RESUMEN
A cell population with progenitor-like phenotype (CD45-CD34+) resident in human white adipose tissue (WAT) is known to promote the progression of local and metastatic breast cancer and angiogenesis. However, the molecular mechanisms of the interaction have not been elucidated. In this study, we identified two proteins that were significantly upregulated in WAT-derived progenitors after coculture with breast cancer: granulocyte macrophage colony-stimulating factor (GM-CSF) and matrix metallopeptidase 9 (MMP9). These proteins were released by WAT progenitors in xenograft and transgenic breast cancer models. GM-CSF was identified as an upstream modulator. Breast cancer-derived GM-CSF induced GM-CSF and MMP9 release from WAT progenitors, and GM-CSF knockdown in breast cancer cells neutralized the protumorigenic activity of WAT progenitors in preclinical models. GM-CSF neutralization in diet-induced obese mice significantly reduced immunosuppression, intratumor vascularization, and local and metastatic breast cancer progression. Similarly, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth. Combined GM-CSF neutralization and MMP9 inhibition synergistically reduced angiogenesis and tumor progression. High-dose metformin inhibited GM-CSF and MMP9 release from WAT progenitors in in vitro and xenograft models. In obese syngeneic mice, metformin treatment mimicked the effects observed with GM-CSF neutralization and MMP9 inhibition, suggesting these proteins as new targets for metformin. These findings support the hypothesis that GM-CSF and MMP9 promote the protumorigenic effect of WAT progenitors on local and metastatic breast cancer. Cancer Res; 77(18); 5169-82. ©2017 AACR.
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Adipocitos/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Células Madre/metabolismo , Células del Estroma/patología , Microambiente Tumoral/inmunología , Adipocitos/patología , Adulto , Anciano , Animales , Apoptosis , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre/patología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 - an aberrant tyrosine kinase - in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs.
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Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Linfoma de Células T Periférico/patología , Antineoplásicos/uso terapéutico , Proteínas de Fusión bcr-abl/sangre , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/uso terapéutico , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Linfoma de Células T Periférico/genética , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
OBJECTIVES: To assess efficacy of bevacizumab in combination with oral chemotherapy in patients with breast cancer with lymphangitic spread to the chest wall (LBC). To identify surrogate biomarkers of response to bevacizumab. PATIENTS AND METHODS: We randomly assigned patients to receive bevacizumab plus either sequential or concurrent oral vinorelbine and capecitabine every 3 weeks. The primary endpoint was time to ultimate progression (TTP); the response rate and overall survival (OS) were secondary endpoints. We performed gene expression profiling on baseline tissue samples collected from triple negative LBC. We assessed circulating endothelial cells (CEC), circulating endothelial progenitors (CEP) and circulating pericyte progenitors (CPP). RESULTS: A total of 66 patients were enrolled. There was no difference in TTP (median TTP 5.3 vs. 4.8 months, p = 0.21) and in OS (median OS 15.8 vs 11.9 months; p = 0.25) when comparing concurrent vs sequential treatment, respectively. Response rate was 25% vs 28% in the concurrent vs sequential arm (p = 1.00), respectively. A set of 16 genes predictive of response to bevacizumab was identified. The counts of CEPs and viable CECs below the median value were associated with an improved overall survival: 26.6 vs 9.5 months for CEPs and 22.6 vs 11.0 months for viable CECs, respectively (p = 0.02). CONCLUSIONS: Oral chemotherapy and bevacizumab (BEVIX) is an active regimen in patients with LBC. We support the importance of using LBC as a biological model for investigating angiogenesis inhibitors. CECs and CEPs biomarkers have been identified as predictive markers of outcome and warrant further investigation.
