ALBERT

Description: We recently described that simvastatin effectively induced apoptosis in myeloma and lymphoma tumor cells by inhibition of proteingeranylgeranylation resulting in the reduction of the anti-apoptosis protein Mcl-1. In addition low concentrations of simvastatin had a chemosensitizing effect in combination with dexamethasone or doxorubicin. Based on these observations we initiated a Phase I study of dose escalating simvastatin combined with chemotherapy in patients with end-stage Myeloma and Lymphoma. Starting dose level of simvastatin was 5 mg/kg/day for 7 days, divided in 2 doses orally, followed by VAD chemotherapy in patients with myeloma and CHOP in patients with lymphoma. Three patients were included per dose level. In the absence of side effects WHO grade III/IV in 3 patients the dose of simvastatin was escalated with 2.5 mg/kg. Twenty-one heavily pretreated patients all refractory to at least 3 lines of chemotherapy (14 myeloma patients and 7 lymphoma patients) were included. No toxicity beyond WHO 2 was recorded with dose level 1–4 (5 mg–12.5mg/kg/day/7days). One patient treated at dose level 5 (15 mg simva/kg/day/7 days) became severely depressed and performed an unsuccessful suicide attempt. Two patients treated at dose level 6 (17.5 mg/kg/day/7 days had severe gastro-intestinal side effects (WHO 3; vomiting, diarrhoea, dehydration), necessitating interrupting simvastatin after 3 and 4 day respectively. The third patient treated at dose level 6 had moderate gastro-intestinal complaints but died on day 13 (2 days after VAD, deeply neutropenic) from overwhelming Gram-Septicaemia although prophylactic antibiotics had been prescribed. Three additional patients were then treated at dose level 5 again without side effects beyond WHO 2. None of the patients complained about muscle pain. No signs of rhabdomyolysis were registered. Although response was not the primary endpoint of the study, it could be evaluated in 16 patients who completed at least 2 cycles of simvastatin with chemotherapy. Six patients (4 myeloma and 1 transformed low grade lymphoma) responded (32 %) including 5 patients with a Partial Response (〉 50 tumor reduction) and 1 patient with a minor response. Four of 8 evaluable (myeloma) patients treated at dose level 4 and 5 responded. Due to toxicity all 3 patients at dose level 6 were not evaluable for response. In patients treated at dose level 4 and 5, in vivo down regulation of Mcl-1 (〉 50 %) was observed in PBMC collected after 7 days of simvastatin treatment. These data show that in vivo downregulation of Mcl-1 by high dose (simva) statin in combination with chemotherpy may be a promising new modality for patients with drug resistant myeloma and lymphoma.

Description: Minor histocompatibility antigens (mHags) that are selectively expressed by hematopoietic cells, including hematopoietic tumor cells, represent attractive targets for T cell-based immunotherapy after MHC-matched allogeneic stem cell transplantation (SCT) to boost the graft-versus-tumor response without causing graft-versus-host disease. We isolated a CD8+ cytotoxic T lymphocyte (CTL) clone from a SCT recipient that recognized a novel HLA-B*0702-restricted mHag with lymphoid lineage-specific expression. In vitro CTL recognition experiments revealed that this mHag was expressed by EBV-transformed B cells, CD40-activated B cells and PHA-stimulated T cells, but not expressed by cells of myeloid origin and non-hematopoietic fibroblasts. Furthermore, testing a panel of HLA-B*0702 transduced cell lines showed that several B lymphoid tumor cell lines express this mHag, which was designated as Lymphoid Restricted Histocompatibility antigen-1 (LRH-1). To define the chromosomal localization of the LRH-1 coding gene, we used genetic linkage analysis using EBV-lymphoblastoid cell lines (LCL) form the Centre d’Etude Polymorphism Humain (CEPH) reference families that have been extensively mapped for thousands of genetic markers. EBV-LCL generated from individuals of six CEPH reference families were retrovirally transduced with HLA-B*0702 and tested for CTL recognition. After determining the LRH-1 segregation pattern, linkage and haplotype analysis revealed that the LRH-1 coding gene maps to a 1.64 Mb region on chromosome 17p13.2 between marker D17S1845 and D17S1584. A database search identified 19 candidate genes that are localized within this region. Real-time RT-PCR analysis revealed that 3 genes showed an expression profile that corresponds to the CTL recognition pattern of which the purinergic receptor gene P2X5 was the most promising candidate. Transfection of P2X5 cDNA cloned from a homozygous antigen-positive individual into 293-HLA-B*0702 cells resulted in efficient recognition by the CTL clone. Testing of different P2X5 transcript variants and deletion constructs revealed that the nucleotide sequence coding the antigenic peptide is located within exon 3 of P2X5 transcript variant 1. In this region, we identified a synthetic nonameric peptide that sensitized antigen-negative donor EBV-LCL to CTL recognition, with half-maximal recognition observed at a peptide concentration of 20 nM. To identify the basis for LRH-1 disparity, exon 3 of the P2X5 alleles of the donor and recipient were sequenced and compared. The antigen-negative donor contained a homozygous C-nucleotide deletion/insertion polymorphism (DIP) resulting in a frame-shift in the protein at the third residue of the epitope. Transfection of the donor P2X5 transcript variant 1 into 293-HLA-B*0702 cells confirmed that the C-nucleotide DIP within the donor allele was associated with resistance to CTL recognition. These results provide the first evidence that mHag can result from differential protein expression as a consequence of a homozygous DIP in a transcript variant of the encoding gene.

