patients aged ≤ 60 years, with CD20-positive DLB-CL and aaIPI score 2 (74 pts) or 3 (38 pts), entered the study protocol and are evaluable. There were 5 early toxic deaths (3 sepsis in the hd-phase, 1 pneumonia and 1 leucoencephalopathy from JC-virus infection after autografting) and one late toxic death due to pneumonia which occurred at 10 mos. after R-HDS. The TRM was 5.3%. Ninety patients (80 %) achieved Complete Remission (CR). At a median follow-up of 24 mos, 90 patients (80%) are alive and 83 (74%) are in continuous CR (CCR), leading to a 5.3-yr event-free survival (EFS) projection of 71%. There was a trend towards a better outcome in aaIPI 2 vs. 3, although the difference was not statistically significant. Conclusions. The CR, OS and EFS rates observed after R-HDS compare favorably with the poor outcome anticipated in aaIPI 2–3 patients managed with conventional chemotherapy. The results of this phase II study prompted an ongoing phase III GITIL multicenter study to compare R-CHOP vs. R-HDS in younger patients with high-risk DLB-CL.

Description: The unfavorable clinical evolution in indolent non-Hodgkin lymphomas suggests defective control of neoplastic growth by the immune system. To address this issue, we evaluated phenotype, function, and maturation profile of CD4+ and CD8+ T cells from peripheral-blood, lymph nodes, or bone marrow of patients with B-cell non-Hodgkin lymphoma (NHL) at diagnosis. T cells from these patients frequently showed an activated but apoptosis-prone phenotype with low frequency of tumor-reactive T cells showing a TH2/Tc2 functional profile in the response to autologous tumor. In peripheral blood or in lymph nodes and bone marrow, and, in comparison to healthy donors, patients' T cells showed a skewed differentiation toward Tnaive and Tcentral memory stages, with low expression of granzyme B and perforin. T-cell culture with autologous tumor in the presence of IL-2, IL-15, and autologous bone marrow–derived cells led to massive T-cell expansion and to differentiation of cytotoxic factor+ CD8+ T cells releasing IFN-γ and killing autologous B-cell tumor in an HLA-class I–restricted fashion. These results suggest impaired T-cell differentiation to effector stage in patients with B-cell NHL, but indicate that T-cell responsiveness to γc cytokines is retained, thus allowing to promote generation of antitumor T cells for immune intervention.

Description: Background The outcome of Diffuse Large B Cell Lymphoma (DLB-CL) has been definitely improved by the addition of the anti-CD20 rituximab to conventional chemotherapy. However, despite the advantages offered by rituximab, results are still disappointing in patients presenting with adverse prognostic factors. For these patients, rituximab-supplemented intensive programs with autograft might represent a suitable option. Thus, a prospective multicenter study has been performed using first-line the Rituximab-supplemented high-dose sequential chemotherapy program delivered with multiple autologous peripheral blood progenitor cell (PBPC) support (R-HDS-maps regimen), in DLB-CL patients with score 2 or 3 according to the age-adjusted International Prognostic Index (aaIPI). Preliminary results have been already reported (ASH 2005; Leukemia2007, 21: 1802). Updated results after a prolonged follow-up are here presented. Methods. R-HDS-maps includes: 3 APO courses; sequential administration of hd-cyclophosphamide (CY), hd-Ara-C, both supplemented with Rituximab, hd-etoposide/Cisplatin, with PBPC harvests following hd-CY and hd-Ara-C; hd-Mitoxantrone/L-Pam + 2 further Rituximab doses; involved-field radiotherapy. PBPC rescue was scheduled following Ara-C, etoposide/Cisplatin and Mitoxantrone/L-Pam. Six GITIL Centers participated in the multicenter study, with patients enrolled between November 1999 and September 2004. Results. Overall, 112 consecutive patients (74 score 2, 38 score 3) entered the study protocol. There were 5 early toxic deaths (3 sepsis, 1 pneumonia, 1 JC-virus leucoencephalopathy) + one late toxic death due to pneumonia, at 10 mos.; two more patients died for secondary myelodysplastic syndrome at 3.4 and 3.6 yrs. As reported in Figures 1–2, at a median follow-up of 59 mos., 86 patients (77%) are alive; 79 (70%) are in continuous CR, with a 5-yr event-free survival (EFS) projections of 69%; no significant differences in OS and EFS between subgroups with Germinal-Center and Activated B-cell phenotype were observed. Conclusions. R-HDS-maps regimen: is feasible in a multicenter setting, with a toxicity similar to that of other dose-intense/high-dose regimens; offers prolonged survival to most patients with aaIPI 2–3 DLB-CL. Its comparative efficacy versus R-CHOP-14 is being tested in an ongoing GITIL phase III study. Figure 1 . Overall Survival of 112 aaIPI DLB-CL Figure 1. . Overall Survival of 112 aaIPI DLB-CL Figure 2. Event-free Survival according to GC and ABC phenotype Figure 2. Event-free Survival according to GC and ABC phenotype

