Comparison of the tumor immune microenvironment and checkpoint blockade biomarkers between stage III and IV non-small cell lung cancer.

BACKGROUND: Adjuvant immune checkpoint blockade (ICB) following chemoradiotherapy and adding ICB to chemotherapy have been key advances for stages III-IV non-small cell lung cancer (NSCLC) treatment. However, known biomarkers like PD-L1 are not consistently indicative of ICB response. Other markers within the tumor immune microenvironment (TIME) may better reflect ICB response and/or resistance mechanisms, but an understanding of how TIMEs differ between stage III and IV NSCLC has not been explored. METHODS: Real-world data from unresectable, stage III-IV, non-squamous, pretreatment NSCLCs (stage III n = 106, stage IV n = 285) were retrospectively analyzed. PD-L1 immunohistochemistry (IHC) was compared to CD274 gene expression. Then, differential gene expression levels, pathway enrichment, and immune infiltrate between stages were calculated from whole-transcriptome RNA-seq. Analyses were stratified by EGFR status. RESULTS: PD-L1 IHC and CD274 expression in tumor cells were highly correlated (n = 295, P < 2.2e-16, ⍴ = 0.74). CTLA4 expression was significantly increased in stage III tumors (P = 1.32e-04), while no differences were observed for other ICB-related genes. Metabolic pathway activity was significantly enriched in stage IV tumors (P = 0.004), whereas several immune-related KEGG pathways were enriched in stage III. Stage IV tumors had significantly increased macrophage infiltration (P = 0.0214), and stage III tumors had a significantly higher proportion of CD4 + T cells (P = 0.017). CD4 + T cells were also relatively more abundant in EGFR-mutant tumors vs. wild-type (P = 0.0081). CONCLUSION: Directly comparing the TIMEs of stage III and IV NSCLC, these results carry implications for further studies of ICB response in non-resectable stage III NSCLC and guide further research of prognostic biomarkers and therapeutic targets.

Integration of tumor extrinsic and intrinsic features associates with immunotherapy response in non-small cell lung cancer.

The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.

Clinical, Genomic, and Transcriptomic Data Profiling of Biliary Tract Cancer Reveals Subtype-Specific Immune Signatures.

PURPOSE: Biliary tract cancers (BTCs) are aggressive cancers that carry a poor prognosis. An enhanced understanding of the immune landscape of anatomically and molecularly defined subsets of BTC may improve patient selection for immunotherapy and inform immune-based combination treatment strategies. METHODS: We analyzed deidentified clinical, genomic, and transcriptomic data from the Tempus database to determine the mutational frequency and mutational clustering across the three major BTC subtypes (intrahepatic cholangiocarcinoma [IHC], extrahepatic cholangiocarcinoma, and gallbladder cancer). We subsequently determined the relationship between specific molecular alterations and anatomical subsets and features of the BTC immune microenvironment. RESULTS: We analyzed 454 samples of BTC, of which the most commonly detected alterations were TP53 (42.5%), CDKN2A (23.4%), ARID1A (19.6%), BAP1 (15.5%), KRAS (15%), CDKN2B (14.2%), PBRM1 (11.7%), IDH1 (11.7%), TERT (8.4%), KMT2C (10.4%) and LRP1B (8.4%), and FGFR2 fusions (8.7%). Potentially actionable molecular alterations were identified in 30.5% of BTCs including 39.1% of IHC. Integrative cluster analysis revealed four distinct molecular clusters, with cluster 4 predominately associated with FGFR2 rearrangements and BAP1 mutations in IHC. Immune-related biomarkers indicative of an inflamed tumor-immune microenvironment were elevated in gallbladder cancers and in cluster 1, which was enriched for TP53, KRAS, and ATM mutations. Multiple common driver genes, including TP53, FGFR2, IDH1, TERT, BRAF, and BAP1, were individually associated with unique BTC immune microenvironments. CONCLUSION: BTC subtypes exhibit diverse DNA alterations, RNA inflammatory signatures, and immune biomarkers. The association between specific BTC anatomical subsets, molecular alterations, and immunophenotypes highlights new opportunities for therapeutic development.

Integrated genomic profiling expands clinical options for patients with cancer.

