Reply to Perner and Rubin

In reply: In response to the letter by Drs Perner and Rubin, the apparent confusion appears to arise from a misunderstanding of the genome map location and/or the colors of the fluorescence in situ hybridization (FISH) probes used. As indicated in the legend to Figure 3 of the article,¹ the green/blue pseudocolored probes map, respectively, to the 5′ and 3′ portions of ERG, whereas the red pseudocolored probe maps telomeric to TMPRSS2. These relationships are shown schematically in Figure 1a. In Figure 3 of the article, the green/blue signals do not appear split apart, indicating, as reported, that the specimen is indeed negative for ERG rearrangement. The distance observed between the red (TMPRSS2) and green/blue (ERG) signals, which map to DNA sequences approximately 3 million base pairs apart on chromosome 21, varies based on the condensation state of the chromatin in the interphase nuclei and on the orientation of the FISH signals with respect to the photographed plane. It is not unusual to observe substantial spacing between the red and green/blue signals in normal nuclei (see Figure 1b). Thus, we believe that the interpretations of the FISH studies are accurate as reported.

Figure 1

Three-color FISH assay of TMPRSS2–ERG rearrangement. (a) Schematic depiction of pseudocolored FISH probes in relation to TMPRSS2 and ERG loci. (b) Three-color FISH analysis of representative normal human lymphocytes. Note that the red (TMPRSS2) signal can be observed at a substantial distance from the green/blue (ERG) signals; the latter two, by their proximity to each other, can appear aqua-colored.