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BackgroundIn patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration.MethodsWe 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.ResultsCirculating 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.ConclusionThe 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 numberNCT03174405.

Summary We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR, TCR) sequences from blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires was associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation and counterregulation by the coreceptors BTLA, Tim-3, PD-1, TIGIT and CD73. Tfh, Th17-like and nonconventional (but not classical anti-viral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development., Highlights • Convergent B cell responses to SARS-CoV-2 epitopes are predominantly naïve • Shared T cell clusters emerge over disease course in recovering COVID-19 patients • A receptor sequence repository from COVID-19 patients is established for public use, It is unclear how immune responses to Sars-CoV-2 differ in patients with mild versus severe COVID-19 disease. Schultheiß et al. define specific immune receptor sequences associated with different disease courses and established an actively updated sequence repository for public use.

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.

TO THE EDITOR: Anergy describes a state of B-cell receptor activation that leads to survival but not to proliferation in chronic lymphocytic leukemia (CLL) cells, and a high proportion of anergic cells correlates with an indolent clinical course. To gain deeper insights into anergic and

Clonal bone marrow (BM) B-cell populations are a common finding in patients with suspected primary central nervous system lymphoma (PCNSL). To assess their clinical significance, benign monoclonal B-cell lymphocytosis (MBL) needs to be differentiated from concomitant BM involvement, since patients with secondary central nervous system lymphoma (SCNSL) generally require more intensive treatment directed also at the component outside the central nervous system (CNS). Here, we retrospectively analyzed BM samplings in 51 patients with suspected PCNSL. We found clonal B cell populations in 8 of 51 cases (16%) by flow cytometry. None of these eight cases had BM involvement by high-grade lymphoma. No lymphoma relapses outside the CNS were recorded. Together, our data points at a significant percentage of patients with suspected PCNSL harboring clonal BM B-cells, which in this patient group always represented benign MBL. Failure to differentiate these populations from systemic lymphoma involvement may result in overtreatment of patients.

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.

The dynamics of immunoaging and the onset of immunoparesis in healthy individuals and cancer patients has been controversially discussed. Moreover, the role of chemotherapy on T cell regeneration needs further elucidation in light of novel immunotherapies that have become standard of care for many elderly cancer patients. We used next-generation immunosequencing to study T cell receptor (TCR) repertoire metrics on 346 blood samples from healthy individuals and cancer patients producing a dataset with around 8.8 million TCR reads. This analysis showed that decline of T cell diversity and increase in T cell clonality is a continuous process beginning in healthy individuals over 40 years of age. Untreated patients with both hematological and solid tumors showed blood TCR repertoires with significantly lower diversity and higher clonality as compared to healthy individuals across all decades. Loss in T cell diversity was essentially driven by a loss in richness in aging healthy individuals, while in cancer patients a loss in repertoire evenness was an additional contributing factor. Interestingly, chemotherapy did not impair the regeneration of blood TCR repertoire diversity to pre-treatment age-specific levels. Surprisingly, even patients over the age of 70 years receiving highly T cell toxic therapies reestablished their pre-treatment T cell diversity suggesting rebound thymic activity rather than recovery of T cell counts by peripheral expansion only. Taken together, these data suggest that human TCR repertoire metrics gradually deteriorate in the aging individual, but age-specific TCR metrics are restored after T cell depleting therapy even in elderly cancer patients.