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Inhibidores de la Angiogénesis/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bevacizumab/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Torácicas/secundario , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Administración Oral , Adulto , Anciano , Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos Fitogénicos/administración & dosificación , Biomarcadores de Tumor/genética , Capecitabina/administración & dosificación , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Pericitos/efectos de los fármacos , Análisis de Supervivencia , Pared Torácica , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Vinblastina/administración & dosificación , Vinblastina/análogos & derivados , VinorelbinaRESUMEN
Human cytomegalovirus (hCMV) infection and its reactivation correlate both with the increased risk and with the worsening of graft-versus-host disease (GVHD). Because scleroderma-like skin lesions can occur in chronic GVHD (cGVHD) in allogeneic stem-cell transplant (HCT) patients and hCMV is relevant in the pathogenesis of systemic sclerosis (SSc), we evaluated the possible pathogenetic link between hCMV and skin cGVHD. Plasma from 18 HCT patients was tested for anti-UL94 and/or anti-NAG-2 antibodies, identified in SSc patients, by direct ELISA assays. Both donors and recipients were anti-hCMV IgG positive, without autoimmune diseases. Patients' purified anti-UL94 and anti-NAG-2 IgG binding to human umbilical endothelial cells (HUVECs) and fibroblasts was performed by FACS analysis and ELISA test. HUVECs apoptosis and fibroblasts proliferation induced by patients' anti-NAG-2 antibodies were measured by DNA fragmentation and cell viability, respectively. About 11/18 patients developed cGVHD and all of them showed skin involvement, ranging from diffuse SSc-like lesions to limited erythema. Eight of eleven cGVHD patients were positive for anti-UL94 and/or anti-NAG-2 antibodies. Remarkably, 4/5 patients who developed diffuse or limited SSc-like lesions had antibodies directed against both UL94 and NAG-2; their anti-NAG-2 IgG-bound HUVECs and fibroblasts induce both endothelial cell apoptosis and fibroblasts proliferation, similar to that induced by purified anti-UL94 and anti-NAG-2 antibodies obtained from SSc patients. In conclusion, our data suggest a pathogenetic link between hCMV infection and scleroderma-like skin cGVHD in HCT patients through a mechanism of molecular mimicry between UL94 viral protein and NAG-2 molecule, as observed in patients with SSc.
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Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Enfermedad Injerto contra Huésped/inmunología , Esclerodermia Sistémica/inmunología , Trasplante de Células Madre , Adulto , Apoptosis , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Reacciones Cruzadas , Infecciones por Citomegalovirus/complicaciones , Endotelio Vascular/inmunología , Femenino , Fibroblastos/inmunología , Enfermedad Injerto contra Huésped/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Imitación Molecular , Esclerodermia Sistémica/complicaciones , Piel/inmunología , Piel/patología , Piel/virología , Tetraspaninas/inmunología , Adulto JovenRESUMEN
Patients with a number of peripheral CD34+ cells ≥20/µL have recently been defined in the literature as "poor mobilizers". We retrospectively reviewed medical records from a total of 248 patients affected by hematological malignancies or solid tumors undergoing peripheral blood stem cell collection following chemotherapy plus G-CSF. On the basis of the CD34+ cell peak in peripheral blood following mobilization therapy, patients were defined as good mobilizers (group A, CD34+ cells ≥20/µL), relative poor mobilizers (group B, CD34+ cells <20 and ≥8/µL) and absolute poor mobilizers (group C, CD34+ cells <8/µL). One hundred and seventy-seven (71%) patients resulted good mobilizers, 35 (14%) patients relative poor mobilizers and 36 (15%) patients absolute poor mobilizers. Target of stem cell collection was ≥2.0×10(6) CD34+cells/kg for each transplantation procedure. All patients in group A, 20 patients in group B (57%) and 1 patient in group C (2.7%) were able to collect ≥2.0×10(6) CD34+cells/kg. The multivariate analysis confirmed that more than three lines of previous chemotherapy and a previous autologous PBSC transplantation negatively affect mobilization of CD34+ cells in peripheral blood. Our data suggest that a number of CD34+ cells ≥20/µL does not always result in a failed stem cell collection and in fact in our patient series more than 70% of the patients defined as poor mobilizers have indeed collected the minimum number of 2.0×10(6) CD34+cells/kg required for a successful transplantation. The use of new agent such as CXCR4 antagonist plerixafor might further improve mobilization efficacy in such patients.
