Multifactorial Remodeling of the Cancer Immunopeptidome by IFNγ.

IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFNγ-treated colorectal cancer patient-derived organoids and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNγ. IFNγ-induced changes in the abundance of source proteins, switching from the constitutive to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is upregulated or downregulated on HLA-I through IFNγ. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFNγ is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics. SIGNIFICANCE: IFNγ remodels the HLA-I-presented immunopeptidome. We showed that peptide-specific factors influence whether a peptide is upregulated or downregulated and identified a preferential loss or downregulation of those with proline near the peptide center. This will help selecting immunotherapy target antigens which are consistently presented by cancer cells.

Computational Image Analysis of T-Cell Infiltrates in Resectable Gastric Cancer: Association with Survival and Molecular Subtypes.

BACKGROUND: Gastric and gastro-esophageal junction cancers (GCs) frequently recur after resection, but markers to predict recurrence risk are missing. T-cell infiltrates have been validated as prognostic markers in other cancer types, but not in GC because of methodological limitations of past studies. We aimed to define and validate the prognostic role of major T-cell subtypes in GC by objective computational quantification. METHODS: Surgically resected chemotherapy-naïve GCs were split into discovery (n = 327) and validation (n = 147) cohorts. CD8 (cytotoxic), CD45RO (memory), and FOXP3 (regulatory) T-cell densities were measured through multicolor immunofluorescence and computational image analysis. Cancer-specific survival (CSS) was assessed. All statistical tests were two-sided. RESULTS: CD45RO-cell and FOXP3-cell densities statistically significantly predicted CSS in both cohorts. Stage, CD45RO-cell, and FOXP3-cell densities were independent predictors of CSS in multivariable analysis; mismatch repair (MMR) and Epstein-Barr virus (EBV) status were not statistically significant. Combining CD45RO-cell and FOXP3-cell densities into the Stomach Cancer Immune Score showed highly statistically significant (all P ≤ .002) CSS differences (0.9 years median CSS to not reached). T-cell infiltrates were highest in EBV-positive GCs and similar in MMR-deficient and MMR-proficient GCs. CONCLUSION: The validation of CD45RO-cell and FOXP3-cell densities as prognostic markers in GC may guide personalized follow-up or (neo)adjuvant treatment strategies. Only those 20% of GCs with the highest T-cell infiltrates showed particularly good CSS, suggesting that a small subgroup of GCs is highly immunogenic. The potential for T-cell densities to predict immunotherapy responses should be assessed. The association of high FOXP3-cell densities with longer CSS warrants studies into the biology of regulatory T cells in GC.

Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy.

Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.

Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment.

BACKGROUND: Patient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment. METHODS: Four microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS. RESULTS: We identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. CONCLUSIONS: Only 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.

Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy.