Despite the fact that granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte c... more Despite the fact that granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used in clinical practice, little is known of their endogenous production, especially in myeloproliferative disorders such as chronic myelogenous leukemia (CML). In order to define serum levels of GM-CSF and G-CSF in subjects affected by CML, the sera of 17 patients with CML in chronic phase treated either with hydroxyurea or interferon-alpha were tested by specific enzyme immunoassays. Fifteen age- and sex-matched healthy volunteers were used as normal controls. Eight out of the 17 patients (44%) with CML showed detectable (> 3 pg/mL) serum levels of GM-CSF (range 3.9-55 pg/mL). Detectable levels (> 50 pg/mL) of G-CSF were observed in 9 of these patients (52%) (range 150-2,830 pg/mL). On the contrary, among the normal controls only one had detectable GM-CSF concentrations, and none had detectable G-CSF concentrations. The highest c...
A system to purge the bone marrow of myeloma cells has been developed in our laboratories with th... more A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody--that recognizes plasma cells and B-cell precursors--and the ribosome-inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno-occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The haemopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.
Several genes with relevant pathogenetic and prognostic value have been identified in patients wi... more Several genes with relevant pathogenetic and prognostic value have been identified in patients with myelodysplastic syndromes. Overexpression of WT1 at diagnosis has been associated with increased progression to acute myeloid leukemia and reduced leukemia free survival. Conversely, few data are available on the prognostic value of BAALC gene overexpression in AML and MDS patients. We evaluated the prognostic value of the combined expression of WT1 and BAALC genes at diagnosis in 86 MDS patients who had been stratified according to IPSS and R-IPSS scoring systems. Our results suggest that in the whole group of patients, low levels of both WT1 and BAALC were associated with a favorable outcome (3-year LFS 74.5%, median not reached), whereas patients presenting high expression levels of both genes had the worst prognosis (3-year LFS 0%, median 12 months, p<0.001). More specifically, molecular profiling was especially useful for intermediate 1 and intermediate-2/high risk groups. This study suggests that evaluating WT1 and BAALC gene expression at diagnosis may improve standard risk stratification and possibly refine the therapeutic approach for MDS patients.
Background: Characterization of genes differentially expressed during the stages of tumour progre... more Background: Characterization of genes differentially expressed during the stages of tumour progression may lead to the identification of "oncoantigens", tumour-associated molecules that play important roles in driving tumour progression and constitute potential targets for preventive antitumour vaccination. Until now, we have identified putative oncoantigens (POAs) as molecules expressed by mammary cells in pre-neoplastic lesions and overexpressed in evident neoplastic lesions. However, many human malignancies, including breast cancer, are organized in a hierarchical network of rare slowly dividing cancer stem cells (CSCs), rapidly dividing amplifying cells and differentiated tumour cells. CSCs constitute the source of the tumour and could be responsible for tumour progression, metastasis, resistance to therapy and recurrence, so preventive vaccination should target them. Thus, analysis of CSCs transcriptional profiling may identify new POAs, more suitable for effective vaccination. Material and Methods: Mammary tumour specimens were obtained from a cell line derived from BALB-neuT breast carcinomas (Ag12). Cells were plated in differentiative conditions to obtain tumour epithelial cells (e0) or under specific conditions to generate mammospheres (p1), which were then disaggregated and plated to obtain second (p2) and third (p3) passage mammospheres. Expression of CSCs markers on mammospheres was checked by cytofluorimetry. Transcription profiling was performed on e0 and p1−3 using Illumina microarray platform MouseWG-6 v2.0. Results: Mammospheres generated from Ag12 displayed clonogenicity, self renewal, CSCs markers and ability to differentiate in mammary epithelial cells and maintained the tumourigenic potential. 452 deregulated transcripts were detected in mammospheres using rank product statistics, comparing e0 with p1−3. To detect CSC vaccination targets, a subset of 183 transcripts (POAs) which expression increased from p1 to p3 were selected by K-mean clustering. Vaccination targets for breast cancer prevention were selected ranking the 183 transcripts on the basis of the relation between their expression and survival in 7 public human breast cancer transcription profiles. The actual protein increase of some of these POAs in p1−3 was confirmed in ELISA and cytofluorimetric experiments. Conclusions: Mammospheres transcription profiling led to the identification of new POAs. Future experiments will validate these POAs in preventive vaccination in BALB-neuT mice.
