Clinical impact of endometrial cancer stratified by genetic mutational profiles, <i>POLE</i> mutation, and microsatellite instability

<div><p>Background</p><p>The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with <i>POLE</i> mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance.</p><p>Materials and methods</p><p>A cohort of 138 EC patients who underwent surgical resection with curative intent was enrolled. Sanger sequencing was used to evaluate mutations in the <i>POLE</i> and <i>KRAS</i> genes. MSI analysis was performed using four mononucleotide repeat markers and methylation status of the <i>MLH1</i> promoter was measured by a fluorescent bisulfite polymerase chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was evaluated by immunohistochemistry (IHC).</p><p>Results</p><p>Extensive hypermethylation of the <i>MLH1</i> promoter was observed in 69.6% ECs with MLH1 deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR genes (<i>P</i> < .0001). MSI-positive and <i>POLE</i> mutations were found in 29.0% and 8.7% EC patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In univariate and multivariate analyses, <i>POLE</i> mutations and <i>MSI</i> status was significantly associated with progression-free survival (<i>P</i> = 0.0129 and 0.0064, respectively) but not with endometrial cancer-specific survival.</p><p>Conclusions</p><p>This study provides significant evidence that analyses of proofreading <i>POLE</i> mutations and MSI status based on mononucleotide repeat markers are potentially useful biomarkers to identify EC patients with better prognosis.</p></div

Association between clinic-pathological features and EC patients stratified by genetic mutational profiles.

<p>Association between clinic-pathological features and EC patients stratified by genetic mutational profiles.</p

Molecular and clinic-pathological features of 138 ECs.

<p>(A) Molecular and clinic–pathological landscape of 138 ECs. Genetic analysis, focusing on frequent hotspot mutations in the POLE gene, and MSI status result in the identification of three molecular subgroups: (1) <i>POLE</i>-mutant, (2) MSI and (3) non-MSI. (B) Progression-free survival and endometrial cancer-specific survival of 138 EC patients stratified by genetic profiles. <i>P</i> values were calculated by the log-rank test.</p

Detection of MSI and distribution of number of MSIs in 138 EC patients.

<p>(A) Example of MSI and non-MSI cases analyzed by four mononucleotide repeat markers (BAT26, NR21, NR27, and CAT25). (B) Association between MSI, <i>POLE</i> mutation, <i>MLH-1</i> promoter methylation and MMR protein expression. The number of mononucleotide repeat markers showing MSI are shown by color.</p

Methylation analysis of the promoter region in the <i>MLH1</i> gene.

<p>(A) Schematic depiction of two regions (5’-region and 3’-region) of the <i>MLH1</i> promoter for methylation and results of a panel of representative fluorescent bisulfite PCR following restriction enzyme analysis. Methylated samples had the new fragment cleaved by the restriction enzyme. (B) The frequencies of <i>MLH1</i> promoter methylation according to <i>MLH1</i> expression status. The top panel shows the results of the <i>MLH1</i>-5’ region, the middle panel shows the <i>MLH1</i>-3’ region and the bottom panel shows partial (i.e. only <i>MLH1</i>-5’ methylation) and extensive methylation (i.e. both <i>MLH1</i>-5’ and -3’ methylation).</p

Univariate and multivariate outcome analyses of 138 EC patients.

<p>Univariate and multivariate outcome analyses of 138 EC patients.</p

Representative examples of immunohistochemistry staining for the four MMR proteins.

<p>Tumors with MLH1-deficiency (dMLH1) show negative expression in both MLH1 and PMS2 IHC, those with MSH2-deficiency (dMSH2) show negative expression in both MSH2 and MSH6 IHC, with PMS2-deficiency (dPMS2) they show negative expression only in PMS2, and tumors with MSH6-deficiency show negative expression only in MSH6.</p

MOESM1 of Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors

Additional file 1: Table S1. Primers for tetra-mononucleotide repeat PCR of tumors (Tetraplex) system