Description: We reported that the clinical efficacy of dendritic cell–based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B+ killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

Description: Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family which signals through VEGF receptor-1. In mice, administration of an adenoviral vector expressing human PlGF accelerates bone marrow recovery following myelosuppression and mobilizes PBPCs. By injecting either murine or human PlGF alone, we failed to detect any PBPC mobilization in mice, whereas the combined injection of PlGF plus G-CSF resulted in a 2- to 4-fold increase of PBPCs. Due to the relevant clinical impact of any procedure capable of improving PBPC mobilization, we tested the mobilizing activity of PlGF (Geymonat S.p.A., Anagni, Italy) as an adjunct to G-CSF in a nonhuman primate model. A cohort of Rhesus Monkeys (n = 4) was initially mobilized with G-CSF alone (100 μg/kg/day, SC, for 5 days) (cycle 1), and after a 6-week wash-out period, received a second mobilization therapy consisting of PlGF (130 μg/kg, IV, for 5 days) plus G-CSF (cycle 2). After an additional 6-week wash-out period, a third mobilization cycle consisting of PlGF (260 μg/kg, IV, for 5 days) plus G-CSF (cycle 3) was administered. Hematopoietic mobilization was evaluated by white blood cell counts (WBCs), committed colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Mobilization parameters were analyzed daily during treatment (days 1 to 5), and 3 and 5 days post-cessation of therapy. As compared to baseline values, a 5-day administration of G-CSF alone induced an average 5-fold increment of the mean (±SD) WBC counts (8,708±2,458 vs 43,523±13,790, P ≤.005). As compared to G-CSF alone, the peak values of WBCs were slightly increased by adding PlGF at 130 μg/kg (60,040±9,508) or 260 μg/kg (49,048±7,120). As compared to pretreatment values, the absolute numbers of circulating CFCs per ml blood were increased on average by 85-, 335-, and 358-fold under G-CSF (11,406±4,093, P ≤.005), G-CSF/PlGF 130 μg/kg (46,283±8,287, P ≤.005), and G-CSF/PlGF 260 μg/kg (60,777±8,563, P ≤.005), respectively. At cycles 2 and 3, the peak levels of CFCs were increased by 4- and 5-fold over cycle 1 (G-CSF alone). As compared to pretreatment values, the absolute numbers of circulating HPP-CFCs per ml blood were increase on average by 17-, 158, and 284-fold after under G-CSF (1,593±405, P ≤.005), G-CSF/PlGF 130 μg/kg (8,557±1,142, P ≤.005), and G-CSF/PlGF 260 μg/kg (12,205±2,172, P ≤.005), respectively. At cycles 2 and 3, the levels of day-5 HPP-CFCs were increased by 5- and 8-fold over cycle 1. Under G-CSF alone, the absolute numbers of circulating LTC-ICs were increased by 53-fold as compared to baseline values (211±41 vs 4±7, P≤.005). The combined G-CSF/PlGF (130 μg/kg) treatment increased LTC-ICs by 389-fold as compared to pretreatment values (3,115±988 vs 8±5, P≤.005), with a 15-fold increase over G-CSF alone. In conclusion, our data demonstrate that in nonhuman primates PlGF strongly synergizes with G-CSF for the mobilization of primitive and committed PBPCs.

Description: Background: Because of severe toxicity, in particular mucositis, neutropenia and thrombocytopenia, myeloablative regimens with stem cell support can be safely delivered only to clinically fit and younger pts, require prolonged hospitalization, and have a limited impact in the therapy of NHL. With the aim of rendering high-dose chemotherapy a well tolerated and widely applicable regimen, we carried out a pilot study using high-dose Zevalin with tandem stem-cell support in a prospective cohort of refractory or relapsed NHL patients. Methods: From June 2004 through June 2006, 29 overall NHL patients entered into the study (DLBCL, n=11; follicular, n=10; mantle cell, n=3; small lymphocytic, n=4; Richter syndrome, n=1). Median age was 62 yrs (29–76). The median number of prior chemotherapy regimes was 2 (1–4). Prior to Zevalin, all patients received 3 cycles of standard-dose salvage chemotherapy (DHAP or CHOP, as appropriate), followed by one cycle of high-dose cyclophosphamide plus rituximab, and one cycle of high-dose cytarabine and rituximab, at patient-adapted doses. Hematopoietic stem cells were harvested from the peripheral blood during the post-cyclophosphamide and/or the post-cytarabine recovery phase, and tested for MRD. Zevalin was administered at 0.8 mCi/kg (n=13 pts) or 1.2 mCi/kg (n=16 pts), respectively, and followed by tandem autografting of CD34+ on day +7 and an on day +14, respectively. The latter procedure was performed late, when the radiation absorbed dose to the reinfused stem cells was estimated to be less than 5cGy. In addition, all patients received on day +7 a limited amount (0.8–4.3 x 106/kg) of CD34+ cells. The aim of this early reinfusion, performed in the presence of myelotoxic levels of body radioactivity, was to foster a rapid albeit transient hematopoietic recovery, thus reducing the extend and duration of severe post-Zevalin pancytopenia. Results: Grade 4 neutropenia was observed in 13 pts (45%), and lasted a median of 4 days only (1–14). Grade 4 thrombocytopenia occurred in 19 pts (65%) for a median duration of 5 days (1–14). Fifteen patients (52%) required platelet transfusions (median 2, range 1–6), and 14 pts (48%) received 1 RBC transfusion each. No extra-hematologic toxicity was observed except for mild nausea in 17% of the patients, and all but 3 patients were cared for as outpatients. The 3 hospital admissions lasted 2, 4 and 11 days respectively, and were required for FUO that resolved upon antibiotic administration. Bone marrow analysis performed at 6 (n=5 pts) and 12 months (n=10 pts) showed in all a normal karyotype and a colony growth comparable to controls (NHL pts autografted following BEAM chemotherapy). After a median follow-up of 12 months, the 2-yr OS rate was 87% for indolent and 85% for aggressive lymphoma pts, respectively, while the EFS rate was 55% and 77%, respectively. Conclusions: High-dose Zevalin with tandem stem-cell transplantation was minimally toxic in this pretreated and elderly patient population, proved fully applicable in an outpatient setting, and showed promising activity. Its upfront inclusion as consolidation step after induction chemotherapy warrants a prospective comparison with R-CHOP, in particular in elderly pts with aggressive NHL.

Description: Abstract 2861 Introduction: The AKT inhibitor Perifosine (Æterna Zentaris GmBH, Germany) has been shown in phase II studies to induce partial responses in a variety of solid tumors. Sorafenib (Nexavar, Bayer) is an oral multikinase inhibitor exerting in vitro and in vivo antiproliferative, antiangiogenic, and proapoptotic effects in a variety of hematological and nonhematological tumors. Our preclinical data demonstrating that the combination of Perifosine and Sorafenib induces gene expression profiling and signaling changes associated with a synergistic cytotoxic activity against lymphoma cell lines in vitro and in vivo, established the rationale for this ongoing phase II study aimed to determine safety and activity of Perifosine/Sorafenib combination therapy in relapsed/refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Methods: Between July 2008 and July 2010, 26 out of 36 planned patients (18 males and 8 females; median age, 42 years; range, 19–73 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 3), follicular lymphoma (FL, n = 3), Waldenstrom macroglobulinemia (WM, n = 1), chronic lymphocytic leukemia (CLL, n = 4), and HL (n = 15) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this trial. Prior to study entry, patients received a median of 5 (range 2 – 11) lines of treatment with autologous SCT performed in 19 (73%) and an additional allogeneic SCT in 10 (38%) patients. At study entry, 7 patients had relapsed and 19 refractory lymphoma. Perifosine (50 mg BID, per os) was initially administered as single agent for 4 weeks to assess tolerability and tumor response. Patients achieving less than partial remission (PR) were given the combination therapy, i.e., Perifosine (50 mg BID, per os) plus Sorafenib (400 mg BID, per os) until disease progression or clinical significant toxicity. Patients achieving ≥PR went off-study and continued with Perifosine (50 mg BID, per os) alone until disease progression or clinical significant toxicity. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. The study was approved by the Institutional Ethical Committee. Results: After a 4-week treatment with Perifosine alone, 22 out of 26 patients achieved