A public-domain image processing tool for automated quantification of fluorescence in situ hybridisation signals

Abstract

This study shows that it is possible to automate the determination of HER2 amplification by the use of open-source software, with results comparable to manual counting. The automated counting decreases the time needed for sample analysis and provides possibilities to enhance inter- and intralaboratory reproducibility of results. The FISH quantification tool (FishJ) is available for download as an ImageJ macro or alternatively it can be utilised through a web interface with an option of uploading FISH images for hybridisation signal counting. Combined with digitisation of FISH samples, the FishJ macro enables gene copy number to be assessed and re-evaluated on any area of a digitised specimen.

‘Genetic heterogeneity' in HER2/neu testing by fluorescence in situ hybridization: a study of 2522 cases

“…Such an approach is difficult and labor intensive. As automated methods of interpreting in situ HER2/neu and 'genetic heterogeneity' hybridization based on image analysis become more widely adopted, [21][22][23] it may become more feasible to determine the prevalence and significance of true tumor heterogeneity in the future. In the meantime, it must be acknowledged that the HER2/CEP17 ratio is a continuous variable, and that the definition of 'HER2 amplification' is, by necessity, dichotomous.…”

State‐of‐the‐art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

“…To detect FISH spots, a 320 objective might be appropriate (28,29); however, if not only the number and intensity of signals are important but their spatial pattern as well, the application of a higher magnification and more importantly high resolution is desirable (30)(31)(32). Considering the prior, several workgroups use 360, 363, or 3100 objectives during the automated i-FISH analysis (21,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). An alternative approach, also increasing speed, is the use of two objectives, one of them for nucleus selection (objective magnification: and the other one for detecting FISH signals (objective magnification: 340-100) (31,44-52).…”

“…The problem emerges especially in case of objectives featured with low DOF (see above). Since this error causes bias in results, image capture in different focus planes and construction of images displaying all FISH signals are basic requirements nowadays (30,32,35,40,44,70,71). Fluorescence microscopy coupled with motorized scanning stage can adequately fulfill this requisite.…”

“…In the field of cancer cytogenetics, automated investigation of HER2 gene amplification was the most often published application, mainly performed on breast cancer specimens (37,40,46,59,85,86,(94)(95)(96)(97)(98)(99)(100). With the introduction of trastuzumab therapy, accurate assessment of HER2 amplification status, i.e., the ratio of HER2 spot signals and centromeric signals of chromosome 17 became essential especially in case of patients with weakly positive (21) HER2 membrane staining.…”

Automated FISH analysis using dual‐fusion and break‐apart probes on paraffin‐embedded tissue sections

“…However, isolation of nuclei is more time-and resource consuming, than analyzing tissue sections and valuable information regarding histological topography is lost. If the sample contains only a small focus of neoplastic cells, their frequency in suspensions of isolated nuclei may drop below the false positivity rate of the FISH analysis.Because of recent advancement of image analysis and computer science, automated FISH analysis is becoming a desired possibility (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). A recent report…”