Analysis of 1p/19q co-deletion using a semi-automated chromogenic in situ hybridization method compared with fluorescence in situ hybridization

Abstract

The 2016 World Health Organization Classification of Tumors of the Central Nervous System explicitly describes the importance of noting 1p and 19q co-deletion status in the pathological diagnosis of oligodendrogliomas. Although 1p/19q co-deletion can be detected using fluorescence in situ hybridization (FISH), this approach has complex sample preparation requirements, and there are challenges in long-term FISH sample storage. In the present study, we therefore developed a semi-automated chromogenic in situ hybridization (saCISH) method, which can be observed under an optical microscope. We used a sample set of 20 cases in which 1p/19q co-deletion analysis had already been performed using FISH. For the saCISH method, we first automated the pre-treatment process using an immunostainer; this was followed by the hybridization of labeled probes. Subsequently, automated immunostaining of the labeled probes was conducted, with missing chromosomes colored in red and reference chromosomes colored in brown. In the prepared samples, we then counted each signal and calculated the ratios of 1p/1q and 19q/19p. The saCISH method had a reduced handling time compared with FISH, from approximately 100 minutes to 35 minutes, and improved the complexity of specimen preparation by automating the pre-processing steps. However, challenges were identified with the saCISH method, including a tendency for red signals to appear brown when signals overlapped, and the potential interference of non-specific pigment deposition when identifying signals. Further improvements in the color development method for labeled probes are therefore necessary.