Replication Timing Aberration of KIF14 and MDM4 / PI3KC 2 β Alleles and Aneuploidy as Markers of Chromosomal Instability and Poor Treatment Response in Ewing Family Tumor Patients

Abstract

Replication timing of allelic gene pairs is strictly regulated according to expression, genome stability, and epigenetic changes, and tumorigenesis may be associated with changes in the allelic replication in various tumors. Our aim was to determine whether such alterations had a prognostic value in Ewing's family tumor (EFT) patients. The KIF14 and MDM4 / PI3KC 2β and the centromeric satellite sequence of chromosomes 8 and 12 were used for replication timing assessments. Aneuploidy was assessed by enumerating the copy numbers of chromosomes 8 and 12. Replication timing and aneuploidy were detected cytogenetically using multicolors fluorescence in situ hybridization assay applied in 135 EFT. Patients with trisomy 8 presented an association with an asynchronous replication pattern (SD) of MDM4 / PI3KC 2β genes ( p = 0.013). Trisomy 12 was associated with a synchronous pattern (DD) of KIF14 probe signals ( p = 0.04). The DD synchronous replication pattern of KIF14 showed a correlation with age ( p < 0.0001), and the SS synchronous replication pattern of the same locus showed a correlation with lung metastatic ( p = 0.012). The subgroup of patients presenting with multiplet signals of MDM4 / PI3KC 2β showed an association with treatment response ( p = 0.045) and age ( p = 0.033). Replication pattern of KIF14 may, significantly, be associated with chromosomal instability as MDM4 / PI3KC 2β may be a considerably new marker of poor treatment response in EFT patients.

Keywords: Ewing sarcoma; aneuploidy; chromosomal instability; poor treatment response; replication timing.

Fig. 1

FISH strategy analysis of KIF14 or MDM4/ P/3KC28 genes replicating pattern in EFT cells at interphase. ( A ) The schematic chromosome 1illustrates the genomic localization and names of the BAC probes encompassing the pericentromeric region and the target genes at 1q32.1. ( B ) Representative FISH scheme are shown for EFT TMA applying the three-color FISH for the replication pattern analysis.

Fig. 2

Schematic representation of timing replicating pattern of the KIF14 or MDM4/ Pl3KC2B hybridization signals at interphase by FISH. ( A ) Configuration of signals in normal (no CNA) cells showing different replication patterns. ( B ) Guide for signal enumeration when the genes dots show up as doublets or multiplet. Note that in cells that showed gain of the genes, only the SS, DD, and M patterns were counted. Abbreviation: CNA*: copy number alteration.

Fig. 3

Frequency of cells showing replication timing (SS, DD, SD, M) of KIF14 and MDM4/ P/3KC2B alleles in the total EFT patients.

Fig. 4

Distribution of cells showing replication timing (SS, DD, SD, M) of KIF14 alleles in EFT patients. The signals were evaluated in cells of EFT patients with CNA and with no CNA for chromosome gains at 1q32.1 and aneusomy of chromosomes 8 and 12.

Fig. 5

Distribution of cells showing replication timing (SS, DD, SD, M) of MDM4/P/3KC2B alleles in EFT patients. The signals were evaluated in cells of EFT patients with CNA and with no CNA for chromosome gains at 1q32.1 and aneusomy of chromosomes 8 and 12.

Fig. 6

Representation of FISH images of 1q32.1 region showing replication pattern in EFT TMA cells at interphase applying the three-color iFISH assay. MDM4/P/3KC2B signals hybridization are seen in green. KIF14 signals hybridization are seen in orange and the control in pink. ( A ) cell with singlets (SS cells) in which neither allele has replicated; Synchronous pattern ( B ) cell with one singlet and one doublet (SD cells), which are Sphase cells in which one allele has replicated while its partner has not ( arrow ), ( C ) cell with doublets (DD cells) in which alleles have replicated; and ( D ) cell with multiplets pattern of signals (arrow).