This document describes a method for accurately evaluating HER-2 amplification in fluorescence in situ hybridization (FISH) images. The method involves extracting nuclei from FISH images, assigning each nucleus a reliability score based on shape and size compliance with a template model, and computing the ratio of HER-2 to CEP-17 markers only using the most reliable nuclei. The method was tested on a dataset of 40 FISH images classified by experts into categories of HER-2 amplification. Using a training set to determine an optimal reliability score threshold maximized classification accuracy evaluated on a test set.

1. Accurate Evaluation of HER-2 Amplification in FISH Images
A. Del Bimbo, M. Meoni, P. Pala
Dipartimento Sistemi e Informatica, University of Firenze, Italy
Context and motivations
• Fluorescence in situ hybridization (FISH) is a cytogenetic technique used to detect and localize the presence or
absence of specific DNA sequences on chromosomes
• A sample application of this technique targets the measurement of the amplification of the HER-2 gene within
the chromosomes, that constitutes a valuable indicator of invasive breast carcinomas
• This requires the application to a tumor tissue sample of two types of fluorescent probes that attach themselves
to the HER-2 genes and to the centromere 17 (CEP-17), respectively
• Estimation of HER-2 amplification is accomplished by measuring the ratio of HER-2 over CEP-17 markers within
each nucleus
• Inaccurate nuclei identification may severely bias the estimation of this ratio
Adaptive threshold Input Image
Distance transform for
markers extraction
Spot extraction
Marked Watershed
Reliability score Ratio evaluation
A model for nuclei reliability
Given a generic nucleus, its reliability score r is evaluated by The reliability score of a generic nucleus is evaluated as:
measuring the compliance of the shape and size of the nucleus
with respect to a template model. (a−at)2 (e−et)2 (c−ct)2
− 2 − 2 − 2
The reliability score of each nucleus is used so as to: r=e σa e σe e σc
• Regions corresponding to a potentially oversegmented
• a is the area of the nucleus and at the area of the template model
nucleus are recursively merged so as to better match the
• e is the eccentricity of the nucleus and et the eccentricity of the template model
actual shape and contour of the nucleus
• c is the convexity of the nucleus and ct the convexity of the template model
• Nuclei can be ordered by decreasing reliability scores so as to Values of σa, σe and σc define the range of allowable deviations from template area and
compute the ratio of HER-2 over CEP-17 markers using only eccentricity. Values of parameters at, et and ct have been estimated by averaging the actual values
the most reliable ones measured on a training dataset.
The Dataset Results
The dataset consists of 40 FISH images extracted from eight 0,9 Classification accuracy is
slides of breast tissue samples. Each image is annotated by an measured through the
expert pathologist with a classification label corresponding to four 0,8 confusion matrix and is
different levels of HER-2 amplification: evaluated for the test dataset
• No amplification (N), 17.5% of the dataset 0,7
using the optimal value of τr
• Borderline (B), 10.0% 0,6 identified on the training
• Amplified (A), 25.0% dataset.
• Strong amplification (A+), 47.5% 0,5
N B A A+
The system automatically classifies a generic image based on the
0,4 N 1.0 0.0 0.0 0.0
average value ρ of the ratio extracted on nuclei with a reliability
B 0.0 1.0 0.0 0.0
score r higher than a threshold τr . 0,3
A 0.0 0.0 0.875 0.125
The optimal value of τr is computed by maximizing classification 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9
A+ 0.0 0.0 0.125 0.875
accuracy over a training dataset.
http://www.dsi.unifi.it/pala/ http://www.micc.unifi.it/projects/her-2/ {delbimbo,meoni,pala}@dsi.unifi.it