gms | German Medical Science

Objective: IDH1 mutations (mut) occur in more than 70% of WHO grade II and III gliomas and secondary glioblastomas (GBM). The most frequent mutation leads to a specific amino acid change at codon 132. Diagnostics of this mutation are 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 an alternative method. Thus, we aimed to (i) validate this new technique in a larger patient cohort, compared it to control tissue, and (ii) investigated if the expression of both, IDH1mut and IDH1 wildtype (wt) correlates with the course of disease and different treatment regimens.

Method: A total of 71 tumor samples were divided into 7 subgroups (control brain tissue, diffuse glioma, anaplastic glioma, secondary glioblastoma ± chemotherapy (CTx), primary glioblastoma ± CTx). 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.

Results: Allele-specific quantitative PCR does work in larger patient cohorts and seems to be an easy and ~10 times more sensitive method for IDH1mut evaluation compared to the ones used so far. The most reliable cut-off value was found with Δconcentration (con.) = conc.(IDH1mut) – conc.(IH1wt). Our results with this new method confirmed previous data. Most astrocytomas and sec. GBM bear the mutation (con. 0.13 to 0.2) whereas most prim. GBM do not (con. ~0.025). The IDH1mut was not detectable in control tissue (con. <0.009±0.002). The difference between control tissue, prim. GBM and astrocytomas/sec. GBM was highly significant (p=0.003). No significant difference was found between grade II, III and secondary grade IV tumors. Radio- and/or CTx do also not alter the IDH1mut expression. Interestingly, IDH1wt was significantly higher expressed in all tumor groups (con. >0.1) compared to control brain tissue (0.007±0.0016).

Conclusions: This assay is able to analyze 100 samples simultaneously in ~1 hour. Recurrent disease and radio-CTx do not alter the general IDH1 status. The high increase of IDHwt expression in all tumor probes is likely due to an upregulated metabolism. In summary, this method is highly sensitive, cost-effective and timesaving and may therefore play an important role in IDH1mut analysis in the future.