ALBERT

Beschreibung: Using the method of culture in vitro, the authors have studied the proliferative and differentiative activities of the erythroblastic tissue in chronic erythremic myelosis. The results of the research allowed the authors to reach the following conclusions: 1. the erythroblastic cells of chronic erythremic myelosis reveal indisputable differentiative activity in vitro; 2. the rate of maturation of the erythremic erythroblasts is slower than normal; 3. the proliferation of the erythroblastic tissue in chronic erythremic myelosis is normal; 4. in the genesis of the erythroblastic hyperplasia in chronic erythremic myelosis, the most evident factor is a defect of maturation of the erythroblasts.

Beschreibung: ThaDD regimen has provided significant results in recurrent/relapsed multiple myeloma (MM) patients (Offidani et al, 2006). In order to further improve those results without significantly increasing toxicity, we decided to include Velcade, synergic as per activity but not toxicity with the other drugs of ThaDD regimen. ThaDD-V was scheduled as follows: Thalidomide 100 mg/day, pegylated liposomal Doxorubicin 30 mg/sm iv days 4; Dexamethasone 20 mg days 1–2, 4–5, 8–9, 11–12 and Velcade 1.3 mg/sm iv days 1, 4, 8, 11 every 28 days (induction therapy). Patients received bortezomib for alternate cycles as following: 1 mg/sm day 1, 8, 15 and dexamethasone 20 mg days 1–2, 8–9, 15–16 and thalidomide 100 mg/day and dexamethasone 40 mg/day for 4 days monthly for a total of six cycles as consolidation therapy. Then patients received thalidomide 100 mg/day until relapse (maintenance therapy). Actually 20 patients (7 M, 13 F; median age 62.5 yrs, range 31–75) are assessable for response and toxicity. Five pts (25%) showed WHO performance status (PS) 〉 1, 9 pts (45%) presented refractory disease, 8 pts (40%) were priorily administered ≥ 2 lines of a treatment and 14 patients (70%) were submitted to one previous autologous stem cell procedure. Seven patients (35%) had extramedullary disease and 7 had unfavourable cytogenetics. Twelve patients scored an ISS ≥ 2 and 11 (55%) were in first remission for ≤ 12 months median duration. No patients were previously treated with Velcade, whereas six patients had received short-term Thalidomide treatment. Response was assessed according to IMVG uniform response criteria. Seventeen of 20 patients responded after at least one chemotherapy cycle reporting 5 (25%) sCR, 3 CR (15%), 8 VGPR (40%) and 1 stable disease. Three patients (15%) had extramedullary progressive disease. In a median follow-up of 12 months, 2 (10%) patients progressed and 1 (5%) died from cardiac infarction. Time to progression and overall survival were 73% and 95% at 12 months. We observed 4 grade 3 thrombocytopenia, 2 grade 3 DVT, 1 grade 3 diarrhoea, 1 grade 3 asthenia, 1 grade 4 infection and 1 grade 3 dermatological toxicity. Six patients developed grade 2 peripheral neuropathy and other three grade 3. In conclusion, ThaDD-V is extremely active in advanced MM patients as demonstrated by the elevated precentage of high quality remission. Nevertheless, patients are at substantial risk of developing neurotoxicity so the protocol was amended as per Velcade dose intensity and Thalidomide dose.

Beschreibung: Maintenance therapy with Thalidomide in MM offer controversial results. De novo or relapsing MM patients presenting at least a minor response after ThaDD were randomized to receive Interferon 3 MU x 3/week or thalidomide 100 mg /d. Both groups were given also Dexamethasone 20 mg/d x 4 days every month. Actually, we have randomized 50 patients in both treatment arms. The two groups were matched for main prognostic factors and response. During maintenance, both the ID and TD regimens improved response obtained by induction in only 10% and 11% of patients, respectively (p=0.832). After a median 2-years maintenance follow-up for both arms, 30 ID patients (60%) relapsed vs 17 (33%) TD ones (p=0.009). Time-to-progression (TTP) was significantly higher in the TD group vs the ID one (p= 0.024). So that TTP amounted to 23% in the ID group vs the 44% in the TD one. In addition, 3-years overall survival (OS) was significantly better in the TD arm with a value of 67% vs 46% of ID (p = 0.030). Both treatment arms were overall fairly well tolerated: fever, anorexia, weight loss, fatigue, liver and heart function abnormalities and hematologic toxicities were significantly more frequent in the ID cohort whereas neurotoxicity was somewhat more frequent in the TD one. This turned into a rate of therapy dropouts rate significantly higher in the ID group than in the TD one (26% vs 8%; p=0.017). Anyway, estimated risk for treatment interruption due to side effects in a 3-years period with thalidomide was only 21% vs 44% for interferon (p =0.014). We concluded that, in MM patients responding to standard induction therapy, low-dose Thalidomide maintenance therapy is feasible even in the long run and offers a significantly longer control over residual disease when compared to standard maintenance regimen.

