Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (α β γ δ ε ξ η ηl θ). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern tech nique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73α protein expression positively correlated with higher risk B-CLL stages (P=0.046). Total p73 mRNA expression was higher (P= 0.01) and p73α protein more frequently detected (P=0.008) in B-CLL compared with normal CD19+—B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the γ (P=0.041), the θ (P ≪ 0.001), and the η variant (P=0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CL

Filgrastim-induced stem cell mobilization in chronic myeloid leukaemia patients during imatinib therapy: safety, feasibility and evidence for an efficient in vivo purging

Therapy with imatinib mesylate is limited by cellular resistance in chronic myeloid leukaemia (CML). Further, the limited availability of matching stem cell donors or an unfavourable risk profile for allogeneic stem cell transplantation (SCT) reduces the number of therapeutic options in a number of patients. To assess the possibility of stem cell mobilization (SCM) during imatinib therapy we performed granulocyte colony-stimulating factor (filgrastim)-induced SCM and subsequent aphaeresis in 15 chronic phase and three accelerated phase CML patients. Aphaeresis was successful in 13 patients (72%) (≥2·0 × 106 CD34+ cells/kg body weight) and five (28%) harvests could be obtained, which were negative for BCR/ABL mRNA as assessed by nested-reverse transcription polymerase chain reaction (RT-PCR). All harvests, except one, were negative after first round RT-PCR, implicating a low level of CML cell contamination. There was no significant change in peripheral BCR/ABL transcript load after SCM as assessed by quantitative real-time RT-PCR. Fifteen patients remained stable in complete cytogenetic remission (CCR) during a median observation period of 9·3 months. One patient achieved a molecular remission shortly after SCM. Another patient who exhibited rising BCR/ABL mRNA levels before SCM achieved CCR after autologous SCT with the generated harvest. One patient with a Philadelphia chromosome-negative, BCR/ABL-positive CML showed a cytogenetic relapse 6 months after SCM. We conclude that filgrastim-induced CD34+ cell aphaeresis under simultaneous imatinib medication is safe and feasible in CML patients. Additionally, we found evidence that this procedure could generate stem cell harvests that exhibit non-detectable levels of BCR/ABL mRNA

Pharmacokinetics and cellular uptake of imatinib and its main metabolite CGP74588

Despite the remarkable clinical response rates to imatinib in the treatment of bcr-abl leukemic patients, pharmacokinetic data on this relatively novel substance are needed to improve our understanding of the emergence of resistance, the interindividual variations of clinical response and the clinical and biologic relevance of its main metabolite N-desmethyl-imatinib. We present here pharmacokinetic data obtained with a newly designed HPLC approach in 97 patients with chronic myeloid leukemia or acute lymphatic leukemia (ALL) under treatment with imatinib that allowed us to calculate the AUC (39.5 μg·h/ml for an oral dose of 400 mg daily), the t1/2 (18.2 h) and the peak concentration (1.92 μ/ml for an oral dose of 400 mg daily) of imatinib in plasma. In a subgroup of patients, the same parameters were analyzed for N-desmethyl-imatinib. We also provide data on the imatinib concentration in the cerebrospinal fluid (CSF) of ALL patients and demonstrate that oral administration of imatinib resulted only in a marginal flux across the blood-brain barrier. Finally, in an in vitro setting, we determined cellular concentrations of imatinib in HL-60 cells and showed an over-proportional uptake both in RPMI medium and in human plasma. Using an arithmetical approach combining all parameters obtained in imatinib-treated patients, we finally provide a conclusive approximation of basic pharmacokinetic data for both imatinib and its main metabolite N-desmethyl-imatinib

Vaccination with autologous non-irradiated dendritic cells in patients with bcr/abl+ chronic myeloid leukaemia

In chronic myeloid leukaemia (CML), dendritic cells (DC) and leukaemic cells share a common progeny, leading to constitutive expression of putative tumour antigens, such as bcr/abl, in DC. In this phase-I/II study, autologous DC were used as a vaccine in patients with chronic phase bcr/abl+ CML, who had not achieved an adequate cytogenetic response after treatment with alpha-interferon or imatinib. Ten patients were enrolled, DC were generated from peripheral blood monocytes and vaccination consisted of four subcutaneous injections of increasing numbers of DC (1-50 x 10(6) cells per injection) on days 1, 2, 8 and 21. Vaccination was feasible and safe. Improvement of the cytogenetic/molecular response, as detected by fluorescence in situ hybridization of peripheral blood mononuclear cells (PBMC), was possibly related to vaccination in four of 10 patients. In three of these patients, T cells recognizing leukaemia-associated antigens became detectable. The proliferative capacity of PBMC in response to autologous DC increased after vaccination in all evaluable patients. We conclude that vaccination with autologous, non-irradiated leukaemic DC is feasible, safe and induces anti-leukaemic T-cell responses in some CML patients. DC vaccination might be useful in CML as postremission therapy, i.e. after treatment with tyrosine kinase inhibitors

Imatinib in Philadelphia chromosome-positive chronic phase CML patients: molecular and cytogenetic response rates and prediction of clinical outcome

Previous clinical trials with the tyrosine kinase inhibitor imatinib in chronic-phase Philadelphia chromosome-positive chronic myelogenous leukemia (CML) resulted in 95% of hematologic and 60% major cytogenetic remissions in patients who failed a previous interferon-alpha-containing regimen. In an identical clinical trial setting with 39 chronic-phase CML patients we achieved comparable cytogenetic response rates after a median follow up of 30.1 weeks, with an almost identical toxicity profile. In order to identify predictive markers for the therapeutical use of imatinib, we monitored apart from standard hematology parameters bcr/abl fusion transcripts by quantitative real-time fluorescence RT-PCR. As previous investigations demonstrated that the plasma protein alpha-1 acid glycoprotein might inactivate circulating levels of free imatinib by protein binding with high affinity, we assessed plasma alpha-1 acid glycoprotein concentrations in our study cohort as well. Median bcr/abl fusion transcripts declined gradually over the entire treatment period and became significantly lowered at month 3 after initiation of imatinib therapy. Further, we observed elevated pretreatment levels of alpha-1 acid glycoprotein in patients who relapsed with leukemia, whereas initial bcr/abl mRNA copy numbers were not of predictive value. In addition, we provide data showing molecular response to this therapy in the vast majority of patients. Finally, our results support the hypothesis, that initially elevated plasma levels of alpha-1 acid glycoprotein might serve as a predictive marker for the clinical outcome of treatment with imatinib

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (alpha, beta, gamma, delta, epsilon, zeta, eta, eta1, theta). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern technique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73alpha protein expression positively correlated with higher risk B-CLL stages (P = 0.046). Total p73 mRNA expression was higher (P = 0.01) and p73alpha protein more frequently detected (P = 0.008) in B-CLL compared with normal CD19+-B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the gamma (P = 0.041), the theta (P < 0.001), and the eta variant (P = 0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CLL