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Fármacos Anti-VIH/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Compuestos Heterocíclicos/administración & dosificación , Neoplasias/terapia , Trasplante de Células Madre de Sangre Periférica , Adolescente , Adulto , Anciano , Bencilaminas , Ciclamas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores CXCR4/antagonistas & inhibidores , Estudios Retrospectivos , Factores de Riesgo , Trasplante AutólogoRESUMEN
Plerixafor, a CXCR4 antagonist, has shown to be effective in increasing the number of circulating stem cells, even in patients failing a previous mobilisation attempt. Recently a number of non-glycosylated recombinant human granulocyte-colony stimulating factor (G-CSF) has been clinically approved for the same indications as the originator G-CSF for comparable safety and efficacy and their reduced cost. In an attempt to provide a less toxic strategy, 14 patients affected by haematological malignancies (non-Hodgkin's lymphoma = 4, Hodgkin's disease = 2 and multiple myeloma = 8), received the combination of biosimilar filgrastim and plerixafor as a first line mobilising strategy. The median number of circulating CD34+ cells on day 4 was 16 (3-42); Plerixafor was administered to all, but one patient who had already 42 CD34+ cells per µL on day 4. On day 5, after plerixafor administration, the median number of circulating CD34+ cells had raised to 60 per µL (14-138). All the patients underwent leukapheresis and were able to collect a number of CD34+ cells ≥ 2.0 × 10(6) /kg in a median number of procedures of one. Although preliminary, these data show the combination of biosimilar filgrastim and plerixafor is effective and provides a non-toxic approach to mobilise stem cells.
Asunto(s)
Trasplante de Médula Ósea , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Compuestos Heterocíclicos/administración & dosificación , Linfoma/cirugía , Mieloma Múltiple/cirugía , Adulto , Anciano , Antígenos CD34/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencilaminas , Terapia Combinada , Ciclamas , Femenino , Filgrastim , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Compuestos Heterocíclicos/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Trasplante Autólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.
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Neoplasias/sangre , Pericitos/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/fisiología , Inhibidores de la Angiogénesis , Animales , Células de la Médula Ósea/citología , Recuento de Células , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Indoles/farmacología , Ratones , Pericitos/citología , Pericitos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Pirroles/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Células Madre/citología , Células Madre/metabolismo , SunitinibRESUMEN
Pegfilgrastim has equivalent efficacy to daily G-CSF in enhancing neutrophil recovery after chemotherapy, but conclusive data concerning its use for peripheral blood stem cell (PBSC) mobilization are lacking. From 2003 to 2008 we used high-dose chemotherapy in 64 lymphoma patients. At mobilization chemotherapy (ESHAP) the first 26 patients used unconjugated G-CSF, while the remaining 38 patients received Pegfilgrastim. At the time of harvest 25 patients collected stem cells after the use of G-CSF and 36 in the Peg group. No statistical by significant differences were observed in median peripheral CD34+ cells mobilized (77 µL versus 71 µL) and in collected PBSC (12.3 × 10(6)/kg versus 9.4 × 10(6)/kg p = 0.76). In the PEG group all patients collected the target PBSC with a single apheresis with a greater proportion of "optimal" mobilizers (83% versus 64%; p = 0.05). In conclusion a single dose of Pegfilgrastim could be a valid alternative to unconjugated G-CSF to mobilize PBSC in lymphoma patients.
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Supervivencia de Injerto , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Linfoma/terapia , Trasplante de Células Madre de Sangre Periférica/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Filgrastim , Humanos , Polietilenglicoles , Proteínas Recombinantes , Trasplante AutólogoRESUMEN
BACKGROUND: Angiogenesis is crucial for tissue repair and cancer progression. We investigated a panel of angiogenic cells, macroparticles and RNA transcripts before, during and after laparoscopic colectomy or open colectomy for colorectal cancer. RESULTS: Viable and apoptotic circulating endothelial cells were significantly increased after open but not after laparoscopic colectomy (p < 0.01). A significant decrease of circulating mRNA coding for VEGFR-C and D and PDGFR-ß was found after laparoscopic but not after open colectomy. METHODS: A total of 24 patients were enrolled. Viable and apoptotic circulating endothelial cells, progenitors and macroparticles were evaluated by flow cytometry. The number of copies of angiogenesis-related RNA transcripts we reevaluated by quantitative PCR. CONCLUSION: Open, but not laparoscopic colectomy, was associated with a significant post-operative increase in circulating endothelial cells, either apoptotic (likely due to surgery-related vascular damage) and viable (likely representing vascular remodeling). Circulating RNA copies coding for some angiogenic genes were significantly decreased after laparoscopic colectomy likely because of the removal of the tumor lesion. This decrease was not observed after open colectomy,were a more pronounced wave of angiogenesis related to wound healing was expected. These results indicate a relevant wave of angiogenesis-related cells and transcripts after open but not after laparoscopic colectomy.