A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (... more A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (ET), and represents a key World Health Organization diagnostic criterion. This hypothesis-generating study (NCT01352585) explored the impact of JAK2(V617F) mutation status on treatment response to anagrelide in patients with ET who were intolerant/refractory to their current cytoreductive therapy. The primary objective was to compare the proportion of JAK2-positive versus JAK2-negative patients who achieved at least a partial platelet response (≤600×10(9)/L) after anagrelide therapy. Of the 47 patients enrolled, 46 were included in the full analysis set (JAK2-positive, n=22; JAK2-negative, n=24). At 12 months, 35 patients (n=14 and n=21, respectively) had a suitable platelet sample; of these, 74.3% (n=26) achieved at least a partial response. The response rate was higher in JAK2-positive (85.7%, n=12) versus JAK2-negative patients (66.7%, n=14) (odds ratio [OR] 3.00; 95% confidence interval [CI] 0.44, 33.97). By using the last observation carried forward approach in the sensitivity analysis, which considered the imbalance in patients with suitable samples between groups, the overall response rate was 71.7% (n=33/46), with 77.3% (n=17/22) of JAK2-positive and 66.7% (n=16/24) of JAK2-negative patients achieving at least a partial response (OR 1.70; 95% CI 0.39, 8.02). There was no significant change in median allele burden over 12 months in the 12 patients who achieved a response. In conclusion, the overall platelet response rate was high in both JAK2-positive and JAK2-negative patients; however, a larger study would be required to confirm the differences observed according to JAK2(V617F) mutation status.
A significant reduction in the proportion and absolute number of circulating Ty (suppressor) lymp... more A significant reduction in the proportion and absolute number of circulating Ty (suppressor) lymphocytes was observed in 15 adult patients with idiopathic thrombocytopenic purpura (ITP). The proportion of cells was also reduced in the spleen of four patients so investigated. This abnormality was not seen in 5 patients cured by splenectomy whilst it persisted in 3 who remained thrombocytopenic after splenectomy. The proportion of Q (helper) lymphocytes in ITP was normal.
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown t... more The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have anti-leukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current anti-leukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here we evaluated the anti-leukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n=6; B-CLL, n=19), and healthy leukocytes. Intracellular NAD+ and ATP levels, mitochondrial transmembrane potential (ΔΨm), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34+ progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD+ and ATP shortage, and induced ΔΨm dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including AML and B-CLL samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Journal of experimental & clinical cancer research : CR, 2002
Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered th... more Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered the study. The mean age of patients was 50 (range 15-60). The induction regimen (FLAG-Ida) included fludarabine (30 mg/sqm), Ara-C (2 g/sqm) on days 1-5, and idarubicin (10 mg/sqm) on days 1, 3, 5. G-CSF (300 mcg/day) was administered s.c. 12 hours before starting fludarabine and was continued for five days. HDT with stem cell rescue was planned for all patients in first CR after one course of high dose Ara-C (HDAC) consolidation and in good clinical conditions. Forty-two (98%) patients were evaluable for response. One patient died during induction (2%). CR was achieved in 35 patients (82%). Twenty-three patients, 66% of those achieving CR, underwent autologous (N = 17) or allogeneic (N = 6) transplantation. With a median follow up of 24 months, the average median duration of CR is 17 months (range 3-66) and the median survival is 20 months (range 1-83). Overall the 5 year projected diseas...
Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologi... more Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologic cells show a strong expression of CD25 (interleukin-2 [IL-2] receptor alpha chain or p55). "Variant" cases of HCL, characterized by hyperleukocytosis, neoplastic elements with a prominent nucleolus and a higher nucleo/cytoplasmic ratio, and an easily obtained bone marrow aspirate, lack surface CD25 determinants. Limited information is available on the expression of the IL-2 receptor beta chain (p75) on normal and neoplastic B cells. In this study, we have assessed by immunofluorescence and mRNA analysis the presence of the IL-2 receptor alpha and beta chains on 12 cases of classic HCL, as well as on 3 variant cases. The results obtained show that, while the alpha chain of the IL-2 receptor is present only on classic HCL, the IL-2 receptor beta chain (p75) is expressed on both the classic and variant form. Unlike hairy cells, only 8 of the 15 B-cell chronic lymphocytic leukemi...
Natural killer (NK) activity, which has been implicated in the immune response against viral infe... more Natural killer (NK) activity, which has been implicated in the immune response against viral infections and neoplasias, is currently evaluated by means of a chromium (51Cr) release assay. However, criticisms have been raised with regard to the reliability and reproducibility of the test. We have developed a different in vitro method for measuring NK activity, based on the inhibition of the target clone growth in plasma clot semisolid medium. This method overcomes the limitations inherent to the 51Cr release test and more closely mimics the in vivo situation. The inhibitory activity revealed by the cloning assay was always greater than the lytic activity in the 51Cr release assay. Moreover, effector/target ratios of 3:1 and 1.5:1 still produced clonal inhibition. B-CLL cells, used as effectors, showed no inhibitory activity and the Raji cell line employed as target was resistant in both techniques. Thus, the clonogenic assay appears to be more sensitive for the evaluation of low leve...
A system to purge the bone marrow of myeloma cells has been developed in our laboratories with th... more A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody--that recognizes plasma cells and B-cell precursors--and the ribosome-inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno-occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The haemopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.
Journal of experimental & clinical cancer research : CR, 2001
Since the social and financial impact of AML therapy is becoming more and more relevant we analyz... more Since the social and financial impact of AML therapy is becoming more and more relevant we analyzed the cost of induction therapy of two different regimens. The first one is part of the widely employed EORTC-GIMEMA AML-10 and consists often days of therapy. The second (FLANG) is a short (three day), Fludarabine, Ara-C, mitoxantrone and G-CSF containing regimen. We first retrospectively analyzed the outcome of 77 consecutive AML patients with comparable clinical and haematological features receiving FLANG (25) or AML-10 (52), between June 1993 and October 1999, and observed equivalent CR rate, as well as DFS and overall survival duration. We then selected 9 non pretreated patients per group who reached CR after one course of therapy. Patients treated with FLANG had a statistically significant earlier platelet recovery compared to those treated with AML-10, fewer days of intravenous antibiotic therapy (14/22, respectively, p < 0.05), and a shorter hospitalization period (22/33 days...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 1999
To compare the toxicity and effects on hematologic recovery and circulating progenitor cell mobil... more To compare the toxicity and effects on hematologic recovery and circulating progenitor cell mobilization of three cytokine regimens administered after high-dose cyclophosphamide (HD-CTX; 6 g/m(2)), given as the first step of a high-dose sequential chemotherapy. Forty-eight patients with breast cancer or non-Hodgkin's lymphoma were randomized to receive granulocyte colony-stimulating factor (G-CSF) alone (arm 1), granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (arm 2), or sequential interleukin-3 (IL-3) and GM-CSF (arm 3). Cytokines were administered as a single daily subcutaneous injection at a dose of 5 to 6 microg/kg/d. Progenitor cells were evaluated in peripheral blood as well as in apheretic product as both CD34(+) cells and granulocyte-macrophage colony-forming units (CFU-GM). Neutrophil recovery was faster in arm 1 as compared with arms 2 and 3 (P <.0001); no significant differences were observed between arms 2 and 3. In arm 3, a moderate acceleration o...