Description: Over 170 000 cord blood units are stored worldwide. For the majority of patients in need of an allogeneic donor, a cord blood unit can be found which is identical or has less than 2 HLA mismatches. However, when partially mismatches cord blood units are used for transplantation, the number of CD34+ cells per kg body weight becomes important and should be higher than 1.7 105/kg (Blood2002;100:1611–1618). For this reason, it is important that only cord blood units with high CD34+ cell content are stored. Our standard selection criteria are volume above 40 mL and TNC of 10x108 or higher. Using these criteria, we routinely store 18% of collected units. The mean CD34+ cell content of stored units is 5.2 106 and TNC is 1.38x109. 94%, 52%, 12%, 4% of cord blood units can be used for patients weighing 10, 25, 50 and 75 kg respectively, based on the criterium of 1.7. 105 CD34+ cells/kg patient weight mentioned above. To optimize CD34+ cell content in the stored units, we compared our present selection method based on TNC and volume (criterium 40/10/?) with a selection method based on volume, TNC and CD34 content. 131 samples with volumes above 40mL and TNC above 8x108 were analysed for CD34+ cell content before processing and subsequently processed in routine. Using this data set, we compared criterium 40/10/? with criteria 40/8/2 (volume:40mL, TNC:8x108, CD34 2x106). We found that both criteria resulted in an equal number of units acceptable for storage. CD34+ cell content increased from 4.5x106 for criterium 40/10/? to 4.8x106 for criterium 40/8/2 whereas TNC dropped from 14.6x109 to 14.1x109. 100%, 47%, 9% and 4 % (criterium 40/8/2) vs 88%, 43%, 9% and 4%(criteria 40/10/?) of cord blood is acceptable for patients weighing 10, 25, 50 and 75 kg resp., demonstrating that the 12% essentially unusable cord blood units are replaced by units which can be used for small children. In conclusion: by including CD34+ cell content in the selection criteria, CD34+ cell content increases so that 100% of the cord blood units can be used for children up to 10 kg. This measure however requires that CD34 measurement is performed on all units with TNC above 8x108 cells.

Description: The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2−/−γc−/− mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2Rβ, (IL-2Rβ) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1−TCRαβ+ CD4+ or CD8+single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA+) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO+ cells. These data suggest that all elements of a functional immune system were present in these animals.

Description: Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (BALL) occur frequently after primary treatment. Therefore, development of additional treatment modalities to eliminate residual tumor cells is needed. Active immunotherapy using dendritic cells (DCs) loaded with tumor-associated antigens is a promising approach to induce specific T-cell immunity in patients with cancer. In previous studies, we described HB-1 as a B-cell lineage-specific antigen that is recognized by donor-derived cytotoxic T lymphocytes (CTLs) on allogeneic B-ALL tumor cells. Here, we investigated the potential use of the HB-1 antigen as an autologous T-cell vaccine target. To determine whether HB-1–specific CTL precursors are present within the T-cell repertoire, we induced expansion of CD8+ T cells using mature monocyte-derived DCs pulsed with the previously identified HB-1.B44 antigenic peptide. In 6 of 8 donors, CD8+ CTL lines have been generated that exert cytotoxicity against target cells exogenously pulsed with peptide or endogenously expressing the HB-1 antigen. From one of these HB-1–specific T-cell lines, we isolated a CD8+ CTL that produces interferon-γ on stimulation with B-ALL tumor cells. Interestingly, the HB-1 antigen also induced CD4+ T-helper responses on activation with protein-loaded mature monocyte-derived DCs. We identified 2 novel epitopes recognized in the context of HLA-DR4 and HLA-DR11 with the use of HB-1–specific CD4+ T-cell clones generated from different donors. These present data, that HB-1 induces both helper and cytotoxic T-cell responses, indicate that the HB-1 antigen is a candidate target to induce T-cell–mediated antitumor immunity in patients.