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Colectomía/métodos , Neoplasias Colorrectales/cirugía , Células Endoteliales/metabolismo , Laparoscopía/métodos , ARN/sangre , Anciano , Apoptosis , Supervivencia Celular , Neoplasias Colorrectales/sangre , Células Endoteliales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tamaño de la Partícula , Periodo Posoperatorio , Periodo Preoperatorio , ARN/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factor C de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/genéticaRESUMEN
The aim of this study was to investigate the efficacy of combined treatment with rituximab and subcutaneous cladribine in patients with newly diagnosed and relapsed chronic lymphocytic leukemia (CLL). Forty-three patients with active CLL or small lymphocytic lymphoma received rituximab 375 mg/m(2) on day 1 and cladribine 0.1 mg/kg subcutaneously on days 2-6. The treatment was repeated every 4 weeks for a total of four cycles. Sixteen patients were pretreated. The overall response rate was 88% (50% complete remission and 38% partial remission). The median time to treatment failure was 37.9 months. Grade 4 neutropenia developed in 5% of patients. The data indicate that combination therapy with rituximab and cladribine is a valuable and safe treatment for patients with CLL.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Terapia Recuperativa , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales de Origen Murino , Estudios de Casos y Controles , Cladribina/administración & dosificación , Tranportador Equilibrativo 1 de Nucleósido/genética , Femenino , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Infusiones Subcutáneas , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Masculino , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rituximab , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
PURPOSE: To assess the efficacy of 2-chloro-2'-deoxyadenosine (2-CdA) given subcutaneously (SC) in combination with rituximab in the treatment of newly diagnosed/pretreated patients with Waldenström macroglobulinemia (WM) and to correlate the response to treatment with biologic findings (immunophenotypic and pharmacogenomic analysis). PATIENTS AND METHODS: From December 2003 to February 2007, 29 patients were enrolled. Intended therapy consisted of a combination of rituximab (375 mg/m(2)) on day 1 followed by 2-CdA 0.1 mg/kg (SC injection) for 5 consecutive days, administered monthly for four cycles. Anemia (n = 16), neurologic symptoms (n = 6), symptomatic cryoglobulinemia (n = 4), and thrombocytopenia (n = 3) represented the reasons for starting treatment. The expression of zeta chain-associated protein kinase 70 (Zap-70) and of seven genes involved in 2-CdA metabolism as markers of response to the combination treatment was evaluated. RESULTS: With a median follow-up of 43 months, the overall response rate observed was 89.6%, with seven complete responses (CR), 16 partial responses, and three minor response, without any difference between newly or pretreated patients (P = .522). The therapy was well tolerated, except for transitory cardiac toxicity (n = 2) and intolerance to rituximab (n = 2). No major infections were observed despite the lack of antimicrobial prophylaxis. No patients developed transformation to high-grade non-Hodgkin's lymphoma nor myelodysplasia. Low expression levels of human concentrative nucleoside transporter 1 (hCNT1) were correlated with the failure to achieve a CR (P = .024), whereas no association with Zap-70 expression was found. CONCLUSION: The combination of rituximab and SC 2-CdA is safe and effective in patients with WM requiring treatment. The pharmacogenomic analysis associated with the study suggests hCNT1 might be beneficial in predicting clinical response to such a combination treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Macroglobulinemia de Waldenström/tratamiento farmacológico , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales de Origen Murino , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Cladribina/administración & dosificación , Supervivencia sin Enfermedad , Esquema de Medicación , Femenino , Genotipo , Humanos , Inmunofenotipificación , Inyecciones Subcutáneas , Italia , Estimación de Kaplan-Meier , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Persona de Mediana Edad , Selección de Paciente , Fenotipo , Estudios Prospectivos , Rituximab , Factores de Tiempo , Resultado del Tratamiento , Macroglobulinemia de Waldenström/inmunología , Macroglobulinemia de Waldenström/metabolismo , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The dependence of tumor growth and metastasis on blood vessels makes tumor angiogenesis a rational target for therapy. Strategies have been pursued to inhibit neovascularization and to destroy existing tumor vessels, or both. These include direct targeting of endothelial cells, and indirect targeting by inhibiting the release of proangiogenic growth factors by cancer or stromal cells. Many patients benefit from antiangiogenic therapies; thus, development of noninvasive biomarkers of disease response and relapse is a crucial objective to aid in their management. A number of non-invasive tools are described with their potential benefits and limitations. We review currently available candidate biomarkers of anti-angiogenic agent effect. Including these markers into clinical trials may provide insight into appropriate dosing for desired biological effects, appropriate timing of additional therapy, and prediction of individual response. This has important consequences for the clinical use of angiogenesis inhibitors and for drug discovery, not only for optimizing the treatment of cancer, but possibly also for developing therapeutic approaches for various other diseases.
