Determination of (fluoro)quinolone antibiotic residues in pig kidney using liquid chromatography-tandem mass spectrometry. I. Laboratory-validated method.

A new LC-MS/MS method has been developed for the multiresidue determination of 11 (fluoro)quinolone antibiotics (FQs), including acidic and amphoteric species, around their maximum residue level (MRL) in pig kidney. The procedure involves a common sample preparation by solid-phase extraction on disposable extraction cartridges followed by a fast reversed-phase liquid chromatography-tandem mass spectrometry analysis. The method was validated according to the Commission Decision 2002/657/CE. The accuracy of the method was satisfactory with recoveries included in the interval 80-100%. The precision results showed mean repeatability and reproducibility coefficients of 7.4% and 11.8%, respectively. Limits of quantification much lower than the MRLs could be obtained.

Determination of (fluoro)quinolone antibiotic residues in pig kidney using liquid chromatography-tandem mass spectrometry. Part II: intercomparison exercise.

A recently in-house validated method for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of eleven (fluoro)quinolone antibiotics (FQs) in pig kidney has been fully validated through an intercomparison exercise. This ring trial involved eight European laboratories and was based on the Commission Decision 2002/657/CE for validation of method and on the IUPAC protocol for method-performances studies. The laboratories data were submitted to a one-way analysis of variance. Satisfactory results were obtained for each FQ with regards to within- and between-laboratory reproducibility and accuracy. The method was validated for the simultaneous qualitative and quantitative determination of the eleven FQs in pig kidney around their maximum residue limit (MRL) as defined in the European Council Regulation 2377/90/EEC.

Characterization of two different cytoplasmic protein tyrosine kinases from human breast cancer.

Two different protein tyrosine kinases were detected in the cytosolic fraction of different human tumor tissues. After partial purification, the two enzymes, which were highly active in breast tumor tissues, were characterized. One of them, soluble tyrosine kinase-1 (STK-1), represents a soluble form of the c-Src protein, which is apparently underphosphorylated on its C-terminal tyrosine residue whereas the other (STK-2) is a 48-kDa protein tyrosine kinase (PTK), which is molecularly and functionally related to the C-terminal Src kinase (Csk). These two protein tyrosine kinases clearly exhibit a different substrate specificity, and are responsible for the high tyrosine kinase activity present in the cytosolic fraction of human breast cancer. In addition, it was observed that STK-1 and STK-2 are also expressed in the breast cancer cell line, CAL-51.

[Prognostic value of quantitative DNA analysis, expressed in integrated optical density, in a series of 415 T1-T2, N0N1, M0 UICC breast cancers]. / Valeur pronostique du contenu en ADN, exprimé dans la densité optique intégrée, dans une série de 415 cancers du sein T1-T2, N0N1, M0 UICC.

From June 1982 to December 1992, 415 patients less than 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), NO (65.1%) N1(34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75), and 58.8% of the patients were menopaused; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade, estradiol receptor content, were used to set up medical adjuvant treatment: hormonotherapy (52%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was done on air dried smears to be analyzed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index, DNA histogram, integrated optical density, DNA malignancy grade, and policy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (P = 0.018), DNA malignancy grade (P = 0.017) have a significant prognostic value on disease free survival estimated by the Kaplan-Meir method. When these parameters were included in a Cox proportional regression hazards model, Scarff-Bloom histological grading (P = 0.002), positives nodes (P = 0.02), optical integrated density (P = 0.045) were significant. Such results need to be updated with a longer follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), has to be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

Protein tyrosine kinase activity in 350 T1/T2, N0/N1 breast cancer. Preliminary results.

Protein tyrosine kinases (PTKs) are a family of enzymes sharing a highly conserved catalytic domain which phosphorylates substrate proteins on tyrosine residues. PTKs play a major role in the transduction of the mitogenic signal and are involved in the control of cell proliferation, differentiation, and transformation processes. PTKs can be subdivided into two major types: membrane associated PTKs consisting essentially of growth factor receptors (receptor tyrosine kinases or RTKs) and cytosolic PTKs involved in the intracellular transduction of mitogenic and differentiation signals. From January 1988 to January 1992, PTK activity was assayed in cytosolic fractions prepared from 350 T1-T2, N0-N1 M0, breast carcinomas. Enzymatic activity was measured using phosphate transfer from [32P]-ATP to poly-Glu-Tyr as an artificial substrate. According to our previously reported pilot study, we chose a cut-off value of 12 pmol 32P incorporated min-1 mg-1 protein, corresponding to the median value. We found positive PTK levels (> or = 12 pmol/min/mg) to be correlated with a loss of differentiation according to Scarff-Bloom grade (p < 0.001), negative PR (p = 0.03) and ER status (p = 0.04). With a median follow-up of 30 months (0-82), patients with a positive PTK level presented a smaller 3-year disease free survival than in the PTK negative group of patients (p = 0.07). In Cox multivariate analysis including pT, pN, Scarff-Bloom grade, PR and ER, PTK activity does not emerge as a significant prognostic factor.

