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Chapter 28
Cytogenetic and molecular
genetic alterations in bone
tumors
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
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FIGURE 28.1 The t(11;22)-translocation in Ewing sarcoma can be detected by A) G-banding, B) FISH or by C) RT-PCR. (A)
Conventional karyotype analysis on a Ewing sarcoma patient sample shows a translocation between chromosomes 11 and 22 (indicated
by arrows). (B) Detection of the EWSR1-FLI1 fusion with fusion signal FISH analysis. In the fusion FISH strategy a red probe flanks one
breakpoint and a green probe the other breakpoint. If the signals are brought together by the translocation, the fusion signal is yellow.
The fusion signal is indicated by a white arrow in the figure. (C) RT-PCR analysis on Ewing sarcoma patient samples shows the EWSR1FLI1 gene fusion on gel lanes 1, 2, 5 and 6. The EWSR1-FLI1 type 1 fusion (328 bp) is detected on gel lines 1 and 2. Samples 5 and 6
show the variant EWSR1-FLI1 gene fusion types. Samples on lines 3, 4, 7 and 8 are negative for EWSR1-FLI1 fusions. (A) and (C) are
reproduced from Current Diagnostic Pathology 2002;8:338–48, by permission of Elsevier Ltd.
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
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FIGURE 28.2 Array CGH method in detection of gene copy number alterations. Genomic DNA is extracted from both a patient and a
normal sample and they are labeled with fluorescent dyes, red (Cy5) and green (Cy3), respectively. The labeled samples are combined
in equal amounts and hybridized on microarray slides, which can contain oligo-, BAC- or cDNA-probes. Samples hybridize to probe spots
in relation to the amount of labeled DNA in the test and the control sample. When the tumor sample has higher copy number compared
to the normal sample the probe spot is red, whereas in case of lower copy number, the spot is green. If no copy number changes are
detected, the spot is yellow. Following the hybridization microarray slides are washed and scanned with confocal laser scanner. The
image analysis, data extraction, data analysis, visualization and, finally, aberration detection are done with appropriate software. The final
result of array CGH interprets which genomic regions in the patient sample are gained and which are lost.
Copyright © 2013 Elsevier Inc. All rights reserved.
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FIGURE 28.3 Small CDKN2A deletions may be missed by FISH but can be detected by array CGH. (A) Narrowest microdeletions
(<190 kb) in 9p21.3 can create false negative results by using commercial FISH probes, as shown in FISH analysis on a Ewing sarcoma
cell line (IOR/RCH). FISH results show that one CDKN2A locus is gained as indicated by three red signals (arrows to left) in the sample.
For experimental details see Savola et al.12. (B) Array CGH analysis with high-resolution oligo-microarray (244,000 probes) on the same
IOR/RCH Ewing sarcoma cell line shows that within the gain of 9p22. 3-p13.3, there is a homozygous deletion at 9p21.3. The size of
homozygous deletion is 58 kb and it harbors only the CDKN2A and CDKN2B genes. (C) Genomic locations of 9p deletions in Ewing
sarcoma patient and cell line samples arranged by their size. The CDKN2A (yellow), CDKN2B (red) and MTAP (green) genes, their sizes
and locations are also indicated in the figure. The smallest overlapping region of deletion (12,2 kb), which is indicated by the purple bar,
is much smaller than the size of a commonly used commercial FISH probe (Vysis p16 FISH probe). This commercial FISH probe is
∼190 kb in size and it covers the region of three genes – CDKN2A, CDKN2B and MTAP - explaining the false negative results seen in
FISH analysis (see Figure 28.3A). (A) and (B) are reproduced from Cytogenetic Genome Research 2007;119:21–26, by permission of S
Karger AG, Basel.
Copyright © 2013 Elsevier Inc. All rights reserved.
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