RESUMEN
PURPOSE: The association of chemotherapy and antiangiogenic drugs has shown efficacy in clinical oncology. However, there is a need for biomarkers that allow selection of patients who are likely to benefit from such treatment and are useful for indicating best drug combination and schedule. EXPERIMENTAL DESIGN: We investigated the predictive potential of six angiogenic molecules/transcripts and nine subpopulations of circulating endothelial cells (CEC) and progenitors (CEP) in 46 patients with advanced breast cancer treated with metronomic cyclophosphamide and capecitabine plus bevacizumab. RESULTS: Median time to progression was 281 days. Baseline CECs higher than the first quartile were associated with an increased time to progression (P = 0.021). At progression, CECs were markedly reduced (P = 0.0002). In the cohort of 15 long-term responders, who progressed later than 1 year after beginning of therapy, circulating vascular endothelial growth factor (VEGF)-A levels measured after 2 months of therapy were significantly reduced, and there were significant trends toward lower levels of PDGF-BB, CEPs, and CECs. At the time of progression, angiogenic growth factors VEGF-A and basic fibroblast growth factor were significantly increased. CONCLUSIONS: Baseline CECs (likely reflecting an active vascular turnover) predicted a prolonged clinical benefit. At the time of relapse, a pattern of decreased CECs and increased angiogenic growth factors suggested a switch toward a different type of cancer vascularization. VEGF-A and basic fibroblast growth factor levels after 2 months of therapy were also useful to identify patients whose disease was likely to progress. These biomarkers are likely to be useful for treatment selection and might be incorporated in design of future studies. (Clin Cancer Res 2009;15(24):7652-7).
RESUMEN
We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin(-)CD34(-)) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34(-) cells are leukemic. CML Lin(-)CD34(-) cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin(-)CD34(-) cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34(+) cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin(-)CD34(-) cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin(-)CD34(-) cells in vitro. Moreover, leukemic CD34(-) cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34(-) leukemic stem cell subset in CML with peculiar molecular and functional characteristics.
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Antígenos CD34/metabolismo , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre Neoplásicas/metabolismo , Piperazinas/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacología , Benzamidas , Células de la Médula Ósea/metabolismo , Células Cultivadas , Análisis por Conglomerados , Citometría de Flujo , Proteínas de Fusión bcr-abl/genética , Perfilación de la Expresión Génica , Humanos , Mesilato de Imatinib , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Cariotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
PURPOSE: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. EXPERIMENTAL DESIGN: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. RESULTS: Sorted DNA/Syto16(+)CD45(-)CD31(+)CD146(+) CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 +/- 171/mL in healthy subjects (n = 37) and 951 +/- 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 +/- 14% in healthy subjects and 43 +/- 23% in cancer patients (P < 0.0001). CEPs were 181 +/- 167/mL in healthy donors and 429 +/- 507/mL in patients (P = 0.00019). Coefficients of variation were 4 +/- 4% (intrareader), 17 +/- 4% (interreader), and 17 +/- 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 +/- 8% (intrareader), 16 +/- 10% (interreader), and 26 +/- 16% (variability over 0-14 days of frozen storage), respectively. CONCLUSIONS: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.
