SPARC, FOXP3, CD8 and CD45 Correlation with Disease Recurrence and Long-Term Disease-Free Survival in Colorectal Cancer

BACKGROUND: SPARC is a matricellular protein involved in tissue remodelling, cell migration and angiogenesis, while forkhead box P3 (FOXP3) protein functions as a transcription factor involved in immune cell regulation. Both SPARC and FOXP3 can play an anti-tumorigenic role in cancer progression. The aim was to determine if SPARC, FOXP3, CD8 and CD45RO expression levels are associated with colorectal cancer (CRC) stage, disease outcome and long-term cancer-specific survival (CSS) in stage II and III CRC. METHODS AND FINDINGS: SPARC expression was initially assessed in 120 paired normal and stage I-IV CRCs. Subsequently, approximately 1000 paired patient samples of stage II or III CRCs in tissue microarrays were stained for SPARC, FOXP3, CD8 or CD45RO. Proportional hazards modelling assessed correlations between these markers and clinicopathological data, including disease outcome and cancer specific survival (CSS). Both SPARC and FOXP3 expression were significantly greater in CRC than normal colon (p<0.0001). High SPARC expression correlated with good disease outcome (≥60 mths without disease recurrence, p = 0.0039) and better long-term CSS in stage II CRC (<0.0001). In stage III CRC, high SPARC expression correlated with better long-term CSS (p<0.0001) and less adjuvant chemotherapy use (p = 0.01). High FOXP3 correlated with a good disease outcome, better long-term CSS and less adjuvant chemotherapy use in stage II (p<0.0037, <0.0001 and p = 0.04 respectively), but not in stage III CRC. High CD8 and CD45RO expression correlated with better disease outcome in stage II CRC, and better CSS, but the differences were not as marked as for SPARC and FOXP3. CONCLUSIONS: These data suggest that high SPARC and FOXP3 are associated with better disease outcome in stage II CRC and may be prognostic indicators of CSS. Further assessment of whether these markers predict patients at high risk of recurrence with stage II CRC and functional studies of these effects are underway

Cancer-specific survival.

<p>The mean SPARC and FOXP3 expression in the primary tumour was measured and correlated with disease recurrence. The number of patients with or without disease recurrence at each time point is displayed below each graph. (<b>A</b>) <b><i>SPARC expression in stage II CRC</i></b><i>.</i> Patients without disease recurrence had significantly higher SPARC levels at all time point up to 60 months and for most points up to 138 months compared to those patients who suffered a disease recurrence. The overall SPARC expression was significantly different between the two groups (p<0.0001). (<b>B</b>) <b><i>SPARC expression in stage III CRC</i></b><i>.</i> SPARC expression was significantly higher in patients without disease recurrence from 84 months and beyond. SPARC was also significantly different between the two groups (p<0.0001). (<b>C</b>) <b><i>FOXP3 expression in stage II CRC</i></b><b>.</b> Higher FOXP3 levels was also significantly associated with less disease recurrence as with (<b>D</b>) <b><i>FOXP3 expression in stage III CRC</i></b><b>,</b> however, the differences were not as marked as in stage II CRC. FOXP3 expression was significantly different between the two groups in both stage II and III (p<0.0001). Each data point represented the mean SPARC or FOXP3 expression level at each time point. (*) p<0.05, (**) p<0.01.</p

T cell density and disease outcome.

<p>When dividing the patients into good and poor disease outcome, FOXP3 expression was significantly greater in patients with a good disease outcome in (<b>A</b>) stage II CRC but not in (<b>B</b>) stage III CRC. Similarly, CD8 was also significantly greater in patients with a good disease outcome in (<b>C</b>) stage II CRC but not in (<b>D</b>) stage III, as with CD45RO in (<b>E</b>) stage II and (<b>F</b>) stage III. Each dot represents one patient and the bars represent the median value ± SE.</p

Tissue microarray analysis.

<p>SPARC and vimentin staining from a single patient. Images demonstrate serial DAB-stained tissue sections for both (<b>A</b>) SPARC and (<b>C</b>) vimentin and computer analysed images of the respective DAB staining (<b>B and C</b>). Blue represents DAB-negative pixels (dark areas), orange DAB-positive pixels and red strongly DAB-positive pixels (light areas). SPARC expression was selectively measured only in the regions of the tissue that were also vimentin-positive.</p

Overall survival.

<p>Kaplan-Meier survival curves were derived to show high and low marker levels in relation to survival. (<b>A</b>) High SPARC levels significantly correlated with better long-term survival. (<b>B</b>) High FOXP3 levels also significantly correlated with better long-term survival. Although high (<b>C</b>) CD8 and (<b>D</b>) CD45RO expression levels were associated with better long-term survival, the separation and significance between the curves were not as great.</p

Adjuvant chemotherapy.

<p>The mean SPARC, FOXP3, CD8 or CD45RO expression levels in patients who did, or did not, receive adjuvant chemotherapy in stage II and III CRC. SPARC was significantly greater in patients with stage III CRC who did not require chemotherapy, whilst FOXP3 was significantly greater in patients with stage II CRC who did receive chemotherapy.</p

SPARC expression in stage II and III CRC using TMAs.

<p>(<b>A</b>) The SPARC to vimentin ratio was significantly greater in CRC tissue compared to normal colonic tissue. (<b>B</b>) No difference in SPARC expression was observed between stage II and III CRC. (<b>C</b>) SPARC was significantly greater in patients with a good disease outcome in stage II CRC, but not in (<b>D</b>) stage III CRC. Each dot represents one patient and the bars represent the median value ± SEM.</p