The distribution of T lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 u... more The distribution of T lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 untreated patients with multiple myeloma (MM) subdivided according to the clinical stage of the disease. A significant reduction (P less than 0.001) of T lymphocytes was observed only in stage II and III patients. The proportion and absolute number of OKT4 positive cells (helper/inducer phenotype) were significantly reduced in all stages of the disease; this quantitative abnormality was more pronounced in advanced disease. While the proportion of OKT8 positive cells (suppressor/cytotoxic phenotype) was increased above normal in all stages, the absolute number (of OKT8 positive cells) was high only in stage I patients; on the contrary in stage II-III patients the total OKT8 count was reduced compared with normal controls. A significantly reduced OKT4/OKT8 ratio was found in both groups of patients (P less than 0.005). Functional studies, carried out on the unfractionated T cells of patient...
Three monoclonal antibodies (MoAb), named 8A, 8F6 and 62B1, reacting with plasma cell-associated ... more Three monoclonal antibodies (MoAb), named 8A, 8F6 and 62B1, reacting with plasma cell-associated antigens, were characterized. 8A was found to be positive throughout the B cell lineage maturation steps from the immature B-committed CD10+ cell to the plasma cells. 8F6 and 62B1 reactivity is restricted to more mature cells and related lymphoid malignancies. In particular 62B1 appears to be limited to hairy cells and plasma cells. The results show that it is possible to obtain reagents reacting with plasma cells by immunizing mice with cells derived from human multiple myelomas. Furthermore, the obtained results suggest that it is possible to elicit antibodies against antigens which are present throughout all the differentiation steps of the B cell lineage. These new MoAb could help in elucidating the phenotype of the plasma cells and the relationships of multiple myelomas with other B cell proliferative disorders.
Attempts have been made by a number of methods to eliminate minimal residual disease from bone ma... more Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.
Despite the fact that granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte c... more Despite the fact that granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used in clinical practice, little is known of their endogenous production, especially in myeloproliferative disorders such as chronic myelogenous leukemia (CML). In order to define serum levels of GM-CSF and G-CSF in subjects affected by CML, the sera of 17 patients with CML in chronic phase treated either with hydroxyurea or interferon-alpha were tested by specific enzyme immunoassays. Fifteen age- and sex-matched healthy volunteers were used as normal controls. Eight out of the 17 patients (44%) with CML showed detectable (> 3 pg/mL) serum levels of GM-CSF (range 3.9-55 pg/mL). Detectable levels (> 50 pg/mL) of G-CSF were observed in 9 of these patients (52%) (range 150-2,830 pg/mL). On the contrary, among the normal controls only one had detectable GM-CSF concentrations, and none had detectable G-CSF concentrations. The highest c...
A system to purge the bone marrow of myeloma cells has been developed in our laboratories with th... more A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody--that recognizes plasma cells and B-cell precursors--and the ribosome-inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno-occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The haemopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.
Several genes with relevant pathogenetic and prognostic value have been identified in patients wi... more Several genes with relevant pathogenetic and prognostic value have been identified in patients with myelodysplastic syndromes. Overexpression of WT1 at diagnosis has been associated with increased progression to acute myeloid leukemia and reduced leukemia free survival. Conversely, few data are available on the prognostic value of BAALC gene overexpression in AML and MDS patients. We evaluated the prognostic value of the combined expression of WT1 and BAALC genes at diagnosis in 86 MDS patients who had been stratified according to IPSS and R-IPSS scoring systems. Our results suggest that in the whole group of patients, low levels of both WT1 and BAALC were associated with a favorable outcome (3-year LFS 74.5%, median not reached), whereas patients presenting high expression levels of both genes had the worst prognosis (3-year LFS 0%, median 12 months, p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001). More specifically, molecular profiling was especially useful for intermediate 1 and intermediate-2/high risk groups. This study suggests that evaluating WT1 and BAALC gene expression at diagnosis may improve standard risk stratification and possibly refine the therapeutic approach for MDS patients.
