Biomedical subjects
Publications and source records attributed to Michele Baccarani.
We assessed the impact of a reduced-dose (10 mg x 3/week for 4 weeks) schedule of alemtuzumab in 10 patients with pretreated cutaneous/peripheral T-cell lymphomas. The overall response rate was 60% (2 complete responses and 4 partial responses). In terms of infectious toxicity, cytomegalovirus reactivation occurred in 1 (10%) patient.
Between July 2003 and March 2004, nine patients with pretreated mycosis fungoides were enrolled into a phase II trial and were treated with temozolomide. The overall response rate was 33%. Further studies are needed to test the efficacy of temozolomide, alone or in combination in earlier stages of this disease.
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BACKGROUND: The objective of the current study was to verify the ability to predict response to imatinib therapy using in vitro assays to evaluate the inhibition of Wilms tumor gene (WT1) expression and colony growth after samples obtained from patients with chronic myelogenous leukemia (CML) before the start of treatment were subjected to short-term incubation with imatinib. METHODS: WT1 transcript levels and colony growth in bone marrow (BM) samples from 23 patients with CML that was later identified as being responsive to imatinib and from 13 patients with CML that was later identified as not being responsive to imatinib were evaluated after incubation of these samples with imatinib at a concentration of 1 microM for 18 hours. In addition, real-time quantitative polymerase chain reaction (RQ-PCR) analysis of WT1 expression was performed during follow-up, and the results were analyzed for associations with cytogenetic response and with BCR/ABL transcript levels as determined using RQ-PCR analysis. RESULTS: Before treatment, it was found that WT1 expression was elevated in BM samples obtained from all patients with CML. WT1 expression and colony growth were reduced significantly after an 18-hour incubation with imatinib in samples obtained from patients who were later identified as responders to treatment, but not in samples obtained from patients who did not experience responses to treatment. Inhibition of WT1 expression in vitro was associated with inhibition of imatinib-induced BCR-ABL tyrosine kinase activity, a finding that also has been made in studies involving certain Philadelphia chromosome (Ph)-positive and Ph-negative cell lines. CONCLUSIONS: Inhibition of WT1 transcript levels after a short period of in vitro exposure of pretherapy BM samples to imatinib was correlated with inhibition of colony growth and may represent the basis for an easy test that is capable of predicting the sensitivity of CML to treatment with imatinib for individual patients.
Since interferon-alpha and imatinib (IM; STI571, Glivec, Gleevec) are effective for the treatment of chronic myeloid leukemia (CML), and their mechanisms of action are different, we designed an exploratory study investigating the effects of a standard IM dose (400 mg/d) and a variable pegylated interferon-alpha (PegIFN) dose (50 microg/wk, 100 microg/wk, and 150 microg/wk). The criteria for dose adjustment were designed so as to ensure the delivery of the IM dose and to protect life quality. There were 76 patients with previously untreated Philadelphia (Ph)-positive CML enrolled in the study. There were 3 patients who discontinued IM and 45 patients who discontinued PegIFN. The severity of adverse events increased with increasing PegIFN dose. The IM dose could be administered to the patients who were assigned to receive 50 microg/wk or 100 microg/wk PegIFN but not to those who were assigned to receive 150 microg/wk. The median administered dose of PegIFN ranged between 32 microg/wk and 36 microg/wk. The cytogenetic response was 70% complete (Ph-neg 100%) and 83% major (Ph-neg > 65%). The BCR/ABL transcript was reduced by at least 3 logs in 68% of complete cytogenetic responders. These data of toxicity, compliance, and efficacy may assist in the design and preparation of prospective studies.
Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V-positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.
Although extracellular nucleotides support a wide range of biologic responses of mature blood cells, little is known about their effect on blood cell progenitor cells. In this study, we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs), and whether activation by their natural ligands, adenosine triphosphate (ATP) and uridine triphosphate (UTP), induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore, stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally, ATP and, to a higher extent, UTP acted as potent early acting growth factors for HSCs, in vitro, because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore, xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions.