Description: PROs provide patients’ own appraisal of their health status and can be used to measure aspects of health that are generally not captured by ‘traditional’ clinical measures such as physician assessments and test results. This study analyzed PRO data from a multicenter, pivotal phase II trial of bortezomib in 202 patients (pts) with relapsed, refractory MM (NEJM2003;348:2609–17). PRO questionnaires (EORTC-QLQ C30 and MY24, FACT/GOG-Neurotoxicity (Ntx) and FACIT-Fatigue subscales) were administered at the screening visit (baseline) and day 1 of cycles 3, 5, 7 and at the end of the study. Results: In this population with relapsed, refractory MM, poorer baseline pre-treatment multi-dimensional quality of life (QoL) scores were significantly correlated (range of r= 0.22 to r= 0.77) with fatigue, pain, dyspnea, appetite loss, neurotoxicity, MM disease symptoms, and treatment side effects. Clinical response to bortezomib (Complete Response or Partial Response) was associated with statistically significant (p

Description: Two functionally distinct subsets of B cells that produce Th1- and Th2-like patterns of cytokines have recently been identified. Interleukin-12 (IL-12) is a critical immunoregulatory cytokine that promotes Th1 differentiation through activation of signal transducer and activator of transcription 4 (STAT4). IL-12 has been reported to induce interferon γ (IFN-γ) production in B cells, but the relevant signaling pathways are poorly documented. Here, in human primary B cells, we found a functional IL-12 receptor (IL-12R) that internalizes following IL-12 binding. IFN-γ and, to a lesser extent, IL-12 positively regulated the IL-12Rβ2 subunit but had no effect on IL-12Rβ1. On examining the effect of IL-12 on STAT4 and T-bet (2 key factors involved in IFN-γ promoter activation), we found that IL-12 induced the phosphorylation and nuclear translocation of STAT4. IL-12-dependent constitutive STAT4 activation was also observed in the Epstein-Barr virus (EBV)-transformed B-cell line RPMI 8866 that spontaneously produces IL-12. T-bet expression has been shown to be dependent on STAT1. IL-12 had no direct effect on STAT1 activation or T-bet expression in primary B cells. In contrast, IL-12-induced IFN-γ led to STAT1 activation, strong expression of T-bet, and IFN-γ expression. IL-12 therefore initiates a cascade of events in B cells, including STAT4 activation, IL-12Rβ2 up-regulation, IFN-γ production, and T-bet up-regulation, potentially leading to Th1-like differentiation. (Blood. 2003;102:4084-4089)