Background: Characterization of genes differentially expressed during the stages of tumour progre... more Background: Characterization of genes differentially expressed during the stages of tumour progression may lead to the identification of "oncoantigens", tumour-associated molecules that play important roles in driving tumour progression and constitute potential targets for preventive antitumour vaccination. Until now, we have identified putative oncoantigens (POAs) as molecules expressed by mammary cells in pre-neoplastic lesions and overexpressed in evident neoplastic lesions. However, many human malignancies, including breast cancer, are organized in a hierarchical network of rare slowly dividing cancer stem cells (CSCs), rapidly dividing amplifying cells and differentiated tumour cells. CSCs constitute the source of the tumour and could be responsible for tumour progression, metastasis, resistance to therapy and recurrence, so preventive vaccination should target them. Thus, analysis of CSCs transcriptional profiling may identify new POAs, more suitable for effective vaccination. Material and Methods: Mammary tumour specimens were obtained from a cell line derived from BALB-neuT breast carcinomas (Ag12). Cells were plated in differentiative conditions to obtain tumour epithelial cells (e0) or under specific conditions to generate mammospheres (p1), which were then disaggregated and plated to obtain second (p2) and third (p3) passage mammospheres. Expression of CSCs markers on mammospheres was checked by cytofluorimetry. Transcription profiling was performed on e0 and p1−3 using Illumina microarray platform MouseWG-6 v2.0. Results: Mammospheres generated from Ag12 displayed clonogenicity, self renewal, CSCs markers and ability to differentiate in mammary epithelial cells and maintained the tumourigenic potential. 452 deregulated transcripts were detected in mammospheres using rank product statistics, comparing e0 with p1−3. To detect CSC vaccination targets, a subset of 183 transcripts (POAs) which expression increased from p1 to p3 were selected by K-mean clustering. Vaccination targets for breast cancer prevention were selected ranking the 183 transcripts on the basis of the relation between their expression and survival in 7 public human breast cancer transcription profiles. The actual protein increase of some of these POAs in p1−3 was confirmed in ELISA and cytofluorimetric experiments. Conclusions: Mammospheres transcription profiling led to the identification of new POAs. Future experiments will validate these POAs in preventive vaccination in BALB-neuT mice.
A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (... more A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (ET), and represents a key World Health Organization diagnostic criterion. This hypothesis-generating study (NCT01352585) explored the impact of JAK2(V617F) mutation status on treatment response to anagrelide in patients with ET who were intolerant/refractory to their current cytoreductive therapy. The primary objective was to compare the proportion of JAK2-positive versus JAK2-negative patients who achieved at least a partial platelet response (≤600×10(9)/L) after anagrelide therapy. Of the 47 patients enrolled, 46 were included in the full analysis set (JAK2-positive, n=22; JAK2-negative, n=24). At 12 months, 35 patients (n=14 and n=21, respectively) had a suitable platelet sample; of these, 74.3% (n=26) achieved at least a partial response. The response rate was higher in JAK2-positive (85.7%, n=12) versus JAK2-negative patients (66.7%, n=14) (odds ratio [OR] 3.00; 95% confidence interval [CI] 0.44, 33.97). By using the last observation carried forward approach in the sensitivity analysis, which considered the imbalance in patients with suitable samples between groups, the overall response rate was 71.7% (n=33/46), with 77.3% (n=17/22) of JAK2-positive and 66.7% (n=16/24) of JAK2-negative patients achieving at least a partial response (OR 1.70; 95% CI 0.39, 8.02). There was no significant change in median allele burden over 12 months in the 12 patients who achieved a response. In conclusion, the overall platelet response rate was high in both JAK2-positive and JAK2-negative patients; however, a larger study would be required to confirm the differences observed according to JAK2(V617F) mutation status.
