Javier Pérez-Calvo

BCR-ABL Induces the Expression of Skp2 through the PI3K Pathway to Promote p27Kip1 Degradation and Proliferation of Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G(1) cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27(Kip1) and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias.

Pilot Clinical Trial of Type 1 Dendritic Cells Loaded with Autologous Tumor Lysates Combined with GM-CSF, Pegylated IFN, and Cyclophosphamide for Metastatic Cancer Patients

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day −7), GM-CSF (days 1–4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ–ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [111In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

Docetaxel Plus Vinorelbine as Salvage Chemotherapy in Advanced Breast Cancer: A Phase II Study

AbstractPrécis Administration of a combined regimen of docetaxel plus vinorelbine every 4 weeks is feasible and shows activity in heavily pretreated patients with advanced breast cancer. Purpose. To determine the activity and tolerance of docetaxel plus vinorelbine in heavily pretreated patients with advanced breast cancer. Methods. Thirty-five metastatic breast cancer patients with ECOG performance status of 0–2 received docetaxel (80 mg/m2 given intravenously) on day 1 and vinorelbine (30 mg/m2 given intravenously) on days 1 and 14, every 4 weeks. The median number of prior chemotherapy regimens was 2 (range: 1–4). Twenty-five patients (71.4%) had been treated previously using intensive therapy approaches with peripheral blood-derived stem cell (PBSC) support, including high-dose chemotherapy (11 patients), multicyclic dose-intensive chemotherapy supported with repeated PBSC infusions (seven patients), or both (seven patients). Twenty-eight patients (80%) received previous chemotherapy for metastatic disease. Adjuvant therapy in the remaining seven patients consisted of high-dose chemotherapy and PBSC support or an anthracycline-containing regimen. Results. The total number of courses was 229, and the median number of courses per patient was 6 (range: 1–16). There was one toxic death (2.8%). Grade 3–4 toxicities included mucositis (17.1%), neutropenia (37.1%), anemia (5.7%), vomiting (2.9%), and asthenia (14.3%). Eighteen patients (58%; 95% CI: 40.6–75.4%) achieved an objective response, including four complete responses (12.9%) and 14 partial responses (45.1%). Overall response rate was 51.4% (95% CI: 34.8–67.9%). After a median follow-up of 20 months (range: 2–42), overall survival was 20 months (95% CI: 16–24), and median time to progression was 13 months (95% CI: 7–19). Conclusion. This combination shows activity and an acceptable toxicity profile in patients with advanced breast cancer.

Idiopathic hyperammonemia following high-dose chemotherapy.

The diagnosis of idiopathic hyperammonemia (IH) is made in the presence of elevated serum ammonia, neurological deterioration of no other obvious aetiology, normal liver function tests and normal amino-acid levels, which rule out enzymatic deficiencies of the urea cycle. Pathological features include presence of Alzheimer type II astrocytes in brain and centrilobular microvesicular steatosis. An acquired deficiency of glutamine synthetase of unknown origin has been suggested as a possible mechanism. Such deficiency would result in waste nitrogen circulating as ammonia and not being converted to glutamine. Immunosuppression and/or high-dose cytoreductive treatment might be responsible for the inactivation of glutamine synthetase. We report here the case of a 52-year-old woman with locally advanced HER2þ breast cancer and extensive breast and axillary involvement after preoperative chemotherapy with anthracyclines and taxanes, who was enrolled onto a phase II study of a novel high-dose regimen of trastuzumab/docetaxel/melphalan/carboplatin. Her post transplant course was complicated with grade 4 colitis with multiorgan failure. After resolution of this toxicity, the patient remained obtunded. Her serum ammonia level was noticed to be high at 266mg/dl, with normal serum GOT GPT, bilirubin and renal function parameters. No urease-producing bacteria were isolated in faeces. Amino-acid analyses (aspartic acid, glutamic acid, hydroxyproline, asparaginase, glycine, glutamine, taurine, histidine, citruline, threonine, alanine, arginine, proline, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan, ornithine and lysine) were normal. Well-known iatrogenic causes of her hyperammonemia, such as valproic acid or asparaginase, were reasonably excluded. Lactulose enemas were unsuccessful, but ammonia-trapping treatment with intravenous and oral sodium benzoate resulted in gradual resolution of the neurological picture and normalization of the ammonia levels over the following 2 weeks. Following discharge, she received locoregional radiotherapy. She remains on maintenance triweekly trastuzumab treatment, free of disease and performance status 0 at last follow-up visit, 10 months after high-dose chemotherapy. Few cases of post transplant IH have been previously described. Mitchell et al. described nine cases, eight after intensive cytoreductive therapy and the ninth, 2 months after an allogeneic BMT. Only three of them survived. The longest series was reported by Davies and colleagues, including 12 patients, 10 of whom died from this complication, despite dialysis and ammonia-trapping therapy. Idiopathic hyperammonemia is a highly lethal complication of BMT. Hyperammonemia needs to be ruled out in the presence of neurological deterioration of metabolic origin. Clinicians involved in the care of BMT patients need to be aware of this rare, albeit potentially fatal, diagnosis.

