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Background: Access to molecular testing for metastatic NSCLC (mNSCLC) patients has been improved by circulating tumor DNA (ctDNA) based liquid biopsies (LB), which can obviate invasive procedures, expedite results, and enable serial testing. Emerging clinical applications for LB include therapeutic efficacy monitoring and minimal residual disease (MRD) detection, which demand a ctDNA assay with high sensitivity, specificity, and appropriate genomic coverage. Here we demonstrate that SafeSEQ next-generation sequencing (NGS) LB delivers equivalent performance to OncoBEAM digital PCR, a sensitive ctDNA approach that has been extensively clinically validated in pivotal trials (e.g., AURA, TIGER-X). Importantly, SafeSEQ delivers significantly expanded genomic coverage to address the need for expanding targeted therapy indications, monitoring treatment response, and detecting MRD. Methods: Whole blood samples (n=176) were collected from mNSCLC patients prior to/during treatment or at disease progression and transported to a CLIA laboratory for OncoBEAM analysis to detect mutations in EGFR (exon 19 del, L858R, T790M, C797S), KRAS (codons 12, 13, 61) and BRAF V600E. Replicate plasma aliquots were analyzed with SafeSEQ to interrogate clinically relevant regions in BRAF, EGFR, ERBB2, KRAS, MET, NRAS, PIK3CA, and TP53. Mutation level concordance between the methods was assessed, where only genomic alterations interrogated by both platforms were considered. For mutations detected by both methods, correlation analysis of mutant allelic frequency (MAF) was performed. Results: Concordance analysis of the mutation results from OncoBEAM and SafeSEQ testing of 176 replicate patient samples demonstrated an overall percent agreement (OPA) of 99.6%, with a positive percent agreement (PPA) of 78.1% and a negative percent agreement (NPA) of 99.9%. The mean MAF for discordant mutations (n=16) was 0.06% (range: 0.04-0.12%). When considering mutations with MAF >0.1%, PPA and OPA increased to 96.0% and 99.9%, respectively. MAF levels for mutations detected by both methods demonstrated a strong linear correlation (R2=0.98). Of 124 patient samples having no mutation detected by OncoBEAM, 75 (60%) showed ≥1 alteration with SafeSEQ. Panel-wide, 76% and 30% of all mutations were detected at Citation Format: Hillary Sloane, Priya Sathyanarayan, Daniel Edelstein, Frederick Jones, Jennifer Preston, Sam Wu, Jenna Los, Lara Duchstein, Johannes Fredebohm, Katharina Wichner, Denise Heim, Frank Holtrup, Hannah Quinn, David Feller-Kopman. Clinical evaluation of NGS-based liquid biopsy genotyping in non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB053.

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor ROR{gamma}t and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7-/-Ror{gamma}t-/-, succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7-/-Ror{gamma}t-/- mice.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

11538Background: Expanded RAS testing with BEAMing has been shown to improve the identification of mCRC patients eligible for anti-EGFR therapy as evidenced by results of 1,218 patients from CRYSTA...

The chemokine receptor CCR7 is a central regulator in the maintenance of cellular homeostasis of mucosal tissues. CCR7⁻/⁻ mice develop autoimmune gastritis and exocrinopathy accompanied by the formation of mucosal tertiary lymphoid follicles. Here we found that CCR7-deficient mice frequently suffered from chronic diarrhea linked with increased gastrointestinal motility and the development of severe anorectal prolapse. Enhanced formation of intestinal lymphoid follicles was associated with an elevated proportion of activated colonic T cells and increased production of the cytokine interleukin (IL)-1β. To uncover the pathomechanisms of diarrhea in CCR7⁻/⁻ mice, colonic epithelial barrier and ion channel activities were analyzed in Ussing chambers. Although overt acute colitis was absent, CCR7 deficiency resulted in reduced electrogenic sodium absorption and colonic chloride secretion. As it is known that IL-1β regulates epithelial sodium channel (ENaC) activity, these data imply a causal link between CCR7 expression, IL-1β level, and Na⁺ malabsorption owing to altered ENaC expression and diarrhea.