Regions of allelic imbalance in the distal portion of chromosome 12q in gastric cancer

Abstract

Aims: To define regions of loss on the distal portion of chromosome 12q in gastric adenocarcinoma.

Methods: Microsatellite analysis on chromosome 12 was performed on 19 human gastric cancer cell lines using 77 markers, 71 of which were within or distal to 12q21; some portions of this region showed extended regions of homozygosity (ERHs) in 10 of 19 gastric cancer cell lines. In addition, microdissected tumour cells from 76 primary gastric adenocarcinomas were examined using 13 markers of interest implicated by the cell line data; 70% of these showed allelic imbalance (AI) at one or more markers in or distal to 12q21.

Results: Mapping ERHs in the cell lines and sites of AI in the tumours identified three regions that contain putative tumour suppressor genes: region A is located within 2.8 Mb between markers D12S1667 and D12S88; region B, within 1.9 Mb between markers D12S1607 and D12S78; and region C, in 0.74 Mb between markers D12S342 and D12S324. Fluorescence in situ hybridisation (FISH) analysis in two cell lines confirmed that two of the ERHs reflected deletions, not amplifications, of D12S81 in region A and D12S340 in region C. FISH analysis of marker D12S1075 within an ERH containing region B in one cell line showed neither amplification nor deletion. AI on 12q was not associated with prognosis, but was associated with ethnicity of the patient.

Conclusions: These results identify regions on chromosome 12 that appear to contain tumour suppressor genes important in the development of gastric cancer.

Figure 1

Comparative genomic hybridisation studies by van Grieken and Kim show losses in the distal portion of chromosome 12q. In the study by van Grieken et al, 17 of 46 gastric cancers showed deletions on the distal portion of 12q; in the study by Kim et al, six of 15 gastric cancers showed such deletions. Figure 1 ▶ was prepared from diagrams in published reports; however, because of the limits of resolution of CGH, the precise breakpoints on 12q cannot be determined with this technique.

Figure 2

The diagram indicates the positions of markers and extended regions of homozygosity (ERH; shown in grey) in 19 gastric cancer cell lines. 1, homozygous; 2, heterozygous. Del, marker was shown to be deleted in FISH experiments. Not del, equivalent numbers of centromeres and non-centromeric probes were seen in FISH experiments. The markers in boldface were used for the analysis of the tumours shown in fig 5 ▶.

Figure 3

Laser capture microdissection images. (A) The stained and coverslipped section is used for the identification of tumour cells (haematoxylin and eosin stain). (B) The next section to that shown in (A) is dark because of the lack of a coverslip (methyl green stain). (C) Tumour cells are specifically removed. (D) The tumour cells have been captured to provide DNA as a template for the polymerase chain reaction.

Figure 4

(A) In laser capture microdissection harvested tumours, which have very little contamination by normal DNA, near complete absence of one of the sets of bands may be seen in the tumours, as shown in the tumour lane in both cases. Marker, D12S1051. (B) Amplification of DNA from normal (N) and tumour (T) cells at marker D12S105 in two normal/tumour pairs. In case 1, tumour DNA shows no difference from normal DNA at this marker. In case 2, allelic imbalance is indicated because the lower set of bands is decreased in density. Faint reactions were not scored. For example, if light bands were visible, but neither upper nor lower bands exceeded the density seen in the lower bands of the tumour in case 2, that reaction would not be scored. (C) Two sets of bands of approximately equal density were amplified from the normal DNA, whereas we sometimes observed a decrease in the density of one band from the tumour DNA, accompanied by intensification of the density of the other band, consistent with a duplication of the portion of the chromosome containing the putative mutant gene. Marker, D12S1128.

Figure 5

Seventy six tumours were subjected to microsatellite analysis using 13 markers within or near regions A, B, or C. These 13 markers and their locations with respect to regions A, B, and C are identified in boldface in the chromosome diagram in fig 2 ▶. At the right side of the figure are percentages of allelic imbalance (% AI), numbers of informative cases (#inf), and observed percentages of heterozygosity (%het). Black squares, AI; open squares, heterozygous markers with no AI; grey squares, non-informative (homozygous); /, no data; *, microsatellite instability.

Figure 6

Some of the tumours showing partial losses from data shown in fig 5 ▶ were subjected to additional analysis, where available material permitted. Black squares, AI; open squares, heterozygous markers with no AI; grey squares, non-informative (homozygous); /, no data; *, microsatellite instability.

Figure 7

(A) Fluorescence in situ hybridisation (FISH) analysis of the cell line KATO-III shows four copies of the chromosome 12 centromere (red) and three copies of sequence homologous to the bacterial artificial chromosome (BAC), which contains marker D12S81 (green), a marker contained in region A. An arrow indicates the centromere of the copy of chromosome 12 in which the marker has been deleted. (B) FISH analysis of cell line MKN-28 shows two copies of chromosome 12, indicated by the centromere probe (red). The sequence homologous to a BAC containing marker D12S1075 is present on both chromosomes 12 (green). D12S1075 is contained within an extended region of homozygosity that encompasses both regions A and B. (C) FISH analysis of cell line KATO-III indicates copies of the centromeric region of chromosome 12 stained red and copies of the DNA containing the marker D12S340, contained in region C, stained green. The arrow indicates the centromere of a copy of chromosome 12 in which the marker has been deleted.