[Modulation of indications of adjuvant hormone therapy with tamoxifen in T1T2/N0N1 breast cancers. Preliminary results of a multicenter study with 695 cases]. / Modulation des indications de l'hormonothérapie adjuvante par le tamoxifène dans les cancers du sein T1T2/N0N1. Résultats préliminaires d'une étude multicentrique de 695 cas.

From 1982 to 1990, patients less than 75 years, without any previous or synchonous carcinoma, suffering from an invasive breast cancer classified as T1T2/N0N1/MO according to clinical TNM staging, were enrolled in this study; 82.4% underwent a breast conservative procedure and 17.2% a modified radical mastectomy followed by a postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade, estradiol receptor content, were used to define three groups of patients. The breakdown of patients is not well balanced: 416 women were included in group I (N-, grade I II, ER+) when there was no adjuvant medical treatment, 110 in group II (N-, grade III, ER+), 169 in group III (N+ < or = 3, grade I II, ER+). Patients from the latter two groups were receiving tamoxifene, 20 mg per day for 2 years; Those women not menopaused received first a pelvic irradiation. With a median follow-up of 35 months (1-138) the overall survival is respectively for the three groups 95%, 96%, 96% (P = 0.5) and the disease free survival 86%, 93%, 90% (P = 0.1). The actuarial local regional remission rate is 94%, 97%, 99% (P = 0.07). Such results need to be updated with a longer follow-up, but they show the ability of adjuvant hormonotherapy to tailor the short term survival thanks to prognostic factors.

Lack of prognostic value of epidermal growth factor receptor in a series of 229 T1/T2, N0/N1 breast cancers, with well defined prognostic parameters.

The prognostic value of epidermal growth factor receptor (EGF-R) was prospectively assessed in a series of 229 clinical T1-T2, N0-N1 breast carcinomas diagnosed between May 1987 and October 1989. EGF-R expression was determined by measuring the specific Bmax of 125I EGF to tumor plasma membrane preparations. Tumor with a Bmax > or = 3 fmol/mg of protein were considered positive with regard to EGF-R expression. With a median follow-up of 34 months, the 3-year overall and disease-free survivals are respectively 92% and 88% for EGF-R < or = 3, and 91% and 86% EGF-R > 3 fmol, showing no significant difference, even when comparing axillary lymph node status. We did not succeed in finding an EGF-R cut-off value which might be significant in univariate analysis. Multivariate analysis of our data indicates that pT (p = 0.001), pN (p = 0.04), and Scarff-Bloom grade (p = 0.04) are the only significant predictors of disease-free survival among the parameters investigated in this study.

Protein tyrosine kinase activity as a prognostic parameter in breast cancer, a pilot study.

Protein tyrosine kinase (PTK) activity was assayed in cytosolic extracts from normal breast tissue, benign tumors, and 84 T1-T2, N0-N1 M0, breast carcinomas. Normal breast tissue extracts yielded an average value of 1.9 +/- 1.1 pmol 32P incorporated/min/mg protein, whereas a mean of 12.5 +/- 6.1 was obtained for cancer samples. With a median follow-up of 34 months, in the series of 40 patients classified N-, PTK positive patients presented a significantly smaller 3-year disease free survival than the PTK negative ones. Multivariate analysis shows that PTK activity emerges as a potential prognostic factor in breast cancer (p = 0.02). These preliminary results will be updated on a bigger cohort of patients.

EGF receptor amplification and expression in human brain tumours.

Human epidermal growth factor receptor (EGFr) gene amplification, rearrangements and expression were studied in tumours of the human nervous system. EGFr gene amplification was studied in 46 brain tumours. Gene expression was analysed by northern blot in 37 tumours and binding of its protein to EGF in 27 tumours. The EGFr gene was simultaneously amplified (with arrangements in 12.5% of gliomas) and overexpressed in 53% (9/17) of malignant gliomas, but never in meningiomas. In five high grade gliomas, amplification was always associated with a high level of receptors. However, since high amounts of EGF receptors found in one glioma were not the result of gene amplification, several systems of deregulation in EGFr production may exist and could be located at translational and/or post-translational levels.

Prognostic value of epidermal growth factor receptor in a series of 303 breast cancers.

