Prognostic Impact of FoxP3+ Regulatory T Cells in Relation to CD8+ T Lymphocyte Density in Human Colon Carcinomas

<div><h3>Background</h3><p>T-lymphocyte infiltration into colon carcinomas can influence clinical outcome, and interactions among T cell subsets may be more informative than either subset alone. Our objective was to examine the prognostic impact of tumor-infiltrating FoxP3⁺ regulatory T cells (Tregs) in relation to cytotoxic CD8⁺ T lymphocytes in patients with colon carcinomas characterized by DNA mismatch repair (MMR) status who participated in adjuvant chemotherapy trials.</p> <h3>Methods</h3><p>FoxP3⁺ and CD8⁺ densities in tumor epithelial and stromal compartments were analyzed by immunohistochemistry and quantified in resected, stage II and III colonic carcinomas (N = 216). Immune marker density was dichotomized at the median and categorized as high <em>vs</em> low. MMR status was classified as MMR deficient (dMMR) or proficient (pMMR). Cox models were adjusted for age, stage, and tumor grade.</p> <h3>Results</h3><p>The density of FoxP3+ infiltration was similar in tumor stroma and epithelia, whereas CD8+ was higher in stroma. The prognostic impact of FoxP3+ and CD8+ T cell infiltration was stronger in stroma <em>vs</em> epithelia, and the density of each marker in stroma was independently associated with improved overall survival (OS). However, the impact of FoxP3+ on survival was dependent upon CD8+ density (<em>P</em> interaction  = .040). Among CD8+_low tumors, FoxP3+_high cases had significantly improved OS compared to FoxP3+_low cases after adjustment for covariates (hazard ratio 0.43; 95% confidence interval 0.19 to 0.95; P = .030). In contrast, FoxP3+ was not prognostic among CD8+_high tumors. FoxP3+ remained prognostic in CD8+_low tumors after further adjustment for MMR or <em>BRAF</em>^V600E mutation status. Additionally, these immune markers identified a pMMR subgroup with a similarly favorable OS as for dMMR tumors.</p> <h3>Conclusions</h3><p>The prognostic impact of FoxP3+ and CD8+ T cell density are inter-dependent, whereby FoxP3+ exerts a favorable influence on survival only in colon cancers with low CD8+ infiltration.</p> </div

Clinicopathologic Characteristics of Study Population by Immune Marker Density (N = 216).

a<p>T cell densities were dichotomized at the median.</p>b<p>FoxP3+ (N = 156).</p>c<p>Histologic Grade: 1 or 2, well or moderately differentiated; 3 or 4, poor or undifferentiated.</p

Multivariable Cox Models for Overall Survival Examining CD8+ and FoxP3+ T-cell Density^a.

a<p>Each model is adjusted for age, stage, grade.</p>b<p>CD8+ and FoxP3+ densities were dichotomized at the median.</p><p>Abbreviations: MMR, mismatch repair.</p

Immune marker expression in colon carcinomas.

<p>Expression of FoxP3+ (a) and cytotoxic CD8+ (b) proteins in T lymphocytes, determined by immunohistochemistry, is shown infiltrating the tumor stroma and epithelia of resected colon carcinomas (<i>left</i>, 20× objective; <i>right</i>, 40× objective).</p

Patient survival according to CD8+ and FoxP3+ T-cell density.

<p>Overall survival (OS) in resected colon carcinomas with (a) high <i>vs</i> (b) low density of cytotoxic CD8+ T-cell infiltration in tumor stroma according to FoxP3+ T-cell density in tumor stroma (<i>P</i> for interaction  = .040). Hazard ratios (HR) are adjusted for age, stage, and tumor grade.</p

Patient survival and comparison of clinicopathologic variables by MMR status and T-cell density.

<p>Overall survival (OS) in deficient MMR (dMMR) <i>vs</i> proficient MMR (pMMR) colon carcinomas according to the density of FoxP3+ (a) or CD8+ (b) T lymphocytes in tumor stroma, showing similar OS among cases with dMMR <i>vs</i> pMMR with high densities of CD8+ or FoxP3+. Comparison of clinicopathological variables (c) between dMMR <i>vs</i> CD8+_high or FoxP3+_high pMMR cancers demonstrates differences in histologic grade and tumor site. Grade: 1 or 2, well- or moderately differentiated; 3 or 4, poor or undifferentiated.</p

Multivariable Cox Models for Overall Survival Examining Stromal FoxP3+ T-cell Density Stratified by CD8+ T-cell Density^a.

a<p>CD8+ and FoxP3+ densities were dichotomized at the median.</p>b<p>pMMR (N = 119).</p>c<p>Within each model, hazard ratios and p values are adjusted for all variables shown.</p

Patient and Lesion Characteristics for Stool Study.

<p>Abbreviation: –, not applicable.</p><p>^a Cases comprised patients with at least one SSP (sessile serrated polyp) ≥1 cm found on screening or surveillance colonoscopy and without synchronous advanced adenomas or CRC.</p><p>^b Control patients had no pathology (no CRC, colorectal polyps, hemorrhagic lesions, or inflammation) on screening or surveillance colonoscopy.</p><p>^c Right-sided location was defined as proximal to the splenic flexure.</p><p>^d Patients found to harbor synchronous polyps (adenomatous or serrated) <1 cm in size were included as cases.</p

Detection of SSP by stool assay of <i>mBMP3</i> and by fecal immunochemical testing (FIT).

<p>FIT sensitivity was evaluated at the conventional cutoff of 100/ml buffer (FIT-100) and at 50 ng/ml (FIT-50). Specificity cutoffs for stool DNA marker <i>mBMP3</i> were selected to match those for both FIT-100 and FIT-50 so that sensitivities could be most meaningfully compared.</p

Tissue levels of aberrantly methylated genes.

<p>Tissue levels of methylated <i>BMP3 (mBMP3)</i> and <i>NDRG4 (mNDRG4)</i> genes are compared in normal colorectal mucosa, n = 20 unique control patients, and sessile serrated polyps (SSP), n = 20 unique case patients. Marker levels are normalized to <i>β-actin</i> (a marker of total human DNA) and expressed in relative units. Levels were substantially and significantly higher in SSP than normal colon for both <i>mBMP3</i> (p<0.0001) and <i>mNDRG4</i> (p<0.0001).</p