PURPOSE: Promising new therapeutic options for follicular lymphoma (FL) include fludarabine plus mitoxantrone (FM) and the mouse/human anti-CD20 antibody, rituximab. We performed a randomized comparative trial of FM with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) front-line chemotherapy with and without sequential rituximab. PATIENTS AND METHODS: All previously untreated CD20(+) FL patients presenting in 15 Italian cooperative institutions from October 1999 were randomly allocated to FM or CHOP. Following clinical or molecular restaging, patients in complete remission (CR) with bcl-2/IgH negativity (CR(-)) received no further treatment; those in CR with bcl-2/IgH positivity (CR(+)) received rituximab, as did those in partial remission (PR) with bcl-2/IgH negativity (PR(-)) or positivity (PR(+)); nonresponders (NR subgroup) were off study. RESULTS: After chemotherapy, the FM arm achieved higher rates of CR (68% [49 of 72 patients] v 42% [29 of 68 patients]; P =.003) and CR(-) (39% [28 of 72 patients] v 13 of 68 patients [19%]; P =.001). Rituximab elicited CR(-) in 55 of 95 treated patients (58%). The final CR(-) rate was higher in the FM arm (71% [51 of 72 patients] v 51% [35 of 68 patients]; P =.01). However, with a median follow-up of 19 months (range, 9 to 37 months), no statistically significant difference was found among the various study arms in terms of both progression-free (PFS) and overall survival (OS). CONCLUSION: These results indicate that FM is superior to CHOP for front-line treatment of FL and that rituximab is an effective sequential treatment option. However, they also confirm that this superiority is unlikely to translate into either better PFS or OS.
BACKGROUND: Mucosa-associated lymphoid tissue (MALT) lymphoma is a specific clinicopathologic entity with gastric and nongastrointestinal site involvement. The authors reported the clinical outcome of patients with Stage IE nongastrointestinal MALT lymphoma treated with a frontline fludarabine-containing regimen or with a regimen containing cyclophosphamide, vincristine, and prednisone (CVP). METHODS: Between 1998 and 2001, 31 patients with Stage IE disease were referred to the Seràgnoli Institute of Hematology and Medical Oncology at the University of Bologna (Bologna, Italy). Presenting sites included periorbital soft tissue (n = 8), lung (n = 5), skin (n = 5), salivary glands (n = 5), lacrimal glands (n = 5), and thyroid (n = 3). Twenty patients were treated with fludarabine and mitoxantrone (FM), and 11 were treated with the CVP regimen. The median follow-up was 3 years. RESULTS: All patients achieved complete responses (CR). Four patients, all treated with CVP, experienced disease recurrence and then achieved a second CR after FM salvage treatment. No tumor recurrence was observed in patients with thyroid, lacrimal gland, or pulmonary lymphoma. The projected 5-year overall survival and disease-free survival rates were 100% and 85%, respectively. CONCLUSIONS: The fludarabine-containing FM regimen provided a relatively effective frontline (or salvage) treatment option for patients with nongastrointestinal Stage IE MALT lymphoma and probably was superior to CVP in terms of efficacy.
BACKGROUND: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. METHODS: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. RESULTS: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). CONCLUSIONS: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.
Human dendritic cells (DC) comprise 2 subsets-plasmacytoid CD123(+) and myeloid CD11c(+) DC-that may have distinct roles in the regulation of immunity after allogeneic hematopoietic stem cell transplantation. In this study, we analyzed the kinetics of CD123(+) DC and CD11c(+) DC reconstitution in 31 patients who underwent transplantation with allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood (PB) stem cells from HLA-identical sibling donors after myeloablative conditioning. Lineage marker-negative HLA-DR(+) CD11c(+) CD11c(+) DC and lineage marker-negative HLA-DR(+) CD123(+) CD123(+) DC, as well as monocytes and lymphoid subsets, were enumerated in donor grafts and in the PB of patients at various time points after transplantation. Reconstitution of both CD11c(+) DC and CD123(+) DC to normal levels occurred within 6 to 12 months and was not affected by the diagnosis, preparatory regimen, or graft composition. However, PB CD11c(+) DC and CD123(+) DC counts were significantly reduced in patients with acute GVHD grade II to IV (at 1 and 3 months) and grade I (at 1 month). Patients with chronic GVHD instead showed reduced CD123(+) DC counts only 6 months after transplantation. Moreover, treatment with steroids (>0.1 mg/kg) was significantly associated with reduced PB CD11c(+) DC and CD123(+) DC counts at all time points after transplantation. In multivariate analysis, only acute GVHD affected DC reconstitution early after transplantation. These results will prompt new studies addressing whether DC reconstitution correlates with immunity against infectious agents or with graft-versus-tumor reactions after PB stem cell allotransplantation.
