Mismatch repair-deficient (MMRd) cancers have varied responses to immune checkpoint blockade (ICB). We conducted a phase 2 clinical trial of the PD-1 inhibitor pembrolizumab in 24 patients with MMRd endometrial cancer (NCT02899793). Patients with mutational MMRd tumors (6 patients) had higher response rates and longer survival than those with epigenetic MMRd tumors (18 patients). Mutation burden was higher in tumors with mutational MMRd compared to epigenetic MMRd; however, within each category of MMRd, mutation burden was not correlated with ICB response. Pre-treatment JAK1 mutations were not associated with primary resistance to pembrolizumab. Longitudinal single-cell RNA-seq of circulating immune cells revealed contrasting modes of anti-tumor immunity for mutational vs. epigenetic MMRd cancers. Whereas effector CD8+ T cells correlated with regression of mutational MMRd tumors, activated CD16+ NK cells were associated with ICB-responsive epigenetic MMRd tumors. These data highlight the interplay between tumor-intrinsic and extrinsic factors that influence ICB response.

<div>Abstract<p>Mismatch repair–deficient (MMRd) cancers have varied responses to immune-checkpoint blockade (ICB). We conducted a phase II clinical trial of the PD-1 inhibitor pembrolizumab in 24 patients with MMRd endometrial cancer (NCT02899793). Patients with mutational MMRd tumors (6 patients) had higher response rates and longer survival than those with epigenetic MMRd tumors (18 patients). Mutation burden was higher in tumors with mutational MMRd compared with epigenetic MMRd; however, within each category of MMRd, mutation burden was not correlated with ICB response. Pretreatment <i>JAK1</i> mutations were not associated with primary resistance to pembrolizumab. Longitudinal single-cell RNA-seq of circulating immune cells revealed contrasting modes of antitumor immunity for mutational versus epigenetic MMRd cancers. Whereas effector CD8⁺ T cells correlated with regression of mutational MMRd tumors, activated CD16⁺ NK cells were associated with ICB-responsive epigenetic MMRd tumors. These data highlight the interplay between tumor-intrinsic and tumor-extrinsic factors that influence ICB response.</p>Significance:<p>The molecular mechanism of MMRd is associated with response to anti–PD-1 immunotherapy in endometrial carcinoma. Tumors with epigenetic MMRd or mutational MMRd are correlated with NK cell or CD8⁺ T cell–driven immunity, respectively. Classifying tumors by the mechanism of MMRd may inform clinical decision-making regarding cancer immunotherapy.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-2-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 247</a></i></p></div>

<div>Abstract<p>Mismatch repair–deficient (MMRd) cancers have varied responses to immune-checkpoint blockade (ICB). We conducted a phase II clinical trial of the PD-1 inhibitor pembrolizumab in 24 patients with MMRd endometrial cancer (NCT02899793). Patients with mutational MMRd tumors (6 patients) had higher response rates and longer survival than those with epigenetic MMRd tumors (18 patients). Mutation burden was higher in tumors with mutational MMRd compared with epigenetic MMRd; however, within each category of MMRd, mutation burden was not correlated with ICB response. Pretreatment <i>JAK1</i> mutations were not associated with primary resistance to pembrolizumab. Longitudinal single-cell RNA-seq of circulating immune cells revealed contrasting modes of antitumor immunity for mutational versus epigenetic MMRd cancers. Whereas effector CD8⁺ T cells correlated with regression of mutational MMRd tumors, activated CD16⁺ NK cells were associated with ICB-responsive epigenetic MMRd tumors. These data highlight the interplay between tumor-intrinsic and tumor-extrinsic factors that influence ICB response.</p>Significance:<p>The molecular mechanism of MMRd is associated with response to anti–PD-1 immunotherapy in endometrial carcinoma. Tumors with epigenetic MMRd or mutational MMRd are correlated with NK cell or CD8⁺ T cell–driven immunity, respectively. Classifying tumors by the mechanism of MMRd may inform clinical decision-making regarding cancer immunotherapy.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-2-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 247</a></i></p></div>

<p>Supplementary Figure S1: Additional characterization of the clinical trial cohort.Supplementary Figure S2: Additional characterization of exome profiles and defining MLH1 methylation status.Supplementary Figure S3: JAK1 alterations are most frequent in endometrial cancer.Supplementary Figure S4: Final cell counts in each scRNA-seq sample.Supplementary Figure S5: Longitudinal transcriptional profiling of circulating immune cells before and after PD-1 blockade.Supplementary Figure S6: Differential abundance of cell neighborhoods after PD-1 immunotherapy.Supplementary Figure S7: Differential abundance of cell neighborhoods in JAK1-mutant tumors.Supplementary Figure S8: Transcriptional profiles of T and NK cells in responders vs non-responders.Supplementary Figure S9: Gene expression changes in T and NK cells following PD-1 immunotherapy.Supplementary Figure S10: Additional transcriptional characterization of CD16+ NK cells in epiR patients.</p>

<p>Supplementary Table S1: Genomic and clinical characteristics of the study cohort.</p>