Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer

Abstract

The tissue kallikrein (KLK) genes are a new source for biomarkers in ovarian cancer. However, there has been no systematic analysis of copy number and structural rearrangements related to their protein expression. Chromosomal rearrangements and copy number changes of the KLK region were studied by FISH with protein levels measured by ELISA. Ovarian cancer and cell lines revealed the KLK region was subject to copy number imbalances or involved in unbalanced translocations and were associated with increased protein expression of KLKs 5, 6, 7, 8, 9, 10 and 11. In this initial study, we introduce the potential for long-range chromosomal effects and copy number as a mechanism for the previously reported aberrant expression of many KLK genes in ovarian cancers.

Figure 1

Summary of FISH findings of the KLK locus in cancer cell lines and ovarian cancer patients. (A) Schematic illustrating the BAC clones used in this study. (B) Summary of FISH studies in cell line and tumors. Chromosome 19 was analyzed using the paint probe WCP19 and BACs that cover most of the 325‐kb KLK genomic region using dual color FISH. Closed circles indicate the presence of the KLK locus located at the resident chromosome 19q site. Open circles indicate the presence of the KLK locus associated with either an intra‐chromosomally or inter‐chromosomal structural rearrangement. Also indicated for each analysis is the ploidy established by chromosomal counts. MCF10A and 22RV1 did not show copy number changes or involvement of translocation. In 70% of cells, T47D also showed a normal diploid pattern, but 30% showed a whole chromosomal gain of 19. LNCAP showed four copies of chromosome 19 with four copies of the KLK locus, showing no net gain over ploidy, but two copies over a normal diploid cell. The remaining cell lines and tumors displayed net gains of the KLK locus by unbalanced translocations.

Figure 2

KLK status in cancer cell lines by sequential SKY and FISH. Shown are representative metaphases of CAOV‐3 (A), MDA‐MB‐468 (B) and MCF‐7 (C) by SKY and FISH using the KLK BACs (red). The affected chromosomes are indicated by arrows and shown in the inset.

Figure 3

Status of the KLK locus in patient ascites. In each case, WCP19 (green) was hybridized with BACs for the KLK locus (red) to metaphase preparations. (A–C) Patient ascites 2. (A and (B) show the presence of chromosome 19 material (green) dispersed throughout the genome (A), with one KLK signal mapping to the resident chromosome 19, with the remaining two copies involved in translocations (B) (arrows). (C) Integrative FISH and SKY analysis of affected chromosomes. Inverted DAPI banding in shows the banding pattern of the metaphase chromosomes hybridized with the SKY paints (left) with FISH of corresponding chromosomes showing chromosome 19 paint (green) and the KLK locus (red) from A/B (left). By SKY complex rearrangements involving two or more chromosomal partners can be seen through the change of color along the length of a contiguous chromosome. The RGB (red–green–blue) display color for chromosome 19 is bright green, with a normal chromosome 19 (top–bottom), followed by five abnormal chromosomes involved in structural rearrangements with other chromosomal partners. The corresponding FISH using the KLK and whole chromosome 19 painting probes confirmed that structurally rearranged chromosomes by SKY also included the KLK locus. (D–G) Patient ascites 3 shows the presence of a KLK signal at the resident chromosome 19 and the presence two isochromosomes 19q (i(19q)) (D,E). The net result is the gain of KLK copy number due to these isochromosomes. (F) Integrative SKY and FISH analysis revealed the involvement of chromosome 19 in translocations with chromosome 3, but without involvement of the KLK locus. (G) Representative SKY metaphase of patient ascites 3. (H,I) Sporadic and untreated primary cancer OCA5. Shown in red is the KLK locus and a BAC clone for the 19q12 region, shown in green revealing the net gain of the KLK locus through whole gains of chromosome 19 and the presence of a multi‐centric (green) ring chromosome containing the KLK locus (red). (I) Integrative SKY and FISH of OCA5. SKY and FISH confirms that no other chromosomes are involved in structural rearrangements on the three chromosomes 19, but the possibility exists for other chromosomal fragments in the ring chromosome. (J) Interphase nuclei of OCA21A shows variability in copy numbers of the KLK locus from cell to cell.

Figure 4

aCGH and FISH validation of copy number imbalances of the KLK locus in BT‐474. (A,B) aCGH for chromosome 19. Regions of gain are highlighted in green with regions of loss indicated by red, and no net change in yellow. (B) Magnification of the KLK locus shows an average ratio approaching +0.5 indicating a net gain of the region. Due to the ploidy of the genome, the net gain suggests one copy over ploidy, thus 1+4n=5 copies of the KLK locus. Side panels: FISH Validation of individual KLKs in BT474. A three‐color FISH approach was used for KLK2 (green), KLK4 (red) and KLK6 (aqua) to BT474 metaphase spreads. Bottom panel: KLK13 validation in BT474. In this three‐color experiment, KLK2 (green), KLK6 (blue) and KLK13 (red) were co‐hybridized and also shown to co‐localize and be present in copy numbers consistent with aCGH findings.