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Células Endoteliales , Citometría de Flujo/métodos , Neoplasias/sangre , Recuento de Células/métodos , Supervivencia Celular , Células Endoteliales/química , Humanos , Microscopía Electrónica , Reproducibilidad de los Resultados , Células Madre , Cuerpos de Weibel-PaladeRESUMEN
Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in different strains of immunodeficient mice has led to preclinical models extensively used to investigate acute leukemia stem cells, biology and drug sensitivity. We studied the engraftment kinetics of AML and ALL cell lines and primary cells in 3 strains of NOD.CB17-Prkdc(scid) (NOD/scid, NS)-related mice (NOD.Cg-Prkdc(scid)B2m(tm1Unc)/J, abbreviated NOD/scid/beta2 null, NSB; and NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ, abbreviated NOD/scid/IL-2Rgamma null, NSG). The engraftment of human malignant cells was investigated by means of clinicopathological criteria, flow cytometry, PCR and immunohistochemistry. In NSG mice, we observed a significantly faster development of leukemia-related symptoms and a higher percentage of leukemia cells in the blood, in the marrow and in the spleen. The leukemia-related angiogenic switch (measured as the number of circulating endothelial cells and progenitors) was faster in NSG compared to NS and NSB mice. These models will be instrumental to studies on leukemia-initiating stem cells, leukemia biology, preclinical treatment studies, and to obtain patient-specific preclinical models to design and investigate patient-tailored therapies.
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Modelos Animales de Enfermedad , Subunidad gamma Común de Receptores de Interleucina/fisiología , Leucemia Mieloide Aguda/etiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Animales , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Trasplante HeterólogoRESUMEN
High altitude and hypoxia are known to induce polycythemia, pulmonary hypertension, and vascular remodeling. The authors investigated a number of blood cell populations in 15 mountain trekkers before and after 12 days spent at >3000 m. Red blood cell and platelet count increased, whereas circulating hematopoietic stem cell (enumerated as CD34bright cells), circulating endothelial cell (CEC) and circulating endothelial progenitor (CEP) count significantly decreased. In particular, the authors observed a decrease in the count of viable CECs, and a decrease in the circulating levels of RNA of the endothelial-specific gene VE-cadherin, whereas the fraction of apoptotic/necrotic CECs was stable. These data suggest a unique pattern of modulation of surrogate markers of vascular remodeling induced by exposure to hypobaric hypoxia.
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Altitud , Células Endoteliales/fisiología , Células Madre Hematopoyéticas/fisiología , Aclimatación , Adulto , Mal de Altura/sangre , Mal de Altura/fisiopatología , Antígenos CD34/metabolismo , Basófilos/citología , Hipoxia de la Célula , Supervivencia Celular , Células Endoteliales/citología , Endotelio Vascular/citología , Recuento de Eritrocitos , Índices de Eritrocitos , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Persona de Mediana Edad , Recuento de PlaquetasRESUMEN
PURPOSE: The purpose of this study was to investigate the incidence and prognostic relevance of tumor cell detection in granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cell collections (PBPCCs) using cytokeratin (CK), maspin (MAS), and mammaglobin (MAM) genes as epithelial cell markers. The population on which the study was conducted was drawn from stage III breast cancer patients undergoing high-dose chemotherapy and autologous transplantation with PBPCCs. EXPERIMENTAL DESIGN: One hundred and ninety-four patients were enrolled in the study and analyzed for tumor cell detection on the basis of 481 PBPCCs gathered before administration of chemotherapy. CK, MAS, and MAM gene expressions were investigated by means of the reverse transcription nested polymerase chain reaction, and those samples expressing CK were further hybridized with a radiolabeled internal probe to reduce false-positive results. Sensitivity and specificity were assessed on 37 controls (12 cell lines, 12 healthy donors, and 13 nonepithelial malignancies). Each of the known prognostic variables (age, stage, lymph node status, receptor status, c-ErbB2 status, and Ki67 status) was then analyzed (both individually and together with CK, MAS, and MAM expression on PBPCCs) in relation to patient overall survival (OS) and relapse-free survival (RFS). RESULTS: After a 3-year follow-up, an estimated 83% (95% confidence interval, 77.1-88.8%) of the patients were alive and an estimated 67% (95% confidence interval, 60.1-74.6%) were free of relapse. One hundred and seventy-six of the 194 patients (91%) had contaminated PBPCCs evidenced by at least one positive sample for any of the markers evaluated. The PBPCC frequency of CK, MAS, and MAM positivity (+) was 71%, 36%, and 16%, respectively. MAM expression on PBPCC was associated with an increased risk of relapse (P = 0.003), whereas CK and MAS expressions were not associated with changes in either RFS or OS. CONCLUSIONS: MAM gene expression on leukapheresis products of high-risk breast cancer patients is an indicator of poor prognosis. The method of evaluation is simple and reproducible and provides new tools for evaluating the role played by tumor cells in apheresis products and their potential in causing metastasis.
