Collagenase type IV improves the quality of HER2 fluorescence in situ hybridization for breast cancers

Autofluorescence often hampers the interpretation of fluorescence in situ hybridization (FISH). This study aimed to develop a novel pretreatment method to reduce autofluorescence and improve HER2 FISH quality for breast cancer diagnosis. Autofluorescence, predominantly originating from the extracellular matrix, was targeted for reduction using enzymatic pretreatment. Formalin-fixed, paraffin-embedded breast cancer sections (4 µm thick) were treated with extracellular matrix enzymes, including type I collagenase, type IV collagenase, and elastase, before standard HER2 FISH. A total of 112 breast cancer samples were assessed for autofluorescence reduction and FISH signal clarity. Type IV collagenase proved most effective in reducing autofluorescence. With standard FISH, 25 (22.3%) of 112 samples exhibited strong autofluorescence, obscuring probe signals. Following type IV collagenase pretreatment, previously indiscernible signals became evaluable. Among these, 1 sample initially difficult to classify was reclassified as HER2 positive based on the HER2/CEP17 ratio and HER2 copy number. In this study, we demonstrated type IV collagenase pretreatment could effectively reduce the extracellular matrix autofluorescence and improve the resolution of probe signals. Therefore, this pretreatment method could enhance the accuracy of the HER2 FISH results.