Cervical carcinoma-associated fibroblasts are DNA diploid and do not show evidence for somatic genetic alterations

Abstract

BackgroundCancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression.MethodsWe combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) in cervical cancer-associated stromal cell fractions (n = 57) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to confirm LOH findings.ResultsSmooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. However, we found no evidence for DNA aneuploidy, somatic genetic alterations in the vimentin-positive stromal cell fractions of any samples, while high frequencies of DNA content abnormalities (43/57) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions. LOH hot-spots on chromosomes 3p, 4p and 6p found were confirmed by microsatellite analysis.ConclusionFrom our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Using flow cytometry and SNP-arrays, stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0061-5) contains supplementary material, which is available to authorized users.

The Genomic and Transcriptomic Landscape of a HeLa Cell Line

“…A large potential LOH region in HeLa chromosome 3 is depicted in detail along with allele frequencies, CN, coverage, SNVs, and rearrangements in Figure 2 (plots for all chromosomes are in Figure S2). Many of the homozygous segments in the HeLa genome correspond to previously reported LOH cervical cancer hotspots, namely on chromosomes 3p, 6p, 11q, and 18q (Mitra et al 1994; Mullokandov et al 1996; Rader et al 1998; Koopman et al 2000; Vermeulen et al 2005; Corver et al 2011). This finding suggests that these LOH events arose during the cervical cancer, prior to cultivation of the HeLa cell line.…”

Genetic alterations and epigenetic alterations of cancer-associated fibroblasts

“…Studies that found a positive somatic alteration in CAFs, without exception, have extracted DNA from archival tissues. In other studies (10,47,136,137), CAFs were isolated from fresh frozen tissues or flow cytometry; no somatic genetic alterations were found, while CAFs from FFPE show a high frequency of LOH and AI. This pattern indicates that FFPE tissues could result in highly fragmented DNA and RNA molecules, which are not suitable for large-scale genetic analysis.…”

Carcinoma-associated fibroblasts: orchestrating the composition of malignancy

“…Recent genetic studies conducted in CAFs derived from human ovarian and breast tumors have demonstrated that unlike epithelial cancer cells, copy number alterations, loss of heterozygosity, and/or the number of TP53 mutations are extremely rare in CAFs (4%-5% of samples). Such rare alterations therefore cannot constitute the basis for the widespread tumor-promoting phenotypes exemplified by CAFs (Qiu et al 2008;Hosein et al 2010;Corver et al 2011).…”