Identification of SNPs associated with response of breast cancer patients to neoadjuvant chemotherapy in the EORTC-10994 randomized phase III trial.

Using cell line panels we identified associations between single-nucleotide polymorphisms (SNPs) and chemosensitivity. To validate these findings in clinics, we genotyped a subset of patients included in a neoadjuvant breast cancer trial to explore the relationship between genotypes and clinical outcome according to treatment received and p53 status. We genotyped 384 selected SNPs in the germline DNA extracted from formalin-fixed paraffin-embedded non-invaded lymph nodes of 243 patients. The polymorphisms of five selected genes were first studied, and then all 384 SNPs were considered. Correction for multiple testing was applied. CYP1B1 polymorphism was significantly associated with pathological complete response (pCR) in patients who had received DNA-damaging agents. MDM2, MDM4 and TP53BP1 polymorphisms were significantly associated with pCR in patients harboring a p53-positive tumor. In the complete SNP panel, there was a significant association between overall survival (OS) and a SNP of ADH1C, R272Q (P=0.0023). By multivariate analysis, only ADH1C genotype and p53 status were significantly associated with OS.

A phase I study of intravenous liposomal daunorubicin (DaunoXome) in paediatric patients with relapsed or resistant solid tumours.

Anthracyclines are widely used in paediatric oncology, but their use is limited by the risk of cumulative cardiac toxicity. Encapsulating anthracyclines in liposomes may reduce cardiac toxicity and possibly increase drug availability to tumours. A phase I study in paediatric patients was designed to establish the dose limiting toxicity (DLT) and maximum tolerated dose (MTD) after a single course of liposomal daunorubicin, 'DaunoXome', as a 1 h infusion on day 1 of a 21 day cycle. Patients were stratified into two groups according to prior treatment: Group A (conventional) and group B (heavily pretreated patients). Dose limiting toxicity was expected to be haematological, and a two-step escalation was planned, with and without G-CSF support. Pharmacokinetic studies were carried out in parallel. In all, 48 patients aged from 1 to 18 years were treated. Dose limiting toxicity was neutropenia for both groups. Maximum tolerated dose was defined as 155 mg m(-2) for Group A and 100 mg m(-2) for Group B. The second phase with G-CSF was interrupted because of evidence of cumulative cardiac toxicity. Cardiac toxicity was reported in a total of 15 patients in this study. DaunoXome shares the early cardiotoxicity of conventional anthracyclines in paediatric oncology. This study has successfully defined a haematological MTD for DaunoXome, but the significance of this is limited given the concerns of delayed cardiac toxicity. The importance of longer-term follow-up in patients enrolled into phase I studies has been underestimated previously, and may lead to an under-recognition of important adverse events.

Separation and determination of liposomal and non-liposomal daunorubicin from the plasma of patients treated with Daunoxome.

Several liposomal formulations of anthracyclines have been developed recently and are currently used in the clinical setting. We describe a technique of separation and quantification of the liposomal and non-liposomal forms of daunorubicin in the plasma of patients treated with DaunoXome, a liposomal formulation of daunorubicin. The method we propose is based upon the property of liposomes to cross reversed-phase C18 silicagel cartridges without being retained, while non-liposomal drug is retained on the stationary phase and is eluted with methanol. Extraction of liposomal and non-liposomal daunorubicin from plasma, therefore, is performed in two steps. This technique is rapid, can be automated in order to handle large series of samples, and the plasma can be frozen after sampling by addition of glycerol. The recovery of liposomal daunorubicin as well as the precision, linearity and accuracy of the technique appear satisfactory for pharmacokinetic purposes.

Pharmacokinetics of liposomal daunorubicin (DaunoXome) during a phase I-II study in children with relapsed acute lymphoblastic leukaemia.

PURPOSE: The pharmacokinetics of DaunoXome were studied during a multicentric phase I-II study performed in children suffering from relapsed acute lymphoblastic leukaemia and treated on a weekly schedule. PATIENTS AND METHODS: A group of 18 patients were studied during the first course of treatment at dose levels between 40 and 120 mg/m2. Blood samples were obtained up to 72 h after infusion. The liposomal and free forms of daunorubicin, as well as daunorubicinol, were separated and quantified by HPLC using fluorometric detection, and data were analysed using a model-independent approach. RESULTS: Unchanged liposomal daunorubicin disappeared from plasma following a monoexponential decay. Its AUC represented 95.8% of the total fluorescent species found in plasma and increased linearly with the dose administered. The elimination half-life was 5.23 h, total plasma clearance 0.344 1/h per m2, and volume of distribution at steady state 2.08 l/m2. Free daunorubicin and daunorubicinol were detected in plasma at all time-points studied. Their AUCs represented, respectively, 2.53% and 1.70% of total fluorescent species and their elimination half-lives were, respectively, 16.6 h and 22.3 h. The daunorubicinol/daunorubicin AUC ratio was 0.82%. CONCLUSIONS: This study is the first to demonstrate that free daunorubicin is present in plasma after DaunoXome administration and that it originates from in vivo release from the liposomes. The pharmacokinetics of free daunorubicin appeared to be comparable to those observed after conventional administration. However, the concentration of daunorubicinol appeared to be lower than that found after conventional administration of daunorubicin.

Evidence for an alpha 2-macroglobulin with complement-inhibiting activity in rat serum.

alpha 2-Macroglobulin (alpha 2M) was purified both from the serum of male rats developing an acute turpentine-induced inflammatory reaction where its concentration is greatly increased (3-4 mg/ml) and from the serum of healthy males where it is present at low levels (15-30 micrograms/ml). A three-step purification procedure involving gel filtration, anion exchange chromatography on DEAE cellulose and negative immunoaffinity was used. A pure native alpha 2M, as assessed by biochemical and immunological tests, was obtained. This alpha 2M differed from other subforms in terms of its electric charge and its complement-inhibiting activity in a complement-dependent immune haemolysis test. Moreover, this inhibitory activity was not affected by complexing with trypsin or modification by interaction with methylamine showing that this newly described property is not linked to the well known antiproteinase function of alpha 2M.

Characterization of one alpha 2-macroglobulin with anticomplementary activity from pregnant women.

In 15 pregnant women during the third term of pregnancy, the immunomodulatory property of alpha 2-macroglobulin (alpha 2M) was initially detected by measuring the inhibitory effect on immune complement-dependent haemolysis of serum alpha 2M fractions obtained by gel filtration. By a two-step chromatography procedure consisting of gel filtration followed by anion-exchange chromatography, different sub-forms of alpha 2M in serum were separated. Amongst them, it was shown that the inhibition of complement activity was almost exclusively linked to one particular subform. Additional studies revealed that the observed effect was not due to proteases bound to alpha 2M during clotting since, by using protease-specific inhibitors, no change was observed in complement inhibition. This subform, though present at very low levels in control sera, appeared in strikingly increased amounts during the third trimester of pregnancy (35 mg/l) and comprised between 3 and 5% of the total alpha 2M. Results show that the increase of alpha 2M anticomplementary activity is linked to the increase in alpha 2M levels in serum.