DTF and SFT cDNA Microarray procedures
42,000 spot cDNA microarrays were used to measure the relative mRNA
expression levels in the tumors. The details of isolating mRNA,
labeling and hybridizing are described elsewhere[11]. The raw data
files are available at Stanford Microarray
Database, the filtered data used for the manuscript are available
at the accompanying web site (https://tma.im/tma_portal/DTF_SFTbreast). Data was filtered using the following criteria: only cDNA spots with a ratio of signal over background of at least 1.5 in both the Cy3 and the Cy5 channel were included; only cDNAs were selected that had an absolute value at least four times greater in at least two arrays than the geometric mean; and only cDNA spots that full-fill these criteria on at least 70% arrays were included. Data was evaluated with unsupervised hierarchical clustering and significance analysis of microarrays (SAM)
Analysis of Breast carcinoma dataset
The gene array data set for breast carcinoma contained 295 tumors arrayed on 25,000 spot oligo nucleotide arrays as described elsewhere[15]. In short: patients were all diagnosed and treated in the Netherlands Cancer Institute for early breast cancer (Stage I and II) between 1984 and 1995. The median follow up for patients alive is 7.8 years. Additional clinical data can be found in Supplemental Table 4.
For DTF and SFT, genes were identified that were highly expressed in either of the two tumor types by using SAM[13]. 1,010 spots satisfied the gene filtering criteria mentioned above in the clustering of the DTF and SFT tumors. The criterion for SAM was set to yield 0.1% false-positive data. A list of 786 clones was obtained, that consistent of 493 genes positively identifying fibromatosis and 293 genes positively identifying SFT. Equal numbers of DTF and SFT clones were chosen for breast carcinoma analysis and clones having the same Unigene locus were removed resulting in 237 unique gene sequences identifying DTF and 246 unique gene sequences identifying SFT. These gene sequences were mapped to spots on the NKI array using Unigene build 172 (release date July 17th 2004) to give 471 unique spots. Gene measurements were mean centered. The resulting dataset was subjected to hierarchical clustering with average linkage clustering.
Overall survival (OS) was defined by death from any cause. In this cohort of young breast cancer patients only 6 patients died of cause other than breast cancer (5 second primaries and 1 cardiovascular). Distant metastasis-free survival (DMFS) was defined by a distant metastasis as a first recurrence event; data on all patients were censored on the date of the last follow-up visit, death from causes other than breast cancer, the recurrence of local or regional disease, or the development of a second primary cancer, including contra-lateral breast cancer. Kaplan-Meier survival curves were compared by the Cox-Mantel log-rank test in Winstat® for MS Excel® (R. Fitch Software, Germany). Multivariate analysis by the Cox proportional hazard method was performed using the software package SPSS® 11.5 (SPSS, Inc.).
Tissue microarray construction
A tissue microarray of fibroblastic conditions was constructed using a
manual tissue arrayer (Beecher Instruments, Silver Spring, MD)
following previously described techniques[23] with modifications.
Briefly, certain specimens, such as skin and fistula tract, contained
tissues whose positional orientation was important for analysis.
Coring of these tissues could lose orientation of the cells within the
core. Therefore, orientation sensitive material was dissected from
the original blocks and re-embedded into the paraffin block used for
tissue arraying. Tissues thus embedded included skin, lung, breast,
granulation tissue and fistula tract (Supplemental Figure 2). After
the embedding process was completed, construction of the tissue array
was performed using single 2 mm cores. In addition, the TMA contained
0.6 mm cores of lobular (14) and ductal (10) breast carcinomas,
fibroadenomas (11), SFT (5), DTF (5), and colorectal carcinomas (2),
scar (1) and keloid (1). All samples were obtained from archived
material at the Stanford University Medical Center Department of
Pathology between 2001 and 2004 with IRB approval. The cores were
taken from areas in the paraffin block that were representative of the
diagnostic tissue.
Immunohistochemistry
Serial sections of 4 µM were cut from the TMA blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. The slides were pretreated with citrate buffer and a microwave step. Staining was then performed using the DAKO EnVision+ System, Peroxidase (DAB), (DAKO, Cambridgeshire, UK). For Apo D (Clone 36C6, 1:40 dilution, Novocastra, Newcastle, UK), CD34 (1:20 dilution, BD Biosciences, San Diego, CA, USA), and BCL2 (1:800 dilution, DAKO Cytomation, Carpinteria, CA, USA) stains. Results were interpreted as follows: staining was interpreted as negative when no more than 5% of the spindled stromal cells showed light staining. A score of "weak positive" was given for light brown staining in greater than 5% of the spindled stromal cells. A score of "strong positive" was given for staining in greater than 50% of the spindled stromal cells. Cores in which no diagnostic material was present were omitted from further analysis. The cores were initially reviewed independently by two pathologists (RW and MvdR) and disagreements were reviewed together to achieve a consensus score. Scoring of the arrays was analyzed using the Deconvoluter software as previously described (ref Chih Long), with each sample receiving the highest score for either of the two cores.
In situ hybridization
In situ hybridization of TMA sections was performed based on a protocol published previously (ref DOG1, Oncogene GIST). Briefly, digoxigenin (DIG)-labeled sense and anti-sense RNA probes are generated by PCR amplification of 400 to 600 bp products with the T7 promoter incorporated into the primers. In vitro transcription was performed with a DIG RNA-labeling kit and T7 polymerase according to the manufacturer's protocol (Roche Diagnostics, Indianapolis, IN, USA). 5um thick sections cut from the paraffin blocks, deparaffinized in xylene, were hydrated in graded concentrations of ethanol for 5 minutes each. Sections were then incubated with 1% hydrogen peroxide, followed by digestion in 10ug/ml of proteinase K at 37oC for 30 minutes. Sections were hybridized overnight at 55oC with either sense or antisense riboprobes at 200ng/ml dilution in mRNA hybridization buffer (DAKO, Cambridgeshire, UK). The following day, sections were washed in 2xSSC and incubated with a 1:35 dilution of RNase A cocktail (Ambion, Austin, TX, USA) in 2xSSC for 30minutes at 37oC. Next, sections were stringently washed in 2X SSC/50% formamide twice, followed by one wash at 0.08X SSC at 50 oC. Biotin blocking reagents (DAKO, Cambridgeshire, UK) were applied to the section to block the endogenous biotin. For signal amplification, a HRP-conjugated rabbit anti-DIG antibody (DAKO, Cambridgeshire, UK) was used to catalyze the deposition of biotinyl tyramide, followed by secondary streptavidin complex (GenPoint kit; DAKO, Cambridgeshire, UK). The final signal was developed with DAB (GenPoint kit; DAKO, Cambridgeshire, UK), and the tissues were counterstained in hematoxylin for 15 seconds.
