Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans

Abstract

Dermatofibrosarcoma protuberans (DFSP) is an aggressive spindle cell neoplasm. It is associated with the chromosomal translocation, t(17:22), which fuses the COL1A1 and PDGFbeta genes. We determined the characteristic gene expression profile of DFSP and characterized DNA copy number changes in DFSP by array-based comparative genomic hybridization (array CGH). Fresh frozen and formalin-fixed, paraffin-embedded samples of DFSP were analyzed by array CGH (four cases) and DNA microarray analysis of global gene expression (nine cases). The nine DFSPs were readily distinguished from 27 other diverse soft tissue tumors based on their gene expression patterns. Genes characteristically expressed in the DFSPs included PDGF beta and its receptor, PDGFRB, APOD, MEOX1, PLA2R, and PRKCA. Array CGH of DNA extracted either from frozen tumor samples or from paraffin blocks yielded equivalent results. Large areas of chromosomes 17q and 22q, bounded by COL1A1 and PDGF beta, respectively, were amplified in DFSP. Expression of genes in the amplified regions was significantly elevated. Our data shows that: 1) DFSP has a distinctive gene expression profile; 2) array CGH can be applied successfully to frozen or formalin-fixed, paraffin-embedded tumor samples; 3) a characteristic amplification of sequences from chromosomes 17q and 22q, demarcated by the COL1A1 and PDGF beta genes, respectively, was associated with elevated expression of the amplified genes.

Figure 1.

Hematoxylin and eosin staining of a representative case of DFSP (STT491) (A), and a cellular fibrous histiocytoma (STT169) (D), with confirmatory immunohistochemical staining for CD34 (B and E), and APOD (C and F). The DFSP is positive for CD34 (B), while the cellular fibrous histiocytoma (E) shows CD34 positivity in endothelial cells only, but not in tumor cells. IHC for APOD is strongly positive in DFSP (C), while the FH fails to stain with antibodies against APOD (F) despite relative high levels of APOD mRNA.

Figure 2.

Hierarchical cluster analysis of gene expression profiles of 36 tumors, including nine DFSPs. Each row represents the relative level of expression for a single gene, centered at the geometric mean of its expression level across the 36 samples. Each column shows the expression levels for a single sample. The red or green colors indicate high or low expression, respectively, relative to the mean. The intensity of the colors indicates the magnitude of deviation from the mean (see color bar). Gray denotes missing data. A: Overview of expression pattern of the 4687 genes used for hierarchical clustering analysis. The black vertical bar highlights a cluster of 465 genes that are characteristically expressed in DFSP. The yellow, green, and blue bars indicate the areas shown in more detail in C, D, and E. B: The same clustering analysis shown in A but with only a subset of the gene expression data displayed, consisting of genes most characteristic of the DFSPs. At the top is the dendrogram depicting the relatedness between tumor samples, with short branches denoting a high degree of relatedness (see correlation coefficient bar). DFSP is shown in blue. Leio, leiomyosarcoma; MFH, malignant fibrous histiocytoma; N.Fasciitis, nodular fasciitis; Fibr. Histiocytoma, cellular fibrous histiocytoma (in green); Synsarc, synovial sarcoma; GIST, gastrointestinal stromal tumor. Gene names are shown on the right. C: Relative mRNA levels in the 36 tumors for a set of 98 genes (correlation coefficient 0.82) that clustered with the PDGFβ gene. D: Expression level of CD34, measured by two separate spots on the arrays. E: Expression level of CD68, measured by three separate spots on the arrays.

Figure 3.

Pairwise comparison of array CGH on three cases of DFSP with DNA isolated from fresh frozen tumor and from formalin-fixed, paraffin-embedded tumor showing nearly identical regions of amplification on chromosomes 17 and 22. In contrast, five cases of solitary fibrous tumor show no significant changes on these chromosomes. Genes are arranged in order along the chromosome. Red indicates gene amplification, green indicates gene deletion. A: Magnified view of start of amplification region on chromosome 17. Note that this area starts at the location of COL1A1, one of the fusion partners in the t(17:22). B: Magnified view of start of amplification region on chromosome 22, starting at the location of PDGFβ, the other fusion partner in the t(17:22).

Figure 4.

Comparison of aCGH data of chromosomes 17 and 22 in four DFSP with mRNA levels of same genes relative to aCGH data and expression levels in SFT.

Figure 5.

Array-based DNA copy number profiles of DFSP STT154 (A) and fibrous histiocytoma 089 (B). Individual cDNAs are arranged according to their known location along human chromosomes, to produce a representation of tumor DNA copy number changes mapped onto the normal human genome. Each vertical line (red or green) indicates the position of a single cDNA clone, and the length of the line corresponds to the log₁₀ fluorescence ratio of tumor versus reference DNA for that locus, reported as a moving average of five adjacent clones. Red lines indicate areas of amplification and green lines indicate areas of deletion. A: The DFSP shows characteristic increase in material on chromosomes 17 and 22. B: The fibrous histiocytoma shows absence of significant amplification of chromosomes 17 and 22. Raw data from all paraffin DNA aCGH experiments can be downloaded from SMD, and figures of all aCGH experiments and histology in this paper can be seen at http://genome-www.stanford.edu/DFSP/.