Uterine Leiomyomas with an Apparently Normal Karyotype Comprise Minor Heteroploid Subpopulations Differently Represented in vivo and in vitro

Abstract

In the present study, we aimed to check whether uterine leiomyomas (ULs) with an apparently normal karyotype in vitro comprise “hidden” cell subpopulations with numerical chromosome abnormalities (heteroploid cells). A total of 32 ULs obtained from 32 patients were analyzed in the study. Each UL was sampled for in vivo and in vitro cytogenetic studies. Karyotyping was performed on metaphase preparations from the cultured UL samples. A normal karyotype was revealed in 20 out of the 32 ULs, of which 9 were selected for further study based on the good quality of the interphase preparations. Then, using interphase FISH with centromeric DNA probes, we analyzed the copy number of chromosomes 7 and 16 in 1,000 uncultured and 1,000 cultured cells of each selected UL. All of the ULs included both disomic cells representing a predominant subpopulation and heteroploid cells reaching a maximum frequency of 21.6% (mean 9.8%) in vivo and 11.5% (mean 6.1%) in vitro. The spectrum of heteroploid cells was similar in vivo and in vitro and mostly consisted of monosomic and tetrasomic cells. However, their frequencies in the cultured samples differed from those in the uncultured ones: while the monosomic cells decreased in number, the tetrasomic cells became more numerous. The frequency of either monosomic or tetrasomic cells both in vivo and in vitro was not associated with the presence of <i>MED12</i> exon 2 mutations in the tumors. Our results suggest that ULs with an apparently normal karyotype consist of both karyotypically normal and heteroploid cells, implying that the occurrence of minor cell subpopulations with numerical chromosome abnormalities may be considered a characteristic of UL tumorigenesis. Different frequencies of heteroploid cells in vivo and in vitro suggest their dependence on microenvironmental conditions, thus providing a pathway for regulation of their propagation, which may be important for the UL pathogenesis.

Cytogenomic Profile of Uterine Leiomyoma: In Vivo vs. In Vitro Comparison

“…In the first stage of the study ( Figure 1 ), we performed a conventional cytogenetic analysis of 32 UL cell cultures ( Figure 2 A,B), of which 20 (62%) showed a normal (described in [ 30 ]), and 12 (38%), an abnormal, karyotype ( Table 2 ). All corresponding cultured myometrial samples had a karyotype of 46,XX.…”

“…L36R) ( Table 2 ). In karyotypically normal ULs, MED12 mutations were detected in four out of nine tumours [ 30 ]. The MED12 mutation frequency did not differ between ULs with a normal and an abnormal karyotype ( p = 1.000, Fisher’s exact test).…”

“…A total of 4 ULs (1, 2, 4, 12) had a c.131G>A substitution in codon 44 (p. G44D) and UL 10 had a c.107T>G substitution in codon 36 (p. L36R) (Table 2). In karyotypically normal ULs, MED12 mutations were detected in four out of nine tumours [30]. The MED12 mutation frequency did not differ between ULs with a normal and an abnormal karyotype (p = 1.000, Fisher's exact test).…”

“…The tissue was minced and digested with collagenase according to the previously described protocol [ 28 , 29 ]. Further culturing until passage 1, harvesting and slide preparation were performed as described earlier [ 30 ].…”

“…For homemade FISH probes, a previously described protocol was used [ 31 ]. For commercial FISH probes, the procedures were performed according to the manufacturer’s recommendations (Abbott Laboratories, Chicago, IL, USA; ZytoVision GmbH, Bremerhaven, Germany; Leica Biosystems, Buffalo Grove, IL, USA) with minor modifications [ 30 ]. The FISH probes used to identify the breakpoints on metaphases from karyotypically abnormal ULs are listed in Table S1 .…”