Prognostic Significance and Gene Expression Profiles of p53 Mutations in Microsatellite-Stable Stage III Colorectal Adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets

Cox regression analysis to determine prognostic significance of <i>p53</i> mutations.

1<p>Adjusted for the <i>p53</i> mutations, age, gender, tumor location, tumor grade, tumor size, tumor depth of invasion, nodal involvement, and adjuvant therapy.</p

IHC expression patterns of up-regulated genes in <i>p53</i> mutant phenotypes of MS CRCs.

<p><b>a</b>, normal colonic epithelium (NCE) demonstrating moderate cytoplasmic (thick arrow) and weak membrane (thin arrow) FUT3 staining (400 µm). <b>b</b>, CRCs exhibiting strong cytoplasmic (thick arrows) and moderate to weak membrane FUT3 staining (thin arrows) (400 µm). <b>c</b>, CRCs with weak cytoplasmic staining (thin arrows) (400 µm). <b>d</b>, NCE demonstrating weak cytoplasmic TRIM29 staining with a focal punctuate pattern (thin arrows) (400 µm). <b>e</b>, CRCs exhibiting moderate to strong cytoplasmic TRIM29 staining with a punctate pattern on the luminal aspect (thin arrows) (600 µm). <b>f</b>, CRCs with weak cytoplasmic TRIM29 staining (thin arrows) (400 µm). <b>g</b>, NCE demonstrating weak cytoplasmic to complete lack of IQGAP3 staining. [Note: Lymphocytes in the stroma show moderate cytoplasmic staining (thin arrows); the adjacent tumor demonstrates moderate cytoplasmic and nuclear immunostaining (thick arrows) (400 µm)]. <b>h</b>, CRCs exhibiting strong cytoplasmic IQGAP3 staining (thick arrows) (400 µm). <b>i</b>, CRCs with lack of staining for IQGAP3 (thick arrows) (400 µm). <b>j</b>, NCE demonstrating moderate cytoplasmic staining of SLC6A8 staining (thin arrows) (600 µm). <b>k</b>, CRCs exhibiting strong cytoplasmic SLC6A8 staining with luminal accentuation (thick arrows) (600 µm). <b>l</b>, CRCs negative for SLC6A8 staining (thick arrows) (600 µm).</p

The prognostic significance of <i>p53</i> mutations in survival of Stage III CRC patients with MSS or MSI-H phenotypes (Kaplan-Meier survival curves).

<p><i>p53</i> mutations were associated with worse patient survival (log-rank, <i>P</i> = 0.025) (<b>A</b>) than wt-<i>p53</i> in the subset of MSS phenotype, but not in the subset of MSI-H phenotype (log-rank, <i>P</i> = 0.695) (<b>B</b>).</p

IHC expression patterns of down-regulated genes in <i>p53</i> mutant phenotypes of MS CRCs.

<p><b>a</b>, NCE demonstrating moderate nuclear and weak cytoplasmic PDLIM3 staining [Note: Lymphocytes in the stroma and the normal epithelial cells show moderate nuclear staining (thin arrows); the adjacent tumor demonstrates strong nuclear and moderate cytoplasmic immunostaining (thick arrows) (400 µm)]. <b>b</b>, CRCs exhibiting lack of PDLIM3 immunostaining (thin arrows) (600 µm). <b>c</b>, CRCs with strong nuclear and weak cytoplasmic PDLIM3 staining (thick arrows) (600 µm). <b>d</b>, NCE demonstrating weak cytoplasmic LPAR6 staining (thin arrows) (600 µm). <b>e</b>, CRCs exhibiting focal weak cytoplasmic LPAR6 staining (thin arrows) (600 µm). <b>f</b>, CRCs with weak to moderate cytoplasmic (thick arrows) and focal nuclear LPAR6 staining (thin arrows) (600 µm). <b>g</b>, NCE demonstrating moderate to strong cytoplasmic PLAT staining (thin arrows) (600 µm). <b>h</b>, CRCs exhibiting focal moderate cytoplasmic PLAT staining (thick arrows) (600 µm). <b>i</b>, CRCs with moderate to strong staining for PLAT (thick arrows) (600 µm).</p

Gene expression profiles of CRCs based on <i>p53</i> status.

<p>Genes and samples were clustered independently by hierarchical clustering. Rows represent genes, and columns represent samples, which are color-coded by <i>p53</i> status (blue and red correspond to wt-<i>p53</i> and <i>p53</i> mutant, respectively. The color scale is shown at bottom right. Values are expressed as log2-ratios of expression in CRCs with <i>p53</i> mutant phenotypes to that in wt-<i>p53</i> phenotypes. (A & B) Clusters of 35 and 49 genes showing consistent up-regulation and down-regulation in <i>p53</i> mutant phenotypes.</p

Validation of deferentially expressed genes by qNPA and qRT-PCR based on <i>p53</i> status in Stage III-MSS CRCs.

<p>Abbreviations: Ch, chromosome number; AHG, Affymetrix human genome plus 2.0 array.</p>1<p>Fold change in differential expression of genes by AHG;</p>2<p>Fold change by qNPA;</p>3<p>Fold change qRT-PCR.</p