Association of loss of epithelial syndecan-1 with stage and local metastasis of colorectal adenocarcinomas: An immunohistochemical study of clinically annotated tumors

The tissue microarray (TMA) was custom built and validated as described [26]. The TMA contained a total of 374 cores, from 14 normal colonic epithelia, 15 adenomas and 158 colorectal adenocarcinomas that were diagnosed at The Cleveland Clinic between 1993 and 1999. The majority of the adenocarcinomas were moderately differentiated and six were poorly-differentiated. Tumors were classified according to standard TNM staging guidelines [33]. To minimize sampling errors, two separate large diameter (1.5 mm diameter) tissue cores of each adenocarcinoma were included in the array, totaling a surface area of 3.5 mm² per case. The areas covered by these cores included the edge of each tumor. Each separate tissue core was assigned a unique TMA location number that was linked to a CCF Institutional Review Board-approved (IRB-5085) database containing a mean 38 months of clinical follow-up. From the 316 cores, 131 of the 158 adenocarcinoma samples were available for scoring. Immunohistochemistry was carried out using an automated Ventana Benchmark system as described [26]. Briefly, a 4-μm thick unstained section of each TMA was placed onto an electrostatically charged glass slide and baked for tissue adherence. Slides were pretreated with the recommended pretreatment solution (Ventana) for tissue deparaffinization and antigen retrieval. Slides were incubated with clone B-A38 mouse monoclonal antibody to human syndecan-1/CD138 (Serotec) at 1: 100 dilution, a secondary biotinylated antibody and a streptavidin amplification step. Antigen detection was carried out by peroxidase/3,3'-diaminobenzidine reaction. CD138 immunoreactivity was scored by two independent observers in a blinded procedure without prior knowledge of the clinical information and was classified for the staining of the tumor or the stromal cells, in either the tumor or the stroma, as 0 (less than 5% of cells); 1+ (5% – 25% of cells); 2+ (25% – 75% of cells), or 3+ (more than 75% of cells). Positive staining in plasma cells served as an internal positive control for syndecan-1. Stromal positivity was evaluated for the non-plasma cell stromal elements. In the less than 10% of cases where the opinions of the two evaluators differed, a consensus agreement was reached by re-review of the slides, thorough discussion and if necessary taking the average score. The fascin staining and scoring method has been described [26].

The expression level of CD138 in relation to clinicopathological factors, Ki67 index, or fascin expression was analyzed using chi². Overall survival was defined as that from the date of the operation to the date of death due to cancer. The Kaplan-Meier method was used to determine the probability of survival and data was analyzed with the log-rank test. StatView for Windows version 5 software (SAS Institute, Cary, NC) was used for the analysis. Median survival times were calculated using Dr. SPSS II for Windows version 11.0.1J (SPSS Japan Inc., Tokyo, JAPAN). In the analyses, a p value of <0.05 was considered significant.

In the normal colon, syndecan-1 was expressed around the basolateral membrane of the normal columnar epithelium and in plasma cells. Syndecan-1 staining was absent from the stroma (Fig. 1a). This staining pattern was unchanged in adenomas (not shown). In contrast, in the majority of the adenocarcinomas, syndecan-1 staining was decreased or absent (Fig. 1b,c). Loss of syndecan-1 from tumor cells was most pronounced in the most poorly differentiated tumors (Fig. 1c). Out of 131 patients, in 65 cases syndecan-1 was expressed on less than 25% of the tumor cells, and in 49 cases between 25% to 75% of tumor cells were stained. Only in 17 cases did more than 75% of the tumor cells stain for syndecan-1 (Table 1). On the tumor cells, syndecan-1 was localized around the entire cell membrane and in many cells appeared to be cytoplasmically located (Fig. 1b). The TMA cores included the tumor borders, however we did not detect any consistent specific association of syndecan-1 immunoreactivity with tumor edges.

In contrast to the normal stroma that was typically negative for syndecan-1 (Fig. 1a), some tumor specimens, without or with tumor expression of syndecan-1, had positive staining for syndecan-1 in the stroma. In total, 16.6% of the tumors had some level of stromal syndecan-1 staining: 8.8% of tumors had weak expression and 7.9% had moderate or strong expression. Fig. 1d shows an example of a tumor with moderate tumor syndecan-1 and strong stromal staining for syndecan-1.

The correlation between syndecan-1 immunoreactivity and several clinico-pathological characteristics was investigated. The TMA had been previously validated as representative for traditional prognostic variables of colorectal cancer, including the correlation of tumor stage and metastasis to local lymph nodes with patient survival [26]. The incidence of low expression of syndecan-1 was significantly higher in males (p = 0.042), in tumors of higher TNM stage (p = 0.045), and in patients positive for lymph node metastasis (p = 0.017) (Table 1). There was no correlation between syndecan-1 status and tumor location (Table 1). In cumulative survival analysis, there was no difference of prognosis associated with either low or high syndecan-1 on the tumor cells or in the stroma. This result was obtained when each scoring category was examined individually, or when the samples were compared as two groups (low, corresponding to 0 and 1+ scores versus high, corresponding to 2+ and 3+ scores) using the Kaplan-Meier method. This result was obtained when the analysis for tumor syndecan-1 was applied either to the whole group of stage I-IV cases (p = 0.398), or to the stage III-IV group alone (p = 0.843) (Fig. 2; for clarity of presentation only the data for the low vs. high scoring groupings is shown). We also examined the 5 year survival rate and median survival times for each of the tumor syndecan-1 scoring categories. Again, there was no significant difference in survival between the scoring categories (Table 2).

We had previously examined the same tissue microarray set of tumors for fascin and Ki67 immunostaining. Positive fascin expression correlated with a poor prognosis in stage III/IV patients and a high Ki67 index correlated with TNM stage [26]. Because syndecan-1 and fascin are components of the same pathway that regulates cell motility in non-transformed cells, the syndecan-1 stained samples were evaluated for possible correlations with either fascin status or Ki67 index, with reference to our previously published datasets for these markers. There was no correlation between low expression of syndecan-1 and high fascin expression in tumor cells, either in the whole group or the stage III/IV patients alone. Neither tumor nor stromal syndecan-1 correlated with Ki67 index (data not shown). However, reduced tumor cell staining for syndecan-1 correlated significantly with strong stromal staining for fascin (p = 0.016; Table 3). An example of a syndecan-1 negative tumor with strong stromal staining for fascin is shown in Fig. 1e and 1f.