Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Oncogene amplification is a critical driver of cancer. Cytogenetic characterization on FFPE samples is a cost-effective way to study oncogene amplification. We present robust and comprehensive instructions for investigating focal gene amplification and FFPE samples.

By examining the FISH signal pattern, it becomes unequivocally clear whether and how a gene locus is amplified. Oncogenes amplified as extra chromosomal DNA are found to be prevalent in human cancer such as lung, breast, and brain cancer. Whole genome sequencing in FISH are the most widely used methods to detect a extra chromosomal DNA.

Applying FISH to FFPE tissues is challenging because cross-linking artifacts and background autofluorescence often undermine data quality. Our protocol includes optimized procedures including protein extraction and digestion, heating naturing and autofluorescence quenching techniques to improve data quality during FISH on FFPE samples, which will give valuable insight into cancer progression in patients. We will study the molecular functions of extra chromosomal DNA and how cancer cells maintain extra chromosomal DNA.

Ultimately, we aim to eliminate them to treat cancer. To begin, obtain the sample-mounted slide after aging it. To pretreat the slide, dip it serially in coplin jars containing appropriate solutions for the specified time.

Digest the tissue with 100 to 200 microliters of proteinase K digestion buffer, and incubate at room temperature for one minute. To stop the digestion, immediately immerse the slide serially into ethanol solutions for two minutes each. Apply the FISH hybridization mix to the slide and cover the sample with a cover slip.

Place the slides onto a hot plate, such as a slide moat hybridization system at 75 degrees Celsius for two to five minutes. Then transfer the slide onto another hot plate set at 37 degrees Celsius to hybridize overnight. Next, dip the slide into the warm wash buffer and carefully remove the cover slip.

Wash and stain the slide using appropriate reagents in the dark. Quickly dip the slide into deionized water for no more than one second and place it on a paper towel. After drying, mount the slide with antifade mounting media.

Place the cover slip and seal it with nail polish before imaging. Using a 60 X oil lens, capture the fluorescent signal in multiple Z-stacks. Finally, perform a maximum 3D projection to achieve the best resolution and apply deconvolution or other background-clearing algorithms.

In the triple negative breast cancer samples, nuclear FISH primarily shows two distinct dots per nucleus, representing HER2 ERBB-2 signals. In contrast, HER2-positive samples display abundant FISH signals, indicating different patterns of gene amplification. Additionally, some nuclei in HER2 positive samples may exhibit clusters, suggestive of extra chromosomal DNA hubs, which are focal points for increased gene amplification.