ALBERT

Description: The absence of mutations in the IgVH genes along with the expression of the cell surface receptor CD38 and the cytoplasmic kinase ZAP-70 are independent molecular markers of unfavourable outcome in CLL patients. Our group has been interested in unravelling the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We have shown that CD38 can positively affect proliferation and survival of CLL cells through binding CD31, the ligand expressed by stromal, endothelial and nurse-like cells (NLC). Starting points of our current investigation are that NLC produce high levels of the attractant stromal cell-derived factor-1 (SDF-1α/CXCL12), CD38 is an essential mediator of migration and homing in murine neutrophils, and ZAP-70 is a key element in CXCR4 (SDF-1α receptor) pathway in T cells. The consequent working hypothesis is that CD38 and ZAP-70 intertwine to influence CLL cell migration. The analysis of a cohort of 56 clinically and molecularly characterized CLL patients demonstrated that CD38+/ZAP-70+ samples migrate significantly better towards CXCL12 than all the other CLL subgroups, including the CD38+/ZAP-70− one, suggesting a functional association between CD38 and ZAP-70. CD38 engagement by agonistic monoclonal antibodies (mAbs) leads to phosphorylation of the activatory tyrosines present in ZAP-70 in 26 CLL cell cases and in an ad hoc modified B line expressing a ZAP-70-myc construct. More importantly, ZAP-70 is required for CD38 signalling, as the CD38 pathway is impaired in CD38+/ZAP-70− CLL cells. Microarray analyses performed on a subgroup of 20 CLL patients indicate the existence of a genetic signature marking CLL samples endowed with a high migratory potential. These studies resulted in the identification of a panel of 24 differently expressed genes, many of which are known to be involved in adhesion and migration. Taken together, the above data point to a direct role for the CD38/ZAP-70 pathway in CLL migration towards CXCL12. Indeed, CXCL12-mediated chemo taxis of CLL cells and of the NALM-6 pre-B cell line can be efficiently blocked by anti-CD38 mAbs in a dose-dependent fashion. Out of the 9 different anti-CD38 mAbs tested, only the ones binding epitopes close to the C terminus of the molecule interfere with migration, while the ones binding closer to the plasma membrane do not. Pre-treatment of CLL and NALM-6 cells results in the almost complete block of the CXCR4 signalling pathway. This was shown by monitoring Ca2+ mobilization, actin polymerization and ligand-induced internalization. Further, upon exposure to CXCL12, CD38 and CXCR4 become physically associated, as shown using confocal microscopy and co-immunoprecipitation approaches. CD38 however, does not internalize upon CXCL12 biding to CXCR4. Collectively, these data suggest that the blocking effects of the anti-CD38 mAbs are due to steric hindrance. In conclusion, we believe that these results offer biological evidence explaining why the combination of CD38 and ZAP-70 results in a more efficient identification of CLL patients with aggressive disease. Further, it may provide a rationale for using anti-CD38 ligands or inhibitors as an adjunct tool for the therapy of aggressive CLL.

Description: High-dose (HD) therapy plus Autologous Stem Cell Transplantation (ASCT) is a milestone treatment program for most hematologic malignancies. Daily subcutaneous injections of G-CSF (filgrastim/lenograstim) until ANC 〉 500/μl are routinely administered following ASCT, in order to accelerate hematopoietic recovery and reduce neutropenic complications. Pegfilgrastim has been shown to have similar efficacy when compared to G-CSF for chemotherapy-induced neutropenia, but little is known about its use in the ASCT setting. We used a 6 mg fixed dose of Pegfilgrastim on day +4 following ASCT in 46 patients (22 M/24 F; median age 56 yrs; r 22-70 yrs) with multiple myeloma (25 pts) and relapsed or refractory Hodgkin’s and non-Hodgkin’s lymphoma (21 pts). Patients received peripheral CD34+ stem cells (median number 4.4 x 106/Kg; r 1.8 – 11.8) harvested after mobilizing chemotherapy (cytoxan, vinorelbine/cytoxan, R-IEV, IGEV, R-ICE) and G-CSF. Standard conditioning regimens (HD-Melphalan or BEAM) were used. Engraftment results were compared to those from a historical control group of 182 patients (median age 56 yrs; r 16-74 yrs) who had received HD-Melphalan or BEAM and ASCT (median CD34+ cells 7.6 x 106/Kg, r 1.8–4.6) supported by G-CSF (5 μg/kg/day from day +5 until ANC〉500/μl). Median number of days to ANC〉500/μl were comparable between the Pegfilgrastim (10, r 8-15) and G-CSF (11, r 7–22) groups, as well as the median number of days to PLT〉20,000/μl (Pegfilgrastim = 12, r 9–20 vs G-CSF= 12, r 7–29). Overall infectious rates, including FUO and documented infections, were of 48% and 39% for Pegfilgrastim and G-CSF groups, respectively (p=NS). Median number of days on i.v. antibiotics were 0 (r 0–18) and 6 (r 0–13) for the Pegfilgrastim and G-CSF groups, respectively. No significant differences in the incidence of bone pain, intensity of transfusion support and length of hospital stay were documented between the two groups. These data indicate that a fixed (6 mg) single-dose of Pegfilgrastim is safe and effective to accelerate engraftment after ASCT. No significant differences with G-CSF were apparent as to engraftment times and overall infectious complications. Growth factor costs were however in favor of Pegfilgrastim (Euro 690 vs 892 for a median of 10.5 G-CSF doses).

Description: CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d− subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d− cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.