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IntroductionIn metastatic colorectal cancer (mCRC), the efficacy of immune checkpoint blockade (ICB) has so far been limited to patients with microsatellite instability high tumors (MSI-H). Unfortunately, most mCRC patients suffer from non-immunogenic microsatellite stable (MSS) tumors. Therefore, new combinatorial strategies are urgently needed to enhance the immunogenicity of MSS tumors to finally increase the number of patients benefiting from ICB.MethodsThe AVETUX trial aimed to combine the PD-L1 antibody avelumab with the standard of care chemotherapy combination FOLFOX and the anti-EGFR antibody cetuximab. Furthermore, we performed a central radiological review of the pre- and on-treatment computed tomography scans to better define the individual response to treatment.Results and DiscussionIn total, 43 patients were treated of which 39 patients were confirmed as RAS/BRAF wildtype in central tissue review and finally response evaluated. A final progression-free survival (PFS) of 11.1 (range: 0.8 to 22.3 months) and a herein updated final overall survival (OS) of 32.9 months (range: 0.8 to 47.1 months) was reached. We observed a strong median depth of response of 67.5% tumor shrinkage and deepness of response correlated significantly with survival. On the other hand, early tumor shrinkage was not an indicator of better outcome at a cut-off of 20% (median values). In a next step, we correlated the individual best radiological response with potential ICB response biomarkers and found that the clonality and diversity, but not frequency of tumor infiltrating lymphocytes (TiLs) and peripheral blood mononuclear cells (PBMCs), strongly correlated with response. In summary, we report the final overall survival of the AVETUX trial and propose T cell clonality and diversity as a potential marker to predict response to chemo-immunotherapy combinations in MSS mCRC by performing a central radiological review.Clinical Trial RegistrationClinicalTrials.gov, identifier (NCT03174405).

Background In patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration.Methods We treated 43 patients with predominantly microsatellite stable RAS/BRAF wild-type mCRC on a phase II trial combining chemotherapy with the epidermal growth factor receptor antibody cetuximab and the programmed cell death ligand 1 (PD-L1) antibody avelumab. We performed next-generation gene panel sequencing for mutational typing of tumors and liquid biopsy monitoring as well as digital droplet PCR to confirm individual mutations. Translational analyses included tissue immunohistochemistry, multispectral imaging and repertoire sequencing of tumor-infiltrating T cells. Detected PD-L1 mutations were mechanistically validated in CRISPR/Cas9-generated cell models using qRT-PCR, immunoblotting, flow cytometry, complement-dependent cytotoxicity assay, antibody-dependent cytotoxicity by natural killer cell degranulation assay and LDH release assay as well as live cell imaging of T cell mediated tumor cell killing.Results Circulating tumor DNA showed rapid clearance in the majority of patients mirroring a high rate of early tumor shrinkage. In 3 of 13 patients expressing the high-affinity Fcγ receptor 3a (FcγR3a), tumor subclones with PD-L1 mutations were selected that led to loss of tumor PD-L1 by nonsense-mediated RNA decay in PD-L1 K162fs and protein degradation in PD-L1 L88S. As a consequence, avelumab binding and antibody-dependent cytotoxicity were impaired, while T cell killing of these variant clones was increased. Interestingly, PD-L1 mutant subclones showed slow selection dynamics reversing on avelumab withdrawal and patients with such subclones had above-average treatment benefit. This suggested that the PD-L1 mutations mediated resistance to direct antitumor effects of avelumab, while at the same time loss of PD-L1 reduced biological fitness by enhanced T cell killing limiting subclonal expansion.Conclusion The addition of avelumab to standard treatment appeared feasible and safe. PD-L1 mutations mediate subclonal immune escape to avelumab in some patients with mCRC expressing high-affinity FcγR3a, which may be a subset experiencing most selective pressure. Future trials evaluating the addition of avelumab to standard treatment in MSS mCRC are warranted especially in this patient subpopulation.Trial registration number NCT03174405.

Epidermal growth factor receptor (EGFR) antibodies may have detrimental effects in patients with metastatic colorectal cancer expressing oncogenic Rat sarcoma (RAS). Since a significant number of patients acquire RAS-mediated resistance during EGFR-directed treatment, understanding the molecular mechanism underlying these antibody-mediated tumor-promoting effects is of relevance to design more resistance-preventive treatment approaches. To test this, we set up a Ba/F3 cellular model system transformed to EGFR/RAS dependency to be able to study proliferation, RAS activity as well as MAPK signaling upon inhibition of wild-type RAS isoforms by therapeutic EGFR antibodies. Here, we show that the EGFR antibodies cetuximab and panitumumab induce paradoxical stimulation and enhance proliferation in cells expressing oncogenic RAS (KRAS G12V). These experiments clearly showed that the stimulatory effect is a direct result of the antibody-EGFR interaction leading to prolonged mitogen-activated protein-Kinase (MAPK) signaling. The effect was also induced by antibody-chemotherapy combinations but always depended on simultaneous low-level ligand-dependent EGFR pathway activation. Moreover, we observed significant growth retardation of RAS mutant cells after antibody withdrawal compatible with a drug-addiction phenotype. Our data suggests that EGFR antibodies paradoxically sustain MAPK signaling downstream of oncogenic RAS thereby driving proliferation of RAS mutant tumors or tumor subclones. The observed drug-addiction encourages fixed-duration or liquid-biopsy-guided drug holiday concepts to preventively clear RAS mutant subclones selected under EGFR-directed therapeutic pressure.

The phosphoinositide 3-kinase–related kinase ATR is a central regulator of the DNA damage response. Its chemical inhibition eliminates subsets of cancer cells in various tumor types. This effect is caused at least partly by the synthetically lethal relationship between ATR and certain DNA repair genes. In a previous screen using an siRNA library against DNA repair genes, we identified PRIM1, a part of the polymerase α-primase complex, as acting synthetically lethal with ATR. Applying a genetic ATR knock-in model of colorectal cancer cells, we confirmed that PRIM1 depletion inhibited proliferation of ATR-deficient cells and excluded artifacts due to clonal variation using an ATR reexpressing cell clone. We expanded these data by demonstrating in different cell lines that also chemical inhibition of ATR or its main effector kinase CHK1 reduces proliferation upon depletion of PRIM1. Mechanistically, PRIM1 depletion in ATR-deficient cells caused S-phase stasis in the absence of increased DNA damage followed by Wee1-mediated activation of caspase 8 and apoptosis. As PRIM1 inactivation sensitizes cancer cells to ATR and CHK1 inhibitors, mutations in PRIM1 or other components of the polymerase α-primase complex could represent novel targets for individualized tumor therapeutic approaches using ATR/CHK1 inhibitors, as has been previously demonstrated for POLD1, the catalytic subunit of polymerase δ.

Using next-generation immunoglobulin (IGH) sequencing and flow cytometry, we characterized the composition, diversity and dynamics of non-malignant B cells in patients undergoing treatment with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib or chemo-immunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR). During ibrutinib therapy, non-malignant B cell numbers declined, but patients maintained stable IGH diversity and constant fractions of IGH-mutated B cells. This indicates partial preservation of antigen-experienced B cells during ibrutinib therapy, but impaired replenishment of the normal B cell pool with naïve B cells. In contrast, after FCR we noted a recovery of normal B cells with a marked predominance of B cells with unmutated IGH. This pattern is compatible with a deletion of pre-existing antigen-experienced B cells followed by repertoire renewal with antigen-naïve B cells. These opposite patterns in B cell dynamics may result in different responses towards neoantigens versus recall antigens, which need to be further defined.