Hyperdiploidy of cultured dermal fibroblasts from patients with aerodigestive squamous carcinoma

Abstract

The incidence of hyperdiploidy in monolayer cultures of dermal fibroblasts from 88 patients with squamous carcinoma of the aerodigestive tract was compared with similar cultures from 39 normal subjects using three different techniques; 1) metaphase assay (MA), 2) flow cytometry of stationary cell cultures (FCM'), and 3) flow cytometry of proliferating dividing cell cultures (FCMd). In vitro hyperdiploidy was considered to be present (IVH+): 1) by MA if more than 4% of metaphases were altered, excluding tetraploidy; 2) by FCMS if more than 8% of cells in stationary cultures were hyperdiploid (i.e., DNA index > l), and 3) by FCMd if more than 6% of cells in logarithmic cultures were hypertetraploid (i.e., DNA index > 2). There was excellent concordance between the three assays, which assigned cell cultures from 55 of the 88 patients with aerodigestive squamous carcinoma (62%) to the in vitro hyperdiploidy positive (IVH+) category. Cell cultures from all 39 normal individuals were hyperdiploidy negative (IVH-).These data suggest that some substantial proportion of patients with aerodigestive squamous carcinoma may have a genetic predisposition for the disease, which can be identified by these assays. The flow cytometry methods were easier to carry out and may be substituted for the metaphase analyses.Key terms: Genetic predisposition, flow cytometry, monolayer culture, metaphase assay In vitro hyperdiploidy (NH) is defined as the presence of cells in culture having more than 46 metaphase chromosomes, as opposed to 92 metaphase chromosomes, or tetraploidy. It has been described in dermal monolayer fibroblast cultures of patients with some heritable tumors (4), including a family with nasopharyngeal cancer (5). We previously compared this assay with another, based on the distribution of DNA by flow cytometry of cells in stationary growth phase (FCM') (7, 8); we now compare the metaphase assay MA), FCM", and a third assay (FCMd), based on the distribution of DNA by flow cytometry of proliferating cells in logarithmic growth phase. The last assay (F'CMd) more closely approaches the proliferative cell culture conditions of the MA. Further, a concordance of all three assays would strengthen the hypothesis of a n underlying genetic disorder in patients with these tumors and enhance the potential value of IVH as a clinical marker of genetic predisposition for certain tumors. MATERIALS ANL) METHODSDermal fibroblast lines were established by standard culture methods (3) using split-thickness skin biopsies from 39 volunteers (23 males, 16 females, ages 16-88 yr) without a family tumor history and from 88 patients with various squamous carcinomas of the aerodigestive tract (50 males, 38 females, ages 18-87 yr). The cultures were maintained for 8-15 weeks prior to the assay (three to six subcultures by trypsinization after the primary explant culture) in plastic petri dishes (growth area 28 cm2) using Eagle's minimum essential medium (13) 585Subjects cm2 plastic petri dishes at an initial density of lo4 cells/ cm2, cultured fo...

Comparison of anatomical location of squamous cell carcinoma within the oral cavity and oropharynx with the incidence of in vitro hyperdiploidy

“…In the present study, as in the previously published studies (Danes et al, 1987a(Danes et al, , 1987b, IVH was considered to be present (IVHt) if all 3 assays on a cell line gave the following data: 1) by metaphase assay (MA), if more than 4% of the metaphases were altered, excluding tetraploidy; 2) by FCMs if DNA index DI > 1 was > 8%, and 3) by FCMd if D I > 2 was >6%. As previously reported (1987b), the standard deviations (SD) for each of the 3 assay values were small for the IVH-and increased for IVH+ cell samples.…”

“…Previously (Danes et al 1987a(Danes et al , 1987b) the association of IVH+ with a certain tumor has been presented as the percentage of total clinical cases having IVH+. In the present study of 76 SCCA patients, 54% (41/76) were IVH+ as compared to 57% (8/14) in a previous small sample study (Danes et al 1987a).…”

“…Total oral cavity I 49 28 53 (FCM'); and 3) flow cytometry of proliferating dividing cell cultures (FCMd) as previously described in detail (Danes et al 1987b).…”