No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas

Abstract

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinomapromoting phenotypes of breast and ovarian CAFs. © 2008 Nature Publishing GroupCorrespondence should be addressed to I.G.C. ian.campbell@petermac.org. AUTHOR CONTRIBUTIONS I.G.C., I.H. and W.Q. designed the study and wrote the paper. W.Q. undertook the bulk of the experimental work including tissue microdissection, SNP genotyping and microsatellite analysis. K.P. provided cell lines, academic support and assisted in manuscript preparation. A.S., E.R.T., M.R. and K.L.G. assisted in SNP genotyping. However, not all studies have identified genetic alterations in CAFs; for example, one study did not find any clonally selected somatic genetic alterations in CAFs separated from fresh breast cancer biopsies using array comparative genomic hybridization (CGH) and SNP array analysis 17 , although these CAFs were epigenetically distinct from those from normal breast tissue, as demonstrated by subsequent genome-wide DNA methylation studies 18 .The evidence for somatic genetic alterations as important mediators of the CAF phenotype is controversial and conflicting. We hypothesized that the contradictory data may in part be a reflection of inherent technical limitations of the various methodologies used. Therefore, we took advantage of innovative SNP array-based technologies 19 to investigate in detail the genomic integrity of CAFs microdissected from fresh frozen primary human ovarian and breast cancers as well as short-term cultures of primary breast CAFs.We assessed the sensitivity of the Affymetrix 500K SNP array platform to detect copy number and LOH in the context of normal DNA contamination in a mixing experiment using tumor epithelial cell DNA from a microdissected primary ovarian cancer that was mixed with various ratios of matched normal DNA. This tumor harbors a complex copy number profile on chromosome 17, including regions of high level copy number gain and regions of LOH with and without associated copy number loss. As shown in Supplementary Figure 1a online, the single copy number gain was clearly visible at 70% tumor DNA, and the high level gain was still discernible at 25% tumor DNA. LOH w...

Origin of carcinoma associated fibroblasts

“…Our data and the consensus of molecular genetic studies of CAFs where technical artifacts were avoided is that somatic alterations in clonal populations of CAFs are at best exceedingly rare. 9,10 Hypothetically, the idea of co-evolution of two cell lineages in the body that carry independent somatic mutations is not implausible. An altered fibroblast that somehow entices the neighboring epithelia to secrete a support signal for other fibroblasts would be expected to consequently expand further.…”

“…Recent evidence shows that unlike cancer cells, where many changes occur via somatic mutations, the changes in cancer neighboring cells do not stem from somatic mutations. 9,10 We will therefore try to consider the reason for this mechanistic disparity between cancer cells and their neighboring cells. "In describing genetic mechanisms, there is a choice between being inexact and incomprehensible" (Nobel prize committee presentation speech of the 1965 award for the discovery of transcription regulation).…”

Genomic (In)stability of the Breast Tumor Microenvironment

“…Elevated frequencies of AI/LOH in fibroblasts laser-microdissected from patients with BRCA1/2 mutations (59.7%) and sporadic tumors (36.7%) suggest that the fibroblast component may contribute to the instability detected in whole stroma (11), yet assessment of fibroblasts isolated from frozen breast stroma did not support these findings (16). In addition, evaluation of the stromal cell types individually failed to detect copy number alterations (13).…”

“…Finally, copy number analysis conducted on DNA from microdissected epithelial and cancer-associated fibroblasts failed to detect any chromosomal alterations. In addition, microsatellite analysis from a region of chromosome 11 considered a hotspot for LOH in breast stroma from earlier studies was also conducted using these DNA samples and LOH was not detected (16). The inability of these studies to detect copy number changes in breast stromal cells has led to the suggestion that the LOH/AI events detected in stroma from FFPE specimens represent technical artifact.…”

“…A number of additional studies, the majority of which were conducted using microsatellites markers and formalin-fixed, paraffinembedded (FFPE) archival specimens, have also detected genomic instability in tumor-associated stroma, furthering the notion that the tumor microenvironment is a dynamic entity that contributes to tumorigenesis through morphologic and molecular alterations (7)(8)(9)(10)(11)(12). In contrast, recent studies using DNA from fresh frozen tissues that failed to detect chromosomal changes in stromal cell, leading some investigators to speculate that the LOH/AI studies conducted on DNA from FFPE specimens do not accurately reflect the chromosomal content of cells of the tumorassociated stroma but rather are the result of technical artifacts resulting from the use of microsatellite assays and fixation methods (13)(14)(15)(16)(17)(18)(19).…”