A significant reduction in the proportion and absolute number of circulating Ty (suppressor) lymp... more A significant reduction in the proportion and absolute number of circulating Ty (suppressor) lymphocytes was observed in 15 adult patients with idiopathic thrombocytopenic purpura (ITP). The proportion of cells was also reduced in the spleen of four patients so investigated. This abnormality was not seen in 5 patients cured by splenectomy whilst it persisted in 3 who remained thrombocytopenic after splenectomy. The proportion of Q (helper) lymphocytes in ITP was normal.
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown t... more The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have anti-leukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current anti-leukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here we evaluated the anti-leukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n=6; B-CLL, n=19), and healthy leukocytes. Intracellular NAD+ and ATP levels, mitochondrial transmembrane potential (ΔΨm), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34+ progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD+ and ATP shortage, and induced ΔΨm dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including AML and B-CLL samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Journal of experimental & clinical cancer research : CR, 2002
Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered th... more Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered the study. The mean age of patients was 50 (range 15-60). The induction regimen (FLAG-Ida) included fludarabine (30 mg/sqm), Ara-C (2 g/sqm) on days 1-5, and idarubicin (10 mg/sqm) on days 1, 3, 5. G-CSF (300 mcg/day) was administered s.c. 12 hours before starting fludarabine and was continued for five days. HDT with stem cell rescue was planned for all patients in first CR after one course of high dose Ara-C (HDAC) consolidation and in good clinical conditions. Forty-two (98%) patients were evaluable for response. One patient died during induction (2%). CR was achieved in 35 patients (82%). Twenty-three patients, 66% of those achieving CR, underwent autologous (N = 17) or allogeneic (N = 6) transplantation. With a median follow up of 24 months, the average median duration of CR is 17 months (range 3-66) and the median survival is 20 months (range 1-83). Overall the 5 year projected diseas...
Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologi... more Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologic cells show a strong expression of CD25 (interleukin-2 [IL-2] receptor alpha chain or p55). "Variant" cases of HCL, characterized by hyperleukocytosis, neoplastic elements with a prominent nucleolus and a higher nucleo/cytoplasmic ratio, and an easily obtained bone marrow aspirate, lack surface CD25 determinants. Limited information is available on the expression of the IL-2 receptor beta chain (p75) on normal and neoplastic B cells. In this study, we have assessed by immunofluorescence and mRNA analysis the presence of the IL-2 receptor alpha and beta chains on 12 cases of classic HCL, as well as on 3 variant cases. The results obtained show that, while the alpha chain of the IL-2 receptor is present only on classic HCL, the IL-2 receptor beta chain (p75) is expressed on both the classic and variant form. Unlike hairy cells, only 8 of the 15 B-cell chronic lymphocytic leukemi...
Natural killer (NK) activity, which has been implicated in the immune response against viral infe... more Natural killer (NK) activity, which has been implicated in the immune response against viral infections and neoplasias, is currently evaluated by means of a chromium (51Cr) release assay. However, criticisms have been raised with regard to the reliability and reproducibility of the test. We have developed a different in vitro method for measuring NK activity, based on the inhibition of the target clone growth in plasma clot semisolid medium. This method overcomes the limitations inherent to the 51Cr release test and more closely mimics the in vivo situation. The inhibitory activity revealed by the cloning assay was always greater than the lytic activity in the 51Cr release assay. Moreover, effector/target ratios of 3:1 and 1.5:1 still produced clonal inhibition. B-CLL cells, used as effectors, showed no inhibitory activity and the Raji cell line employed as target was resistant in both techniques. Thus, the clonogenic assay appears to be more sensitive for the evaluation of low leve...
A system to purge the bone marrow of myeloma cells has been developed in our laboratories with th... more A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody--that recognizes plasma cells and B-cell precursors--and the ribosome-inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno-occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The haemopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.
