In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 2218-2218
Abstract:
Diagnostic of the IDH1 mutation (IDH1mut) is so far based on DNA sequencing and immunohistochemistry (IHC), methods limited in terms of sensitivity and ease of use. Recently, measuring the IDH1mut level by real time PCR was introduced as alternative method. Therefore, we aimed to (i) validate this new technique in a larger patient cohort, compare it to control tissue, and (ii) investigate if the expression of both, IDH1mut and IDH1 wildtype (IDH1wt) correlates with the course of disease and different treatment regimens. A total of 113 tumor samples were obtained intraoperatively from 84 glioma patients with diagnosis of diffuse glioma (WHO°II), anaplastic glioma (WHO°III), secondary glioblastoma +/- chemotherapy (CTx), primary glioblastoma +/- CTx (WHO°IV). Tumor samples were snap frozen and processed for sectioning, RNA and protein isolation. The quantitative expression of IDH1 mRNA was assessed using real-time PCR with specific primers for IDH1mut and -wt; protein expression was verified by Western Blot analysis and IHC. Additionally, 19 samples of LGG and their consecutive HGG were analyzed at different time points of disease. Allele-specific quantitative RT-PCR does work in larger patient cohorts and seems to be an easy and ∼10 times more sensitive method for quantitative IDH1mut evaluation compared to the ones used so far. Our results with this new method confirmed previous data showing that most astrocytomas and secondary GBM bear the mutation (conc. 0.13 to 0.2) whereas most primary GBM do not (conc. ∼0.025). Quantitative analysis revealed that IDH1mut expression is upregulated in sGBM (mean±SEM: 3.52±0.55) compared to lower grade glioma (II° = 1.54±0.22; III° = 1.67±0.23). By contrast, IDH1wt expression is upregulated in all glioma grades (con. & gt;0.1) compared to control brain tissue (0.007± 0.0016). Western Blot analysis showed a high concordance to both, sequencing and RT-PCR results in qualitative analysis of IDH1mut status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in sGBM. Taken together, recurrent disease and radiochemotherapy do not alter the general IDH1 status. Moreover, we found an increase of IDH1mut gene expression in sec. GBM compared to lower grade glioma indicating a pivotal role of IDH1mut not only in gliomagenesis but also in glioma progression. Citation Format: Marco Timmer, Moritz Perrech, Gabriele Röhn, Lena Dreher, Roland Goldbrunner. Qualitative and quantitative analysis of IDH1 mutation in progressive gliomas by allele-specific quantitative RT-PCR and Western blot analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2218. doi:10.1158/1538-7445.AM2015-2218
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2015-2218
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2015
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3