Characterization of shared neoantigens landscape in Mismatch Repair Deficient Endometrial Cancer

Abstract

Endometrial cancer (EC) with Mismatch Repair deficiency (MMRd) is characterized by the accumulation of insertions/deletions at microsatellite sites. These mutations lead to the synthesis of frameshift peptides (FSPs) that represent tumor-specific neoantigens (nAg) proved to be shared across patients/tumors with MMRd. In this study, we explored the feasibility of a nAg-based cancer vaccination design in EC with MMRd. We adopted a whole exome sequencing approach and ad hoc bioinformatics pipelines to characterize FSPs in 35 patients with EC. A mean of 146 mutated mononucleotide repeats (MNRs) was identified with enrichment in the patients' group with MLH1 impairment. A high coverage emerged from the comparative analysis of the EC FSPs with the content of the previously validated NOUS-209 vaccine. We obtained pieces of evidence of FSPs translation as expressed proteins from Ribo-seq, supporting the potential as the target of vaccination. The development of a nAgs-based vaccine strategy in MMRd EC may be further explored.

Fig. 1. Workflow of the experimental design of the study.

In the figure is reported the workflow utilized to perform the look-up analysis in the 35 EC MSI patients and in the AN3CA cell line. EC endometrial cancer, MSI-h microsatellite instability-high, MNR mononuclear repeats, FSP frameshift peptides, WES Whole Exome Sequencing.

Fig. 2. Landscape of mutated MNR identified in the 35 MSI-h EC cohort.

a Blue bars indicate the total number of mutated MNRs identified in the cohort of 35 EC patients, according to the IHC MMR results, with mean for each group. b Boxplots represent the distribution and the abundance of the out-of-frame indels detected at mutated MNRs. The EC patients are shown as NOU-1 to NOU-35. c Scatterplot reporting the distribution of genes with respect to their frequency of mutation in the analyzed cohort of 35 patients and in the TCGA EC MSI cohort. Only the genes targeted by FSMs shared ≥50% of the cohort of 35 MSI EC are reported MNR mononuclear repeats, EC endometrial cancer, MSI microsatellite instability, IHC immunohistochemistry.

Fig. 3. Enrichment of vaccine-targeted FSPs in the MSI-H EC cohort.

The figure shows the profile of the NOUS-209 targeted FSPs identified in the cohort of patients: a total number of mutations encoding NOUS-209 FSPs found in the 35 patients; b sharedness of the 163 mutations found in at least one patient; c cumulative length in amino acids of the identified FSPs in each patient; d number of potentially immunogenic epitope identified for the patient. The EC patients are shown as NOU-1 to NOU-35. EC endometrial cancer; MSI-h microsatellite instability-high, FSP frameshift peptides.

Fig. 4. Frequency of NOUS-209 FSPs mutations compared to other mutations.

Kernel Density Estimation plot reporting the distribution of the frequency of observation of NOUS-209 detected mutations (light blue) compared to the distribution of all the other mutations at MNR loci (blue). The density distribution was estimated by using the density function included in R 4.2.1. Statistical significance was determined with the Kolomogorv–Smirnoff test. EC endometrial cancer, FSP frameshift peptides.

Fig. 5. Detection of NOUS-209 FSMs in AN3CA cell line.

a Venn diagram showing the overlap among the FSPs detected in the 35 patients (brown) with Exomeseq (pink) and Ribo-seq (green) mutations detected in the AN3CA MSI EC cell line. b log2 fold change of the RNA expression of mutated vs the wild type counterparts for the 38 loci confirmed by Ribo-seq analysis. The expression is estimated by using normTPM measure (details in “Methods” section). c Immunogenicity of 14 FSPs in a cohort of MSI CRC vaccinated with NOUS-209. Ex vivo IFN-γ ELISpot on patients' PBMCs stimulated with pools of overlapping peptides covering the sequences of the tests FSPs. The number of SFCs per 106 PBMCs is reported. DMSO (peptide diluent represented the negative control).