BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer

Abstract

Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, RET and neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- and interchromosomal rearrangements has been suggested as a cause of the disease. However, many phenotypically similar tumors do not carry an activated RET or NTRK-1 gene or express abnormal ret or NTRK-1 transcripts. Thus, we hypothesize that other cellular RTK-type genes are aberrantly expressed in these tumors. Using fluorescence in situ hybridization-based methods, we are studying karyotype changes in a relatively rare subgroup of PTCs, i.e., tumors that arose in children following the 1986 nuclear accident in Chernobyl, Ukraine. Here, we report our technical developments and progress in deciphering complex chromosome aberrations in case S48TK, an aggressively growing PTC cell line, which shows an unusual high number of unbalanced translocations.

Fig. 1

FISH-based analysis of chromosomal alterations in a case of post-Chernobyl childhood thyroid cancer. A)-D) SKY analysis reveals a large number of translocations most of which are unbalanced (A: pseudo-RGB image, B: chromosome classification image, C: inverted DAPI counterstain image, D: SKY karyotype table). E) Dual color FISH analysis of S48TK metaphase spreads with BACs flanking NTRK1. The insert shows enlarged images of two copies of a der(1) (left: pseudo-RGB composite of FISH signals and DAPI counterstain; right: the inverted DAPI picture). F) Analysis of S48TK metaphase spreads with BACs flanking TMP3. The inserts show enlarged images of the two marker chromosomes, M1 and M2 (left: pseudo-RGB composite of FISH signals and DAPI counterstain; right: the DAPI picture). G) Various abnormal chromosomes found in S48TK metaphase spreads carry material originating from chromosome 9 (shown in white in the classification images). These SKY images show the pseudo-RGB picture to the left, an inverted DAPI image in the middle and the corresponding classification images to the right. The arrow points at a small segment of chromosome 9 material inserted into a der(8)t(8;15) marker chromosome. H) Chromosome arm painting (CAP) probes delineate the short (red) and long (green) arm of chromosome 9 in normal metaphase chromosomes. In S48TK metaphase chromosomes, these probe pools highlight the chromosome 9-derived material on marker chromosomes. The arrow points to a small segment of chromosome 9q material on the der(8)t(8;15) marker chromosome. I) Schematic illustrating the use of pooled BAC probes each covering several megabases on chromosome 9 for mapping of translocation at the single band resolution. J) Pools (9–10 + 9–11) specifically bind to the small segment of interest (arrow). [The images shown in D) and G) - J) are reproduced from Weier et al. [34] with permission from S. Karger AG, Basel, CH.]

Fig. 2

Schematic diagram of the BAC-FISH pooling strategy for delineation of chromosomal rearrangements on chromosome 9. Clones are arranged on 96-well plates such that each row contains clones which map within a region a few megabasepairs. Arrows point from rows on the plate to the target chromosome bands. The profiles on the right depict copy number changes along chromosome 9 in case S48TK, with a region of copy number overrepresentation indicated by a hatched box near the chromosome 9 ideogram.