Three-dimensional reconstruction and morphologic measurements of human embryonic hearts: a new diagnostic and quantitative method applicable to fetuses younger than 13 weeks of gestation.

2 figures supplémentaires par rapport à la version 1International audienceImprovements in the diagnosis of congenital malformations explain the increasing early termination of pregnancies. Before 13 weeks of gestation, an accurate in vivo anatomic diagnosis cannot currently be made in all fetuses with current imaging instrumentation. Anatomopathologic examinations remain the gold standard to make accurate diagnoses, although they reach limits between 9 and 13 weeks of gestation. We present the first results of a methodology that can be applied routinely, using standard histologic section, thus enabling the reconstruction, visual estimate, and quantitative analysis of 13-week human embryonic cardiac structures. The cardiac blocks were fixed, embedded in paraffin, and entirely sliced by a microtome. One of 10 slices was topographically colored and digitized on an optical microscope. Cardiac volume was recovered by semiautomatic realignment of the sections. Another semiautomatic procedure allowed extracting and labeling of cardiac structures from the volume. Structures were studied with display tools, which disclosed the internal and external cardiac components and enabled determination of size, thickness, and precise positioning of ventricles, atria, and large vessels. This pilot study confirmed that a new 3-dimensional reconstruction and visualization method enables accurate diagnoses, including in embryos younger than 13 weeks. Its implementation at earlier stages of embryogenesis will provide a clearer view of cardiac development

The prognostic value of erythrocyte polyamine in the post-nephrectomy stratification of renal cell carcinoma specific mortality.

International audiencePURPOSE: The polyamines spermine and spermidine are ubiquitous polycationic structures which are essential for cell proliferation and differentiation. Circulating polyamines, spermine and spermidine, represent valuable prognostic markers in prostate cancer, acute leukemia and supratentorial malignant glioma. We tested whether spermine and spermidine could improve the prognostic ability of several established predictors of cancer specific mortality after partial or radical nephrectomy for renal cell carcinoma. MATERIALS AND METHODS: Testing was performed on 399 patients with stages T(1-4), N(0-2), M(0-1) renal cell carcinoma who were treated with radical or partial nephrectomy at a single institution between 1990 and 2007. Univariable and multivariable Cox regression models tested the prognostic ability of spermine and spermidine levels in cancer specific mortality predictions. Covariates consisted of TNM stage, Fuhrman grade, tumor size and symptom classification. Harrell's concordance index (c-index) quantified accuracy and 200 bootstrap resamples were used to correct for overfit bias. RESULTS: The 5-year cancer specific mortality-free survival of patients with spermine levels 3 or less, 3.1 to 8, 8.1 to 13 and greater than 13 nmol/8x10(9) erythrocytes was 88.8%, 75.8%, 40.2% and 21.8%, respectively. Similarly the 5-year cancer specific mortality-free survival of patients with spermidine levels 12 or less, 12.1 to 15, 15.1 to 21 and greater than 21 nmol/8x10(9) erythrocytes was 79.0%, 56.6%, 53.2% and 27.4%, respectively. On multivariable analyses addressing cancer specific mortality after surgery spermine (p = 0.007) and spermidine (p = 0.04) achieved independent predictor status. Consideration of spermine and spermidine also improved the accuracy of established cancer specific mortality predictors by 2.2% (p <0.001). CONCLUSIONS: Spermine and spermidine may significantly improve the prognostic value of established cancer specific mortality predictors after partial or radical nephrectomy for all stages of renal cell carcinoma. Independent external validation of our findings is required

Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 µM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 µM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 µM and 14 µM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 µM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities

The prognostic value of erythrocyte polyamines in the preoperative evaluation of patients with renal cell carcinoma.

International audienceINTRODUCTION: Polyamines, spermine and spermidine, are ubiquitous polycationic structures, which are essential for cell proliferation and differentiation. We tested whether spermine and spermidine could improve the prognostic ability of six established preoperative predictors of cancer-specific mortality (CSM) after partial or radical nephrectomy for renal cell carcinoma (RCC). MATERIALS AND METHODS: Overall, 385 patients with clinical stages T(1-3), M(0-1) RCC were treated with radical or partial nephrectomy at a single institution between 1990 and 2007. Kaplan-Meier plots depicted CSM after stratification according to spermine and spermidine levels (dichotomised to above and below the median value). Univariable and multivariable Cox regression models tested the prognostic ability of continuously coded spermine and spermidine levels in preoperative CSM predictions. Covariates consisted of pre-treatment T stage, M stage, age, gender and symptom classification. RESULTS: The 5-year CSM-free survival of patients with spermine levels 4.5 nmol/8x10(9) erythrocytes were, respectively, 79.5% and 65.0%. Similarly, the 5-year CSM-free survival of patients with spermidine levels 9.0 nmol/8x10(9) erythrocytes were, respectively, 81.1% and 63.7%. In multivariable analyses addressing CSM after surgery, both spermine (p< or =0.002) and spermidine (p< or =0.001) achieved independent predictor status and improved the accuracy of established preoperative CSM predictors by 2.1% (p<0.001). CONCLUSIONS: Circulating polyamine levels may significantly improve the prognostic value of established determinants of CSM in patients with RCC of all stages prior to nephrectomy. External validation of our findings is required prior to implementation in clinical practice

Valeur pronostique des polyamines érythrocytaires dans le cancer du rein. Étude chez 418 patients [Prognostic value of erythrocyte polyamines levels in renal cell carcinoma. Prospective study in 418 cases]

International audienceOBJECTIVES: Polyamines: Spermine (Spm) and Spermidine (Spmd), are essential for cell proliferation and differentiation. A measurement of erythocytes polyamines (EPA) was developed in our institution. Our objective was to evaluate this marker as a new prognostic factor in renal cell carcinoma. PATIENTS AND METHODS: A blood sample was prospectively taken before surgery, among 418 patients who had an enlarged nephrectomy (n=318) or a partial nephrectomy (n=100) to quantify EPA rates by using the HPLC technique. The qualitative and quantitative variables have been compared using chi(2) and Student statistical analyses. The survivals have been normalized by the Kaplan Meier and Cox methods. RESULTS: The average age of our population was 64 years (21-88). The average decline was 41 months (1-214). The median size of tumors was 6.5cm (1-24). The median rate of Spm and Spmd were respectively 4.7 (1-83) and 9 (2-86)nmol/8.10(9) erythrocytes. Spm and Spmd were linked to the T stage (p=0.001), and the ECOG (p=0.001 and 0,008). Spm was not linked at N and M stages but at the Fuhrman grade (p=0.001). Spmd was linked to the N, M stages (p=0.04). With univariate analysis, the tumor diameter, the TNM stage, the Fuhrman grade as well as Spm and Spmd (p<0.0001) were predictors of specific survival. With multivariate analysis, some prognostic factors remained independent: the TNM stage, the ECOG and Spmd, a continuous variable (p=0.0001), pushing the rank of Fuhrman out of the model. When Spm and Spmd were dichotomized in quantitative variables, they were both independent factors. CONCLUSION: The EPA is a new prognostic tool, before surgery, which will be tested for its integration into prognostic normograms