125I-EGF (epidermal growth factor) binding assay was used in tumoral specimens concerning 303 clinical T1-T2, N0-N1 breast carcinoma diagnosed between May 1987 and October 1989. Binding assay for epidermal growth factor receptor (EGFR) was performed using single saturating concentration of 125I-EGF incubated with membrane preparations in the presence or absence of unlabelled EGF. A median value of 3 fmol EGF binding capacity per mg of membrane was obtained and then selected as the threshold value to define positive and negative EGFR tumour samples. According to this definition, 50.8% of the samples were EGFR positive. We noted an inverse relationship between the expression of EGFR and that of oestrogen receptor, and a decreased EGFR expression with tumour differentiation. With a rather short median follow-up (16 months), the multivariate analysis shows that progesterone receptor appears as the only powerful predictor of disease-free survival (P = 0.002), taking into account that 70% of the patients received an adjuvant medical treatment.

Estimation of epidermal growth factor receptor in 177 breast cancers: correlation with prognostic factors.

Steroid receptors (ER, PR) and EGF-receptor were determined on 177 loco-regional primary breast cancers. Binding assay for EGF receptor was performed using a single saturating concentration of 125I-EGF incubated with membranes in the presence or absence of unlabeled EGF. With threshold values of 5 fmol/mg and 10 fmol/mg for EGF-R and steroid receptors respectively, we noted an inverse relationship between the expression of EGF-R, and ER and PR. EGF-R expression is decreased with tumor differentiation.

[Prognostic value of estradiol and progesterone receptors in stage I and II breast cancer with an adjuvant treatment]. / Etude de la valeur pronostique des récepteurs à l'estradiol et à la progestérone dans les cancers du sein stade I, II UICC, soumis à un traitement adjuvant.

Forty-seven stage T1, 225 stage T2, treated from January 1977 to December 1982 were studied. The median followup is 31 months (18-92) and the median age 57.5 years (26-92). On the 247 axillary clearances performed, there were 53% N- and 47% N+. The dextran coal method was used for the receptors dosage with R 2858 as ligand for estradiol and R 5020 for progesterone: positivity threshold was set at 10 fmoles. The RE+/RP+ group represents 40%, the RE+/RP- group 30%, the RE-/RP- group 7%, the RE-/RP- group 23%. The radio-surgical combination was systematic, with or without conservative treatment, followed by an adjuvant chemotherapy (15%), an hormonotherapy (34%), a chemohormonoprophylaxy (23%), within a protocol balancing the systemic treatment according to the following poor prognosis factors: axillary clearance positivity, grade 3 SBR, cytological grade 3. The crude actuarial survival is 96 +/- 4% (RE+ RP+); 73 +/- 15% (RE+ RP-); 78 +/- 12% (RE- RP-) and the disease-free survival was 84 +/- 9% (RE+ RP+); 68 +/- 15% (RE+ RP-), 67 +/- 14% (RE- RP-) with a significant difference between RE+ RP+ and RE+ RP- (P less than 10(-3)) and RE+ RP+ and RE- RP- (P less than 10(-5)). The crude actuarial survival and the disease free survival are studied according to menopausal status, Scarff-Bloom grade, and N+/N- axillary status. For N- patients, there is no significant difference for the disease-free survival, 84 +/- 14% (RE+ RP+); 85 +/- 11% (RE+ RP-); 79 +/- 15% (RE- RP-), as there is no difference for the five year disease-free survival between N- RE+ RP+ patients (84 +/- 14%) and N+ RE+ RP+ patients (84 +/- 13%).

[Estradiol and progesterone receptors in loco-regional breast cancer. Clinical and prognostic correlations]. / Récepteurs à oestradiol et à progestérone dans le cancer du sein loco-régional. Corrélations cliniques et pronostiques.

Estradiol (ER) and progesterone (PR) receptor assay was performed in breast cancer specimens from 133 women, 31 to 83 years of age. Mean ER levels increase significantly from one ten year age group to the next, ranging from 59.9 + 46.2 femtomoles/mg cytosolic protein in the 30-40 year group to 627 + 479 fmol/mg protein in the 89-90 year group. Mean ER values were found to be 50 + 20.4 fmol/mg protein in premenopausal patients and 176.8 + 55 after menopause. PR concentrations were similar in pre and post-menopausal patients (62.2 + 25.8 and 73.8 + 33.8 fmol/mg protein respectively. Receptor level was not correlated with T.N.M. clinical staging or lymph node involvement. Analysis of ER and PR levels according to histologic (Scarff and Bloom) grading showed a positive correlation between receptor concentration and cellular differentiation. Disease-free survival rate two years after initial therapy was significantly higher in ER positive cases (p = 0.01), although 79% of ER negative patients received adjuvant systemic therapy. In PR positive cases, this difference nearly disappears (p = 0.09), even though 70% of PR negative patients were submitted to adjuvant systemic therapy. Chemotherapy is therefore justified in RE negative patients who carry a poor prognosis.