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Gemtuzumab ozogamicin (GO), an anti-CD33 monoclonal antibody conjugated to calicheamicin, is effective as single agent in the treatment of poor risk acute myeloid leukaemia (AML) patients. We treated with GO in combination with cytarabine as continuous perfusion nine elderly AML patients, either untreated (five cases), or with relapsed/refractory disease (four cases). Five patients achieved a complete remission (CR), four were resistant. One patient died while in CR due to CNS haemorrhage, two relapsed and two are still in CR. The median CR duration was 10 months. The median overall survival was 6 months (1-19 months). The most common adverse event was myelosuppression, as expected. No hepatic veno-occlusive disease was recorded. Notably, in four cases we observed a grade III/IV bleeding, including gasto-intestinal bleeding, epistaxis, CNS haemorrhage, and ocular bleeding. Larger prospective studies are now warranted in order to better define the possible role of this regimen in the treatment of elderly AML patients.
The endoplasmic reticulum (ER) is the site where proteins destined to either secretion or different subcellular compartments assemble and the major storage of intracellular Ca(2+). The ER stress resulting from a variety of toxic insults leads to apoptosis. Here, we showed that the apoptotic death triggered by STI571, an inhibitor of the p210 bcr-abl tyrosine kinase, in murine myeloid progenitors transducing the p210 bcr-abl tyrosine kinase of Chronic Myeloid Leukemia (CML) proceeds from ER stress. The Bcl-2 dowmodulation and inactivation induced by the binding to its antagonist: Bad, the release of caspase 12 from the ER membranes in its active form and of Ca(2+) from the ER pool addressed towards ER a sensor of STI571-induced pro-apoptotic signal.
STI571 is the most innovative drug for the cure of Chronic Myeloid Leukemia. It inhibits, in fact, the disease causative event, the p210 bcr-abl tyrosine kinase, and addresses clonal myeloid progenitors to apoptotic death. Here, we demonstrated that STI571 also induces growth arrest by activating the Chk2-Cdc25A-Cdk2 axis, a pathway complementary to p53 in the activation of G(1)/S cell cycle checkpoint. In vitro exposure to STI571 of 32D murine myeloid progenitor cell clones transducing a temperature-sensitive p210 bcr-abl construct was associated with Chk2 phosphorylation and activation, Cdc25A degradation and persistent Cdk2 inhibitory phosphorylation, preventing, in turn, cell transition to and progression throughout the S phase of cell cycle. Chk2 and Cdc25A are both components of a complex network that integrates signals involved in regulated cell cycle progression, DNA repair and cell decision between life or death. Chk2 gene mutations or decreased expression, leading to its protein loss of function on Cdc25A target, and Cdc25A overexpression have been linked to poor prognosis of human cancers. In CML, they might further enhance the proliferative advantage and genomic instability of clonal myeloid progenitors featuring a class of poor prognosis patients eventually resistant to STI571.
Patients with aggressive non-Hodgkin's lymphoma (NHL) who relapse after initial therapy have a poor prognosis and with standard dose salvage therapy the outlook remains poor. In this work we examine the patient characteristics and outcome of patients with aggressive NHL treated with HDT and autologous transplantation at our Institute from 1982 to 1999. A retrospective analysis was performed examining patient characteristics, prior chemotherapy regimens, pretransplant disease status, HDT regimen, source of stem cells, time for hematopietic recovery, complications of transplantation, response rates, overall survival (OS) and relapse-free survival (RFS). One hundred and thirty-four patients with aggressive NHL were treated with estimated 10-year OS and RFS rates of 50% and 66%, respectively. Disease status (sensitive vs. refractory) pre-HDT was the most powerful predictive parameter for OS and RFS, at both univariate and multivariate analysis. For the entire cohort, transplant-related mortality was only 3.5% without evidence of second malignancies. Our results confirm that HDT with autologous transplantation is associated with a durable RFS in a remarkable proportion of aggressive NHL patients with very low global early and late toxicity. Improved patient selection, transplant timing, ongoing improvements in supportive care, and selected phase III trials should increase outcomes further.
Despite aggressive approaches, including second transplant, donor lymphocyte infusion and several new agents, the prognosis of acute lymphoid leukemia (ALL) patients relapsing after stem-cell transplantation (SCT) remains poor. Monoclonal-antibodies (moAb) could provide a useful tool in this setting. In particular, anti-CD52 moAb is useful in lymphoid malignancies. We thus treated as compassionate with campath-1H 3 ALL patients relapsed after SCT. In 2 cases we observed a reduction of peripheral blood and/or bone marrow blasts. In 1 case a GVHD grade reduction was observed. Larger trials are required in order to define the role of campath-1H in ALL.
Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78+/-10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.