Description: G-CSF is used to enhance neutrophil recovery after autologous peripheral blood stem cell transplantation (aPBSCT), even if the optimal timing and dose of G-CSF has not been established. Recently Peg-Filgrastim has been approved for clinical use. The clearance of Peg-Filgrastim is neutrophil-mediated with a sustained duration of action with prolonged serum concentration during neutropenia. In order to verify the efficacy and feasibility of use of Peg-Filgrastim, we administered to 10 patients (A Group, 3F, 7M, median age 46y) submitted to aPBSCT for haematological malignancies (6 NHL, 3 MM, 1 HD) a single 6 mg dose of Peg-Filgrastim subcutaneously on day +1 and we compared the engraftment of these patients to data of a group of 30 patients (B group 9F, 21M, median age 46y, 18 NHL, 6 MM, 2 POEMS syndrome, 1 Plasmacell leukemia, 3 HD) submitted to aPBSCT not receiving G-CSF. Patients were matched for sex, age, disease, disease status at transplant, conditioning regimen, transplant procedure and they received comparable CD34+ cell dose. We evaluated haematological engraftment, and other clinical outcomes, all results are expressed as a median value in the table. Peg-Filgrastim was well tolerated in all but 1 patient reporting bone pain. Neutrophil engrafment was significantly faster (72 hours) in the Peg-Filgrastim group: days +10 and +12 (500 and 1000 cells/μL) vs days +13 and +15.5 of control group; faster neutrophil engraftment was also clear analyzing number of days with absolute neutrophil count (ANC) 500/μL 10 (7–11) 13 (7–18) p 1000/μL 12 (7–15) 15.5 (10–38) p 20 x 10³/μL 11 (8–24) 12 (9–18) p=0.4 Days to Plts 〉 50 x 10³/μL 24 (12–39) 14 (10–60) p=0.39 Days to Plts 〉 100 x 10³/μL 40 (14–120) 18 (12–210) p=0.06 Days to Reticulocytes 〉1% 15 (11–16) 13.5 (10–30) p=0.59 Days to Hgb 〉10 g/dl 33 (10–84) 21 (10–210) p=0.48 Days of fever 5 (2–10) 5 (2–13) p=0.92 Days of antibiotic therapy 7 (5–20) 9.5 (5–18) p=0.72 Bloodstream infections 4/10 6/30 p=0.2 Number of pRBCu 1 (0–2) 0 (0–2) p=0.02 Number of SDu 1.5 (1–3) 1 (0–2) p=0.022 Days of hospitalization 25 (19–30) 23 (20–34) p=0.89

Description: Recent studies demonstrate that bisphosphonates - anti-resorptive drugs - have direct anti-tumour effect in many tumour cell lines, including hematopoietic ones. The aim of this study was to evaluate the apoptotic effect of Zoledronate in cells from B chronic lymphocytic leukemia (B-CLL) and low-grade lymphoma (LGL) patients. Samples of 19 B-CLL or LGL patients were incubated with Zoledronate 100 μM in RPMI medium supplemented with fetal bovine serum 10% at 370C for 12h. Apoptosis using Annexin V assay, by flow cytometry (FC), was observed in 8 out of 19 (42,1%) patients despite previous treatment. Multidrug resistance (MDR) phenotype was performed using rhodamine-123 efflux assay by FC. Our results demonstrate that Zoledronate 100 m M can induce apoptosis in B malignant lymphocytes despite previous treatment and MDR phenotype. So, patients previously treated with distinct therapeutic agents or not can potentially benefit from the anti-tumour effect of Zoledronate. This is the first work demonstrating the anti-tumour effect of Zoledronate in newly obtained cells from patients with B-CLL and LGL. These results in association with evidences from recent studies suggest that Zoledronate may have an important and complementary role in hematological malignant diseases.

Description: Introduction: Cerebrovascular disease (CVD) is a multifactorial disease caused by the interaction of genetic and environmental factors. The atherosclerotic plaque, the pathological hallmark of CVD, is an inflammatory process, where pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα). TNFα secretion shows a high degree of interindividual variability, which is at least partly genetically determined. We have analysed the prevalence of −238 G/A and −308 G/A polymorphisms in the regulatory region of the TNFα gene promoter in CVD. Patients and methods: Genotypic analyses were performed on 308 consecutive unrelated patients diagnosed with ischemic CVD, 147 women and 161 men, mean age 70±0.8 years, who were diagnosed according to the Trial of Org 10172 in Acute Stroke Treatment. All included cases were age and sex matched to a control from the same geographic area who had no history of vascular disease. Patients and controls completed a questionnaires including blood pressure, diabetes status, total serum cholesterol level and smoking history. The TNFα variants were detected by PCR using primers containing a single base-pair mismatch to introduce a restriction site into the wild-type nucleotide sequences after amplification. PCR products were digested with NcoI and MspI to detect −308 and −238 variants, respectively. The strength of the association of the polymorphisms with the occurrence of CVD was estimated by calculation of the OR and its 95%CI by exact method. P values less than 0.05 were considered significant. Logistic regression analysis was applied to estimate the risk in a multivariable predictive model with dependent variable (case/control) and all independent variables significant in the bivariate analysis. SPSS 9.0 was used for the statistical analysis. Results: Genotype analysis showed a significant higher prevalence of the G/A and A/A genotypes of −238 G/A TNFα in patients (p〈 0.01;OR= 2.16;95%CI= 1.40–3.34). The prevalence of the A allele was also significantly increased in the group of CVD patients than in the controls (13.6% vs 7.0%; p