Beschreibung: 5-Azacytidine (AZA) is an hypomethylating agent approved in US for the treatment of all FAB subtypes of myelodysplastic syndromes (MDS). Some recent reports have raised the question of a possible efficacy of AZA in selected patients with acute myeloid leukaemia (AML). In September 2007, we started a retrospective study aiming to register and analyse all Italian patients with MDS or AML who had received AZA for the treatment of their disease outside of clinical trials, on the basis of a national patient named program. Among a total of 246 patients treated in 31 different Italian Institutions since 2005, 55 AML diagnosed according to WHO criteria were collected. Median age was 72 years (range 29–87) and 28 patients were male. Poor karyotype was present in 11 patients (20%), while 14 patients (25%) had secondary AML. Median time from diagnosis was 5 months (range 0–72). Eighteen patients (33%) received AZA as front-line treatment, as they were considered not eligible for intensive chemotherapy due to age, co-morbidities or poor performance status. Thirty-seven patients (67%) were pre-treated with growth factors (3 patients) or with one or more lines of chemotherapy (11 and 23 patients, respectively); most of the pre-treated patients (22 out of 34) had received high dose chemotherapy, including autologous or allogeneic stem cell transplantation. Low dose chemotherapy had been employed in the remaining cases. The median number of monthly AZA cycles administered was 4 (range 1–22). Thirty-nine patients (71%) received AZA at the fixed dose of 100 mg/d s.c., 16 patients (29%) received a dose of 75 mg/sqm/d s.c.. A seven-day per month schedule was employed in 43 patients (78%), while 11 patients (20%) received AZA for more than 7 days and one patient for 5 days. Twenty-nine patients (52.8%) received AZA alone, twenty-six (47.2%) in various combinations with growth factors (1), valproic acid +/− ATRA (21) or gentuzumab-ozogamycin (4).The most relevant toxicities observed (grade 3–4) were represented by further myelosuppression (15%), infections (24%: in particular, 1 fungal lung infection, 3 pneumonia and 1 septic shock) and gastrointestinal adverse events (20%). The overall response rate was 35.3% (18/51): 8 patients achieved a complete remission (CR) (15.7%), while a partial response (PR) was observed in 5 patients (9.8%). Five haematological improvements were also seen (9.8%). Response rate was significantly higher in untreated patients compared to pre-treated ones (p=0.02). A statistically significant difference (p=0.04) in response rate in favour of 75 mg/sqm/d versus 100 mg fixed dose was also observed. The actuarial probability of overall survival (OS) at 16 months was 45% for patients responding to AZA and 10% for those non responding (p=0.0027). In conclusion, our data show that: AZA can induce significant responses in about one third of AML patients; the “standard” dose of 75 mg/sqm/d seems to be more effective than 100 mg/d (one single vial) fixed dose; AZA is more effective in de novo as compared to pre-treated (refractory and/or relapsed) disease; AML patients responding to AZA have a significant survival advantage compared to non responders.

Beschreibung: Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of several environmental mutagens, carcinogens, and anticancer drugs. GST polymorphisms resulting in decreased enzymatic activity have been associated with several types of solid tumors. We determined the prognostic significance of the deletion of 2 GST subfamilies genes, M1 and T1, in patients with acute myeloid leukemia (AML). Using polymerase chain reactions, we analyzed theGSTM1 and GSTT1 genotype in 106 patients with AML (median age, 60.5 years; range, 19-76 years). The relevance ofGSTM1 and GSTT1 homozygous deletions was studied with respect to patient characteristics, response to therapy, and survival. Homozygous deletions resulting in null genotypes at theGSTM1 and GSTT1 loci were detected in 45 (42%) and 30 (28%) patients, respectively. The double-null genotype was present in 19 patients (18%). GST deletions predicted poor response to chemotherapy (P = .04) and shorter survival (P = .04). The presence of at least one GST deletion proved to be an independent prognostic risk factor for response to induction treatment and overall survival in a multivariate analysis including age and karyotype (P = .02). GST genotyping was of particular prognostic value in the cytogenetically defined intermediate-risk group (P = .003). In conclusion, individuals with GSTM1 or GSTT1 deletions (or deletions of both) may have an enhanced resistance to chemotherapy and a shorter survival.

