IDH1 mutation analysis in low cellularity specimen: A limitation of diagnostic accuracy and a proposal for the diagnostic procedure

Abstract

Recently, new techniques for detecting IDH1 mutations have been developed. Most studies assessed the mutation status in glioma tissue without consideration of the size of the samples. We assessed the mutation status of IDH1 in simulated small biopsied tissue from 5 low grade gliomas, prepared by grid cutting procedure with direct sequencing, IDH1 immunohistochemistry (IHC), multiplex PCR with single base extension (SBE) assay and PNA-clamping method, and then analyzed the agreement between the methods. Kappa values were 0.53 (direct sequencing), 0.59 (multiplex PCR with SBE assay), and 0.69 (PNA-clamping method). Discrepant results between the methods were observed in lower cellularity samples. Twelve out of 25 cases were classified as wild type by direct sequencing, even with IDH1 IHC-positive cells, whereas 6, 8, and 11 of IHC-negative cases were classified as mutant cases by other 3 methods. In conclusion, newly developed sensitive methods, such as the PNA-clamping method and multiplex PCR with SBE assay, are practically useful in addition to the conventional IDH1 IHC in small biopsied samples.

Introduction

IDH1 mutation is a milestone of glioma research [1] considering its significance in subtype stratification [2], [3] and prognosis [4], [5], and the assessment of R132H mutation status of the IDH1 gene has been recognized as one of the major biomarkers of glioma [6], [7], [8]. Practically, DNA sequencing and mutation-specific IHC in formalin-fixed, paraffin-embedded tissue are widely used [9], [10], [11], but several other PCR-based techniques [12], [13], [14], [15], [16], [17], such as peptide nucleic acid-clamping-based detection and multiplex PCR with single base extension assay, are being developed for IDH1 mutations. Perizzolo et al. identified the IDH1 mutation in a mixture of 5% mutant allele vs. 95% wild type allele by multiplex PCR with a single base extension assay [12]. Comparison between IHC and direct sequencing with other methods has been described in various previous studies [13], [18], [19]. In particular, in specimens with small size or with very low tumor cellularity, as in the tumor border of low-grade glioma, these two techniques seem to be complementary [18].

Most previous studies have analyzed the mutation status of various specimen types without consideration of their size or tumor fraction. The technical progress of IDH mutation detection methods is expected to be practically useful in small sized specimens, especially in stereotactic neurosurgical biopsies. The sensitivity of different IDH1 detection methods was demonstrated by mutant/wild type DNA serial dilution in most previously studied cases [12], [13], [17], but the application of those results is difficult in real clinical practice, because it is practically impossible to define the “real” IDH1 mutation status since the microscopically-assessed tumor fraction may not be reflected in the amount/quality of DNA needed for proper analysis, especially in small biopsied tissues with a low tumor fraction.

The aim of this study was to evaluate the diagnostic utility of the newly developed methods, peptide nucleic acid (PNA)-clamping method and multiplex PCR with single base extension assay, compared to the conventional methods in small tissue samples simulating small biopsied tissue of various tumor cell fractions using grid-cutting methods.