Genomic analysis of paired tumor-normal samples and clinical data can be used to match patients to cancer therapies or clinical trials. We analyzed 500 patient samples across diverse tumor types using the Tempus xT platform by DNA-seq, RNA-seq and immunological biomarkers. The use of a tumor and germline dataset led to substantial improvements in mutation identification and a reduction in false-positive rates. RNA-seq enhanced gene fusion detection and cancer type classifications. With DNA-seq alone, 29.6% of patients matched to precision therapies supported by high levels of evidence or by well-powered studies. This proportion increased to 43.4% with the addition of RNA-seq and immunotherapy biomarker results. Combining these data with clinical criteria, 76.8% of patients were matched to at least one relevant clinical trial on the basis of biomarkers measured by the xT assay. These results indicate that extensive molecular profiling combined with clinical data identifies personalized therapies and clinical trials for a large proportion of patients with cancer and that paired tumor-normal plus transcriptome sequencing outperforms tumor-only DNA panel testing.

Clinical validation of the tempus xT next-generation targeted oncology sequencing assay.

We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor-normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.

Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery.

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.

RNA Sequencing of the Tumor Microenvironment in Precision Cancer Immunotherapy.

RNA sequencing (RNA-seq) provides an efficient high-throughput technique to robustly characterize the tumor immune microenvironment (TME). The increasing use of RNA-seq in clinical and basic science settings provides a powerful opportunity to access novel therapeutic biomarkers in the TME. Advanced computational methods are making it possible to resolve the composition of the tumor immune infiltrate, infer the immunological phenotypes of those cells, and assess the immune receptor repertoire in RNA-seq data. These immunological characterizations have increasingly important implications for guiding immunotherapy use. Here, we highlight recent studies that demonstrate the potential utility of RNA-seq in clinical settings, review key computational methods used for characterizing the TME for precision cancer immunotherapy, and discuss important considerations in data interpretation and current technological limitations.

Integrating RNA expression and visual features for immune infiltrate prediction.

Patient responses to cancer immunotherapy are shaped by their unique genomic landscape and tumor microenvironment. Clinical advances in immunotherapy are changing the treatment landscape by enhancing a patient's immune response to eliminate cancer cells. While this provides potentially beneficial treatment options for many patients, only a minority of these patients respond to immunotherapy. In this work, we examined RNA-seq data and digital pathology images from individual patient tumors to more accurately characterize the tumor-immune microenvironment. Several studies implicate an inflamed microenvironment and increased percentage of tumor infiltrating immune cells with better response to specific immunotherapies in certain cancer types. We developed NEXT (Neural-based models for integrating gene EXpression and visual Texture features) to more accurately model immune infiltration in solid tumors. To demonstrate the utility of the NEXT framework, we predicted immune infiltrates across four different cancer types and evaluated our predictions against expert pathology review. Our analyses demonstrate that integration of imaging features improves prediction of the immune infiltrate. Of note, this effect was preferentially observed for B cells and CD8 T cells. In sum, our work effectively integrates both RNA-seq and imaging data in a clinical setting and provides a more reliable and accurate prediction of the immune composition in individual patient tumors.

Clinical validation of the Tempus xO assay.

We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification.

Myeloperoxidase deficiency preserves vasomotor function in humans.

AIMS: Observational studies have suggested a mechanistic link between the leucocyte-derived enzyme myeloperoxidase (MPO) and vasomotor function. Here, we tested whether MPO is systemically affecting vascular tone in humans. METHODS AND RESULTS: A total of 12 135 patients were screened for leucocyte peroxidase activity. We identified 15 individuals with low MPO expression and activity (MPO(low)), who were matched with 30 participants exhibiting normal MPO protein content and activity (control). Nicotine-dependent activation of leucocytes caused attenuation of endothelial nitric oxide (NO) bioavailability in the control group (P < 0.01), but not in MPO(low) individuals (P = 0.12); here the MPO burden of leucocytes correlated with the degree of vasomotor dysfunction (P = 0.008). To directly test the vasoactive properties of free circulating MPO, the enzyme was injected into the left atrium of anaesthetized, open-chest pigs. Myeloperoxidase plasma levels peaked within minutes and rapidly declined thereafter, reflecting vascular binding of MPO. Blood flow in the left anterior descending artery and the internal mammary artery (IMA) as well as myocardial perfusion decreased following MPO injection when compared with albumin-treated animals (P < 0.001). Isolated IMA-rings from animals subjected to MPO revealed markedly diminished relaxation in response to acetylcholine (P < 0.01) and nitroglycerine as opposed to controls (P < 0.001). CONCLUSION: Myeloperoxidase elicits profound effects on vascular tone of conductance and resistance vessels in vivo. These findings not only call for revisiting the biological functions of leucocytes as systemic and mobile effectors of vascular tone, but also identify MPO as a critical systemic regulator of vasomotion in humans and thus a potential therapeutic target.