Beschreibung: Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy. Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (〉150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (〉40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents. Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Pinto:Takeda, Celgene, Roche, TEVA: Honoraria; Takeda: Research Funding.

Beschreibung: Abstract 4289 The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells. To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive. We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines. In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Chronic Lymphocytic Leukemia (CLL) represents the most frequent adult leukemia, and remains incurable with current standard therapies. Natural Killer (NK) cell count is predictive of CLL disease progression and their dysfunction in mediating cytokine release and direct or antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells is well documented. Detailed mechanistic insight into the etiology of NK-cell dysfunction in CLL patients is currently lacking. CLL B-cells overexpress HLA-E, the natural ligand for heterodimer CD94/NKG2A receptor complex that is expressed on the surface of NK cells, and this interaction suppresses NK cell activation. While NKG2A/CD94/HLA-E interaction is known to assist NK cells in recognizing "self", tumor cells utilize this mechanism to evade effector cell killing. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mab) we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in patients with CLL. We hypothesized that limiting the interaction of HLA-E/NKG2A will reverse NK cell anergy and result in increased direct cytotoxicity of CLL cells. Our results confirm the over expression of HLA-E on CLL B-cells and demonstrate NKG2A expression on CD16+ NK cells from CLL patients. Next, we examined the effect of anti-NKG2A mab on NK cell direct cytotoxicity. Treatment of NK cells, from both healthy donor and CLL patients, with anti-NKG2A mab increased direct cytotoxicity over isotype control on targets at various effector to target ratios of 25:1 (54% vs. 46%, p〈 0.05, n= 12), 12:1 (43% vs. 35%, p

Beschreibung: Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi). BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p

Beschreibung: Abstract 3887 Poster Board III-823 Background Peripheral neuropathy (PN) is a non-hematologic side effect frequently reported in elderly patients treated with bortezomib-melphalan-prednisone (VMP). To address this issue, both bortezomib-melphalan-prednisone-thalidomide (VMPT) and VMP dosing regimens were changed; and bortezomib schedule was modified from twice weekly to weekly administration. Aims To determine incidence and risk factors of bortezomib-associated PN in twice weekly or weekly bortezomib infusion schedules. Methods Patients (N=511) older than 65 years were randomly assigned to receive VMPT followed by maintenance with bortezomib and thalidomide or VMP. Initially, patients were treated with nine 6-week cycles of VMPT (induction: bortezomib 1.3 mg/m2 days 1,4,8,11,22,25,29,32 in cycles 1-4 and days 1,8,22,29 in cycles 5-9; melphalan 9 mg/m2 days 1-4; prednisone 60 mg/m2 days 1-4 and thalidomide 50 mg days 1-42; maintenance: bortezomib 1.3 mg/m2 every 15 days and thalidomide 50 mg/day as maintenance) or VMP (bortezomib, melphalan and prednisone at the same doses and schedules previously described without maintenance). In March 2007, the protocol was amended: both VMPT and VMP induction schedules were changed to nine 5-week cycles and bortezomib schedule was modified to weekly administration (1.3 mg/m2 days 1,8,15,22 in cycles 1-9). Baseline grade ≥ 2 PN was an exclusion criteria. Results 254 VMPT patients and 257 VMP patients were evaluated in intention-to-treat: 141 patients received twice weekly infusion of bortezomib and 370 once weekly. The overall incidence of PN was 37% in the VMPT patients and 27% in the VMP patients (p=0.01) while the grade ≥ 3 was quite similar (8% and 5%. p=0.19). When VMPT and VMP groups were combined, the incidence of PN was significantly higher in patients who received twice weekly infusion of bortezomib: the incidence of all grade PN was 45% in the twice weekly group and 27% in the once weekly group (p=0.0002), including a grade ≥ 3 PN incidence of 16% and 3% (p