Journal of experimental & clinical cancer research : CR, 2001
Since the social and financial impact of AML therapy is becoming more and more relevant we analyz... more Since the social and financial impact of AML therapy is becoming more and more relevant we analyzed the cost of induction therapy of two different regimens. The first one is part of the widely employed EORTC-GIMEMA AML-10 and consists often days of therapy. The second (FLANG) is a short (three day), Fludarabine, Ara-C, mitoxantrone and G-CSF containing regimen. We first retrospectively analyzed the outcome of 77 consecutive AML patients with comparable clinical and haematological features receiving FLANG (25) or AML-10 (52), between June 1993 and October 1999, and observed equivalent CR rate, as well as DFS and overall survival duration. We then selected 9 non pretreated patients per group who reached CR after one course of therapy. Patients treated with FLANG had a statistically significant earlier platelet recovery compared to those treated with AML-10, fewer days of intravenous antibiotic therapy (14/22, respectively, p < 0.05), and a shorter hospitalization period (22/33 days...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 1999
To compare the toxicity and effects on hematologic recovery and circulating progenitor cell mobil... more To compare the toxicity and effects on hematologic recovery and circulating progenitor cell mobilization of three cytokine regimens administered after high-dose cyclophosphamide (HD-CTX; 6 g/m(2)), given as the first step of a high-dose sequential chemotherapy. Forty-eight patients with breast cancer or non-Hodgkin's lymphoma were randomized to receive granulocyte colony-stimulating factor (G-CSF) alone (arm 1), granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (arm 2), or sequential interleukin-3 (IL-3) and GM-CSF (arm 3). Cytokines were administered as a single daily subcutaneous injection at a dose of 5 to 6 microg/kg/d. Progenitor cells were evaluated in peripheral blood as well as in apheretic product as both CD34(+) cells and granulocyte-macrophage colony-forming units (CFU-GM). Neutrophil recovery was faster in arm 1 as compared with arms 2 and 3 (P <.0001); no significant differences were observed between arms 2 and 3. In arm 3, a moderate acceleration o...
The distribution of T lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 u... more The distribution of T lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 untreated patients with multiple myeloma (MM) subdivided according to the clinical stage of the disease. A significant reduction (P less than 0.001) of T lymphocytes was observed only in stage II and III patients. The proportion and absolute number of OKT4 positive cells (helper/inducer phenotype) were significantly reduced in all stages of the disease; this quantitative abnormality was more pronounced in advanced disease. While the proportion of OKT8 positive cells (suppressor/cytotoxic phenotype) was increased above normal in all stages, the absolute number (of OKT8 positive cells) was high only in stage I patients; on the contrary in stage II-III patients the total OKT8 count was reduced compared with normal controls. A significantly reduced OKT4/OKT8 ratio was found in both groups of patients (P less than 0.005). Functional studies, carried out on the unfractionated T cells of patient...
Three monoclonal antibodies (MoAb), named 8A, 8F6 and 62B1, reacting with plasma cell-associated ... more Three monoclonal antibodies (MoAb), named 8A, 8F6 and 62B1, reacting with plasma cell-associated antigens, were characterized. 8A was found to be positive throughout the B cell lineage maturation steps from the immature B-committed CD10+ cell to the plasma cells. 8F6 and 62B1 reactivity is restricted to more mature cells and related lymphoid malignancies. In particular 62B1 appears to be limited to hairy cells and plasma cells. The results show that it is possible to obtain reagents reacting with plasma cells by immunizing mice with cells derived from human multiple myelomas. Furthermore, the obtained results suggest that it is possible to elicit antibodies against antigens which are present throughout all the differentiation steps of the B cell lineage. These new MoAb could help in elucidating the phenotype of the plasma cells and the relationships of multiple myelomas with other B cell proliferative disorders.
Attempts have been made by a number of methods to eliminate minimal residual disease from bone ma... more Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.
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Papers by Marco Gobbi