Fig. 5

Interpretation of mosaic intratumoral heterogeneity (ITH) by comparison to a statistical model of truncation artifact. a The mosaic ITH is measured in all tissue samples and benchmarked with the readout of the two cell lines: it is defined as the ratio of the number of individual heterogeneous cells (i.e., cells with positive/negative FISH status in a cluster of cells of negative/positive FISH status), divided by the total number of cancer cells in a tissue. For a HER2-positive sample (mean HER2 loci/cell ≥ 6 or HER2/CEP17 ≥ 2), mosaic ITH cells correspond specifically to LH cells of spatial association analysis, like the ones marked by the + symbol in Fig. 4f. For a HER2-negative and equivocal sample (mean HER2 loci/cell < 6 and HER2/CEP17 < 2), mosaic ITH cells correspond to HL cells of spatial association analysis, like the ones marked by the × symbol in Fig. 4f. For both cell lines and tissues, the apparent mosaic ITH of a homogeneous population can be artificial when it is due to truncation artifacts. Indeed, truncated cells having a random position of the nucleus with respect to the cut contain a number of HER2 dots that follows a binomial law of probability. The full line represents a model simulation that describes such truncation artifacts in a homogeneous population of cells as a function of varying HER2 loci number. Data points lying above the model curve can be considered as exhibiting true mosaic ITH (see an image of b), while points on the curve represent a false mosaic ITH, as due to truncation artifacts (see an image of c). b The automatic scoring FISH image of a true mosaic ITH. Arrows indicate cells that differ from their neighborhood. c The automatic scoring FISH image of an artificial mosaic ITH case. Less heterogeneous cells are observed. Scale bars: 20 µm. d HER2 overexpression versus HER2 amplification for all tissues with additional heterogeneity information. It shows one patient with true mosaic ITH (the same as shown in d) and one patient with cluster heterogeneity (data of Fig. 4f) among the 20 patients tested. Thresholds for positivity are indicated by the dotted lines. Colors in FISH images: blue = DAPI, red = HER2, green = CEP17