A phase II trial of high-dose chemotherapy (HDCT) supported by hematopoietic stem-cell transplantation (HSCT) in germ-cell tumors (GCTs) patients failing cisplatin-based chemotherapy: the Multicentric TAXIF II study.

BACKGROUND: High-dose chemotherapy (HDCT) is an effective salvage treatment of germ-cell tumors (GCTs) patients. In the first salvage setting, 30%-70% of patients may achieve durable remissions. Even when HDCT is administered as subsequent salvage treatment, up to 20% of patients may still be definitively cured. However, patients with refractory/relapsed disease still have a very poor long-term prognosis, requiring earlier intervention of HDCT. PATIENTS AND METHODS: This phase II trial was addressed to nonrefractory patients failing Cisplatin-based chemotherapy. Inclusion criteria included seminomatous GCT in relapse after two lines of chemotherapy, nonseminomatous GCT in relapse after first or second lines, partial remission after first line, primary mediastinal GCT in first relapse. Patients received two cycles combining Epirubicin and Paclitaxel (Epi-Tax), followed by three consecutive HDCT, one using a Paclitaxel/Thiotepa (Thio-Tax) association and two using the 5-day Ifosfamide-Carboplatin-Etoposide regimen. The main objective was to determine the complete response rate. RESULTS: Forty-five patients were included between September 2004 and December 2007: 44 received the first HDCT cycle, 39 two HDCT cycles, 29 could receive the whole protocol. Sixteen patients did not receive the entire protocol, including eight (17.7%) for toxic side-effects. Two patients (4.4%) died of toxicities, and 17 (37.7%) of disease progression. With a median follow-up time of 26 months (range, 4-51), the final overall response rate was 48.8% (including a complete response rate of 15.5% and a partial response/negative serum markers rate of 26.6%) in an intent-to-treat analysis. The median progression-free survival (PFS) and overall survival (OS) times were 22 months [95% confidence interval (CI) 2-not reached] and 32 months (95% CI 4-49), respectively. The 2-year PFS was a plateau setup at 50% (95% CI 32-67) and the 2-year OS was 66% (95% CI 44-81). CONCLUSION: The TAXIF II protocol was effective in nonrefractory GCT patients failing Cisplatin-based chemotherapy. The toxic death rate remained acceptable in the field of HDCT regimens. TRIAL REGISTRATION NUMBER: NCT00231582.

Skin ulcers related to chronic graft-versus-host disease: clinical findings and associated morbidity.

BACKGROUND: According to the National Institutes of Health classification of chronic graft-versus-host disease (cGVHD), skin ulcers after allogeneic haematopoietic stem-cell transplantation (HSCT) are recorded as having the maximal severity score but published data are scarce. OBJECTIVES: To describe skin ulcers related to cGVHD with an emphasis on clinical findings, associated morbidity, management and evolution. PATIENTS AND METHODS: A multicentre retrospective analysis was carried out of patients with a diagnosis of cGVHD skin ulcers. RESULTS: All 25 patients included in the study had sclerotic skin cGVHD and 21 had lichenoid skin lesions associated with the sclerotic skin lesions. Thirteen patients had severe cGVHD without considering the skin, because of the involvement of an extracutaneous organ by cGVHD. The median time from HSCT to the onset of ulcers was 44 months. In addition to scleroderma, initial skin lesions at the site of ulcers were bullous erosive lichen in 21 patients and bullous erosive morphoea in four patients. Fifteen patients had an inaugural oedema. Ulcers were mostly bilateral with a predilection for the lower limbs. They were frequently colonized but few infections occurred. Four patients died during a median follow-up period of 55 months. CONCLUSIONS: Chronic graft-versus-host disease skin ulcers occur in patients with sclerodermatous skin cGVHD, are associated with severe cGVHD, often start with bullous lichenoid lesions or bullous morphoea and seem to cause more morbidity than mortality, given the low rate of mortality observed in our series of patients.

Mutations of the von Hippel-Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma.

The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL.

Early evaluation of natural killer activity in post-transplant acute myeloid leukemia patients.

The focus of this study was to investigate NK cell reconstitution early after hematopoietic stem cell transplantation (HSCT). We were particularly interested in acute myeloid leukemia (AML) since patients with this disease may display an altered NK cell function. The function and the phenotype of donor-derived NK cells obtained from 35 allografted patients 30 and 60 days after HSCT for AML or other-than-AML hematological malignancies has been assessed. NK functional status was investigated by measuring the degranulation capacity (externalization of CD107a) of NK cells against human K562. We also concomitantly determined the concentration of selected cytokines known to modulate NK function and/or receptor expression. At day 30, donor-derived AML and non-AML NK cells could efficiently degranulate when exposed to leukemic K562 targets. At day 60, we observed a reduced NK degranulation potential in AML patients only. Decreased NK activity in AML patients was concomitant to NKp46 and NKp30 down-regulation. AML NK cells were chronically exposed to low IL-2 levels following HSCT. TGF-beta(1) was undetectable in all patients. In AML, the functional activity of donor-derived NK cells is remarkable at day 30 but may strongly decrease two months after HSCT. Therefore, in this condition, early NK immune-modulation might improve HSCT outcome.

[Prolonged acute pancreatitis after bone marrow transplantation]. / Une pancréatite aiguë prolongée après allogreffe de moelle.

Acute pancreatitis is not infrequent after allogenic marrow transplantation. Several causes can predispose to pancreatitis, including Graft-Versus-Host Disease (GVHD), a condition which is probably underestimated. In the literature, few description of pancreatic GVHD can be found. Pancreatic GVHD diagnosis can be difficult if pancreatic involvement occurs without other typical manifestations of GVHD. We report the case of a woman, 54 years old, suffering from prolonged, painful pancreatitis two months after allogenic bone marrow transplantation for acute myeloid leucemia. Pancreatic GVHD diagnosis was performed after five weeks on duodenal biopsies despite the absence of diarrheoa. The patient dramatically improved within few days on corticosteroids.

The sensitivity of renal cell carcinoma cells to interferon alpha correlates with p53-induction and involves Bax.

Interferon alpha (IFN-alpha) is an approved treatment in metastatic renal cell carcinoma (RCC). The underlying mechanisms are far from being clear, but are presumed to be a combination of stimulation of cell-mediated cytotoxicity, direct antiproliferative activity and antiangiogenic effects. Recently, the role of p53 in the cellular response to IFN-alpha has been proposed in other tumor models (hepatoblastoma). We therefore studied the expression of p53 during IFN-alpha treatment using two freshly established RCC cell lines RCC5 and RCC7. While IFN-alpha treatment significantly enhanced the expression of p53 in RCC7, no changes were observed in RCC5. Cell viability under IFN-alpha remained unchanged in both cell lines. Following gamma-irradiation, a p53-activating stimulus, an enhanced cell death was observed in IFN-alpha-treated RCC7 but not in RCC5. We further demonstrate that there were no changes in Bcl-2- and Bax-expression, two target genes regulated by p53. However, intracellular staining revealed that cell death induced by IFN-alpha and gamma-irradiation was preceded by a shift of Bax to the mitochondria in RCC7. Our results suggest a role of p53 and its downstream target Bax, in the control of RCC sensitivity to IFN-alpha.

Induction and large-scale expansion of CD8+ tumor specific cytotoxic T lymphocytes from peripheral blood lymphocytes by in vitro stimulation with CD80-transfected autologous melanoma cells.

Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.

Induction of tumor-specific cytotoxic T lymphocytes by immunization with autologous tumor cells and interleukin-2 gene transfected fibroblasts.

In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-beta chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient's tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.