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Neoplasias de la Mama/sangre , Leucaféresis , Proteínas de Neoplasias/sangre , Uteroglobina/sangre , Adulto , Anciano , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Células Cultivadas , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Humanos , Queratinas/sangre , Antígeno Ki-67/sangre , Metástasis Linfática , Mamoglobina A , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Modelos de Riesgos Proporcionales , Receptor ErbB-2/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Factores de Tiempo , Resultado del TratamientoRESUMEN
No markers are currently available to indicate the angiogenic profile of a specific malignant disease nor to predict response to antiangiogenic therapies. Nevertheless, many different antiangiogenic drugs are presently being tested in many clinical trials, with an obvious scarcity of useful endpoints for treatment outcome beside survival. By means of a quantitative reverse transcription-PCR approach, we measured VE-cadherin (VE-C), Tie-2, vascular endothelial growth factor receptor 2 and CD133 RNA in the blood of 14 healthy controls, 3 pregnant women, and 84 newly diagnosed (or relapsed) cancer patients. Circulating VE-C RNA was increased in pregnant women and cancer patients (P = 0.0002). VE-C RNA was particularly increased in patients affected by hematological malignancies and decreased to normal values in patients achieving complete remission. Conversely, circulating RNA levels of other endothelial or progenitor cell-specific markers Tie-2, vascular endothelial growth factor receptor 2, and CD133 were not significantly increased in either pregnant women or cancer patients. Comparison of various surrogate angiogenesis markers indicated a switch toward increased plasma vascular endothelial growth factor (VEGF) levels, viable circulating endothelial cells, and circulating VE-C RNA levels in patients affected by hematological malignancies. Taken together, our data indicate that the quantitative evaluation of circulating VE-C RNA is a specific and highly promising tool with which to investigate the angiogenic phenotype of cancer patients.
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Cadherinas/genética , Endotelio Vascular/patología , Neoplasias Hematológicas/sangre , Neoplasias/sangre , Neoplasias/irrigación sanguínea , Neovascularización Patológica/sangre , ARN Neoplásico/sangre , Antígenos CD , Apoptosis/genética , Endotelio Vascular/metabolismo , Femenino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Embarazo , ARN Neoplásico/genética , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
Treatment of acute leukaemia in adult Jehovah's Witnesses (JW) is challenging because of 'a priori' refusal of most physicians to apply diagnostic and therapeutic procedures to haematological abnormalities resembling acute leukaemia. Rejection of blood transfusions by individuals of this faith is usually blamed to justify this attitude, thus leading to severe personal, medical and psychological distress related to the lack of care. We therefore intended to verify whether a standard (tailored) chemotherapy, without the use of prophylactic blood product transfusions, could be applied during treatment of acute leukaemia under such circumstances. Eleven consecutive JW adult patients with acute leukaemia, all of whom had been denied care in other institutions, were treated at the European Institute of Oncology (EIO) in Milan, Italy. Five had acute lymphoblastic leukaemia (ALL) (one bcr/abl positive), six had acute myeloid leukaemia (AML) with immunophenotype and/or cytogenetic intermediate-high risk features, except one patient with acute promyelocytic leukaemia (APML). Standard induction chemotherapy [cytosine arabinoside (ARA-C) and daunorubicin (DNR) for AML, vincristine (VCR), DNR and prednisone (PDN) for ALL, all-trans retinoic acid (ATRA) and DNR for APML] with the antracycline dose of at least 30 mg/sqm were used. All patients experienced severe anaemia after induction chemotherapy despite erythropoietin. Median haemoglobin nadir for patients with ALL and AML was 4.5 g/dL (range 1.3-6.9) and 5.1 g/dL (range 2.6-6.8), respectively. Median platelet nadir counts for all patients was 14.5 x 10(9))/L (range 1-24). One patient died during induction probably due to haemorrhage. Four of five patients with ALL achieved a complete remission (CR) (including the bcr/abl case) while among patients with AML only the one with APML achieved CR. Three patients (APML = 1 and ALL = 2) are still alive and disease-free. This small series of adult patients with leukaemia illustrates difficulties in treating patients who are practising JW, yet nevertheless provides a significant argument against the prejudicial decision leading to evasion of treatment in these patients.