Phase 1–2 pilot clinical trial in patients with decompensated liver cirrhosis treated with bone marrow–derived endothelial progenitor cells

&NA; The aim of this nonrandomized, open label, phase 1 clinical trial was to evaluate the safety and the feasibility of the treatment with autologous bone marrow–derived endothelial progenitor cells (EPC) in decompensated liver cirrhosis. In addition, the changes in liver function and hepatic venous pressure gradient (HVPG) and their relation with the characteristics of the cellular product were analyzed. Twelve patients with Child‐Pugh ≥8 liver cirrhosis underwent bone marrow harvest for ex vivo differentiation of EPC. The final product was administered through the hepatic artery in a single administration. Patients underwent clinical and radiologic follow‐up for 12 months. The phenotype and the ability to produce cytokines and growth factors of the final cellular suspension were analyzed. Eleven patients were treated (feasibility 91%). No treatment‐related severe adverse events were observed as consequence of any study procedure or treatment. Model for end‐stage liver disease score improved significantly (P 0.042) in the first 90 days after cells administration and 5 of the 9 patients alive at 90 days showed a decreased of HVPG. There was a direct correlation between the expression of acetylated‐low density lipoprotein and von Willebrand factor in the cellular product and the improvement in liver function and HVPG. The treatment with EPCs in patients with decompensated liver cirrhosis is safe and feasible and might have therapeutic potential. Patients receiving a higher amount of functionally active EPC showed an improvement of liver function and portal hypertension suggesting that the potential usefulness of these cells for the treatment of liver cirrhosis deserves further evaluation.

Respuesta precoz a idursulfasa en un paciente de 31 años de edad con síndrome de Hunter

The response to Enzyme Replacement Therapy (ERT) in Hunter syndrome (MPS II) occurs early in most of the patients after its initiation and continues during the first 12-18 months. However, almost all the patients with MPS II have severe forms of the disease and death occurs prematurely. More than 90% of subjects die before 25 years, and only a minority will survive after the age of 30. There is very limited information on early response to ERT among adult patients with Hunters syndrome. We report the case of a 31 year-old male with MPS II, with a remarkably severe joint disability, but mild cognitive impairment, who was treated with idursulfase for six months. The pattern of response observed, was similar to what can be expected in younger patients. The amelioration in joint mobility observed in this case suggests that older patients with advanced articular involvement may benefit from idursulfase, even when therapy is started in later stages of the disease.

A single prior course of BCNU-cisplatin chemotherapy has a significant deleterious effect on mobilization kinetics of otherwise untreated patients.

Summary:Extensive prior treatment with cytotoxic agents is associated with impaired mobilization of hematopoietic cells. To assess the effect of a single course of standard-dose chemotherapy (CT), we compared the results of filgrastim-induced mobilization among two sequential groups of grade III–IV malignant glioma patients included in a hematopoietic transplantation program. The first group (21 patients) had never been treated with CT until 2 days after surgery, when they received a course of 100 mg/m2 BCNU (IV) and 100 mg intracarotid cisplatin for cytoreduction (not for mobilization). At 1 month after this CT, they were mobilized with 12 μg/kg filgrastim. The second group (22 patients) was mobilized with the same dose of filgrastim directly after the surgery, without having ever received any prior CT. The blood level of CD34+ cells was significantly lower in the CT-treated patients, both on the fourth day of filgrastim (15 vs 36 cells × 106/l; P=0.01) and on the fifth (25 vs 58 cells × 106/l; P=0.003), as it was the number of CD34+ cells collected per apheresis (1.3 vs 3.5 × 106/l; P<0.0005). The toxic effect of a single course of BCNU-cisplatin CT led to significant impairment of the filgrastim-induced mobilization response.

Pulmonary embolism after local urokinase instillation in a Hickman catheter.

Bone Marrow Transplantation total body irradiation before pharmacological immunosuppression after marrow transplantation. Blood 1997; 89: 3048– 3054. 5 Giralt S, Estey E, Albitar M et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative chemotherapy. Blood 1997; 89: 4531–4536. 6 Slavin S, Nagler A, Naparstek E et al. Non-myeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic disease. Blood 1998; 91: 756–763. 7 Berger R, Bernheim A, Gluckman E et al. In vitro effect of cyclophosphamide metabolites on chromosomes of Fanconi anemia patients. Br J Haematol 1980; 45: 565–571. 8 Zanis-Neto J, Ribeiro RC, de Medeiros CR et al. Bone marrow transplantation for patients with Fanconi anemia: a study of 24 cases of a single institution. Bone Marrow Transplant 1995; 15: 293–298. 9 Flowers MED, Zanis-Neto J, Pasquini R et al. Marrow transplantation for Fanconi anemia: conditioning with reduced

Factors determining the actual received dose intensity in a program of multicyclic dose-intensive alternating chemotherapy with sequential stem cell support.

Dose intensity has been related to clinical outcome in several solid tumors. We studied the influence of clinical and cellular parameters on dose intensity received in a series of 53 patients with metastatic breast cancer or advanced ovarian cancer. They received courses of cisplatin 120 mg/m2 plus etoposide 600 mg/m2 alternating every 14 days with ifosfamide 8 g/m2 plus paclitaxel 200–350 mg/m2. Blood stem cell support was administered after every course except for the first one. Patients with excellent mobilization underwent immunomagnetic selection of CD34+ cells. We found a significant inverse correlation between the CD34+ cell dose infused and the delay for the administration of the next cycle. A CD34+ cell dose between 1.5 and 5 × 106/kg per cycle was found to be feasible and was followed by a median delay of 1day (not different from doses above 5 × 106/kg). Three factors independently predicted the actually received dose intensity in a multiple regression model (R2 = 0.4): previous autologous transplantation, eligibility for immunomagnetic selection (excellent response to mobilization) and median CD34+ cell dose received along the treatment.