Immunological profiling of molecularly classified high-risk endometrial cancers identifies POLE-mutant and microsatellite unstable carcinomas as candidates for checkpoint inhibition

Abstract

High-risk endometrial cancer (EC) is an aggressive disease for which new therapeutic options are needed. Aims of this study were to validate the enhanced immune response in highly mutated ECs and to explore immune profiles in other EC subgroups. We evaluated immune infiltration in 116 high-risk ECs from the TransPORTEC consortium, previously classified into four molecular subtypes: (i) ultramutated POLE exonuclease domain-mutant ECs (POLE-mutant); (ii) hypermutated microsatellite unstable (MSI); (iii) p53-mutant; and (iv) no specific molecular profile (NSMP). Within The Cancer Genome Atlas (TCGA) EC cohort, significantly higher numbers of predicted neoantigens were demonstrated in POLE-mutant and MSI tumors compared with NSMP and p53-mutants. This was reflected by enhanced immune expression and infiltration in POLE-mutant and MSI tumors in both the TCGA cohort (mRNA expression) and the TransPORTEC cohort (immunohistochemistry) with high infiltration of CD8⁺ (90% and 69%), PD-1⁺ (73% and 69%) and PD-L1⁺ immune cells (100% and 71%). Notably, a subset of p53-mutant and NSMP cancers was characterized by signs of an antitumor immune response (43% and 31% of tumors with high infiltration of CD8⁺ cells, respectively), despite a low number of predicted neoantigens. In conclusion, the presence of enhanced immune infiltration, particularly high numbers of PD-1 and PD-L1 positive cells, in highly mutated, neoantigen-rich POLE-mutant and MSI endometrial tumors suggests sensitivity to immune checkpoint inhibitors.

Keywords: Checkpoint inhibition; endometrial cancer; high-risk; molecular classification; tumor-infiltrating lymphocytes.

Figure 1.

Infiltration of CD3⁺, CD8⁺, CD103⁺ and CD27⁺ cells in POLE-mutant, MSI, NSMP and p53-mutant endometrial cancers. (A) Representative immunohistochemical stainings of CD3⁺, CD8⁺, CD103⁺ and CD20⁺ cells. (B) Average number of positively stained intratumoral cells for each of the markers in the above panel, counted per core, corrected for the number of cells present. (C) Average number of positively stained cells for each of the markers in the above panel, counted per core within the infiltrative margin, corrected for the number of cells present. The numbers of cases analyzed for each molecular subgroup are listed below the x-axis. Boxes represent the interquartile range (IQR), with the upper whisker indicating the 75th percentile and the lower whisker the 25th percentile. The median and mean values are indicated by a horizontal line and cross, respectively. Abbreviations: POLE, POLE-mutant; MSI, microsatellite unstable; NSMP, no specific molecular profile; p53, p53-mutant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Infiltration of TIA-1⁺, T-Bet⁺, CD20⁺ and CD45RO⁺ cells in POLE-mutant, MSI, NSMP and p53-mutant endometrial cancers. (A) Representative immunohistochemical stainings of CD45RO⁺, CD27⁺, T-Bet⁺ and TIA-1⁺ cells. (B) Average number of positively stained intratumoral cells for each of the markers in the above panel, counted per core within the tumor center, corrected for the number of cells present. (C) Average number of positively stained cells for each of the markers in the above panel, counted per core within the infiltrative margin, corrected for the number of cells present. The numbers of cases analyzed for each molecular subgroup are listed below the x-axis. Boxes represent the interquartile range (IQR), with the upper whisker indicating the 75th percentile and the lower whisker the 25th percentile. The median and mean values are indicated by a horizontal line and cross, respectively. Abbreviations: POLE, POLE-mutant; MSI, microsatellite unstable; NSMP, no specific molecular profile; p53, p53-mutant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Infiltration of PD-1⁺ and PD-L1⁺ cells in POLE-mutant, MSI, NSMP and p53-mutant endometrial cancers. (A) Representative immunohistochemical stainings of PD-1⁺ and PD-L1⁺ cells. (B) Average number of PD1⁺ cells counted per core within the tumor center, corrected for the number of cells present. (C) Percentage of PD-L1⁺ tumor-infiltrating immune cells within the tumor core and infiltrative margin core. (D) Average number of PD1⁺ stained cells counted per core within the infiltrative margin. The numbers of cases analyzed for each molecular subgroup are listed below the x-axis. Boxes represent the interquartile range (IQR), with the upper whisker indicating the 75th percentile and the lower whisker the 25th percentile. The median and mean values are indicated by a horizontal line and cross, respectively. Abbreviations: POLE, POLE-mutant; MSI, microsatellite unstable; NSMP, no specific molecular profile; p53, p53-mutant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Immunofluorescent stainings of PD-1, PD-L1 and myeloid markers. Representative image of a POLE-mutant endometrial cancer stained with keratin (green)–CD163 (blue)–CD68 (red) in (A), and PD-1 (green)–PD-L1 (blue) in (B). The two triple immunofluorescent stainings from A and B, performed on sequentially cut slides, are layered in (C), with single channel markers for the inset in (D), with keratin (green), PD-L1 (blue), CD68 (red) and CD163 (yellow), demonstrating the co-localization of PD-L1 with myeloid markers.

Figure 5.

Expression of immune markers in according to tumor molecular subtype in TCGA series. RSEM normalized RNAseq data were log2 transformed and analyzed according to tumor molecular subtype. Boxes represent the interquartile range (IQR), with the upper whisker indicating the 75th percentile and the lower whisker the 25th percentile. The median and mean values are indicated by a horizontal line and cross, respectively. Abbreviations: POLE, POLE-mutant; MSI, microsatellite unstable; NSMP, no specific molecular profile; p53, p53-mutant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Predicted number of HLA-A2-binding neoantigens across the four molecular subgroups in The Cancer Genome Atlas endometrial cancer cohort. (A) Comparison between the number of predicted HLA-A2 binding neoantigens in POLE-mutant, MSI, NSMP and TP53-mutant subgroups based on RNAseq. (B) Comparison between patients with high and low infiltration (based on CD8A expression from RNAseq, relative to median within the group) of lymphocytes within POLE-mutant, MSI, NSMP and TP53-mutant subgroups. The numbers of cases analyzed for each molecular subgroup are listed below the x-axis. Boxes represent the interquartile range (IQR), with upper whisker indicating the 75th percentile and the lower whisker the 25th percentile. The median and mean values are indicated by a horizontal line and cross, respectively. Abbreviations: POLE, POLE-mutant; MSI, microsatellite unstable; NSMP, no specific molecular profile; p53, p53-mutant. *p < 0.05, **p < 0.01, ***p < 0.001.