Risikofaktoren der Umgraduierung des Prostatakarzinoms zwischen Stanzbiopsie und Prostatovesikulektomie: Korrelation der immunhistologischen CD57- und PTEN-Expression an Stanzbiopsien mit einer postoperativen Hochgraduierung

Der Stanzbiopsie im Allgemeinen und der Graduierung als einer der stärksten unabhängigen Prognosemarker des Prostatakarzinoms im Besonderen kommt eine zentrale Bedeutung in der Therapieplanung des Prostatakarzinoms zu. Eine Möglichkeit der Stanzgüteprüfung stellt die Korrelation von Stanzbiopsie und Prostatovesikulektomie dar. In der Arbeit wurden Risikofaktoren der Umgraduierung zwischen Stanzbiopsie und Prostatovesikulektomie untersucht. Ferner wurde die immunhistochemische Expression von CD57 und PTEN an der Stanze mit verschiedenen Prognosefaktoren des Prostatakarzinoms korreliert

Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies

Additional file 1. Sequences of oligonucleotides and probes.Nucleotide sequences and labeling of all oligonucleotides and probes used in this study

Risikofaktoren der Umgraduierung des Prostatakarzinoms zwischen Stanzbiopsie und Prostatovesikulektomie:Korrelation der immunhistologischen CD57- und PTEN-Expression an Stanzbiopsien mit einer postoperativen Hochgraduierung

Der Stanzbiopsie im Allgemeinen und der Graduierung als einer der stärksten unabhängigen Prognosemarker des Prostatakarzinoms im Besonderen kommt eine zentrale Bedeutung in der Therapieplanung des Prostatakarzinoms zu. Eine Möglichkeit der Stanzgüteprüfung stellt die Korrelation von Stanzbiopsie und Prostatovesikulektomie dar. In der Arbeit wurden Risikofaktoren der Umgraduierung zwischen Stanzbiopsie und Prostatovesikulektomie untersucht. Ferner wurde die immunhistochemische Expression von CD57 und PTEN an der Stanze mit verschiedenen Prognosefaktoren des Prostatakarzinoms korreliert

Amplification of the <i>EGFR</i> gene can be maintained and modulated by variation of EGF concentrations in <i>in vitro</i> models of glioblastoma multiforme

<div><p>Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (<i>EGFR</i>) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM <i>in vitro</i> models, rapid loss of <i>EGFR</i> gene amplification is a common observation during cell culture. To maintain <i>EGFR</i> amplification <i>in vitro</i>, heterotopic GBM xenografts with elevated <i>EGFR</i> copy number were cultured under varying serum conditions and EGF concentrations. <i>EGFR</i> copy numbers were assessed over several passages by quantitative PCR and chromogenic <i>in situ</i> hybridization. As expected, in control assays with 10% FCS, cells lost <i>EGFR</i> amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated <i>EGFR</i> copy numbers could be maintained over several passages <i>in vitro</i>, levels of <i>EGFR</i> amplification were variable and dependent on the EGF concentration in the medium. <i>In vitro</i> cultures of GBM cells with elevated <i>EGFR</i> copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of <i>EGFR</i> driven glioblastoma and assist in the development of new improved therapies.</p></div

<i>EGFR</i> amplification is maintained over several <i>in vitro</i> passages, but lost in the 10% FCS control and with high EGF concentrations.

<p>qPCR analysis of A) xHROG22, B) xHROG33, C) x HROG59 cell lines grown under all culture conditions at passage 2 (grey bars), passage 6 (dark grey bars) and passage 10 (light grey bars). Black bars represent the respective PDX prior to cell culture establishment. D) qPCR analysis of the 10% FCS control (white bars) and the 30 ng/ml EGF cell line (grey bars) of xHROG33 over several <i>in vitro</i> passages. Error bars represent the standard deviation of triplicate analyses. * p<0.05, Tukey test.</p

2C CISH analysis confirms qPCR data of <i>EGFR</i> amplification.

<p>2C CISH analyses of xHROG22, xHROG33 and xHROG59 cell lines established under different conditions as indicated at passage 10 of cell culture; 400x magnification.</p

EGF withdrawal restores <i>EGFR</i> amplification.

<p>A and D) qPCR analysis of xHROG33 (A) and xHROG59 (D) cultured with 30 ng/ml EGF until passage 10 (grey bars) and without EGF for 5 additional passages (dashed bars), respectively. Error bars represent the standard deviation of triplicate analyses; B and E) 2C CISH analysis of paraffin embedded samples of xHROG33 (B) and xHROG59 (E) after 5 passages post EGF withdrawal (P15), C and F) EGFR immunohistochemistry staining of paraffin embedded samples of xHROG33 (C) and xHROG59 (F) after 5 passages post EGF withdrawal (P15). 400x magnification. * p<0.05, Tukey test.</p

EGFR protein expression is concordant with genomic <i>EGFR</i> amplification.

<p>EGFR immunohistochemistry staining of paraffin embedded xHROG22, xHROG33 and xHROG59 cells grown under different conditions as indicated at passage 10 of cell culture; 200x magnification.</p

Analysis of <i>EGFR</i> amplification by 2C CISH and qPCR and corresponding EGFR protein expression by immunohistochemistry in GBM PDX tissue of HROG22, HROG33 and HROG59.

<p>A) 2C CISH of HROG22 (400x magnification), B) EGFR IHC of HROG22 (200x magnification), C) 2C CISH of HROG33 (400x magnification), D) EGFR IHC of HROG33 (200x magnification), E) 2C CISH of HROG59 (400x magnification), F) EGFR IHC of HROG59 (200x magnification); red signals: centromere of chr. 7; green signals: <i>EGFR</i> in the 2C CISH analyses; G) qPCR analysis of GBM PDX tissue, error bars represent the standard deviation of triplicate analyses.</p

MOESM2 of Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies

Additional file 2. Overview of patient characteristics diagnosed with WHO°I–III tumors. Summary of molecular alterations, cryoperiod of the samples prior to implantation and the outcome of PDX establishment attempts