Background For many years the standard approach to staging of the mediastinum in non-small cell lung cancer (NSCLC) has been surgical using cervical mediastinoscopy, left anterior mediastinotomy or video assisted thoracoscopic surgery (VATS). More recently endobronchial ultrasound (EBUS) and endoscopic ultrasound (EUS) have been reported. We conducted a randomised phase III trial to compare surgical staging versus endoscopic staging. The primary endpoint was detection of mediastinal nodal metastasis (N2/3); secondary endpoints were complication and futile thoracotomy rates. Methods Consecutive patients with potentially resectable (suspected) NSCLC in whom invasive mediastinal staging was indicated based on CT or PET-CT findings were randomly assigned to either Arm A, surgical staging or Arm B, combined EBUS/EUS (followed by surgical staging if endoscopic findings were negative for malignancy). Surgical staging involved mediastinoscopy and/or mediastinotomy and/or VATS. Subsequently, in the absence of mediastinal disease, thoracotomy with systematic lymph node sampling was performed. 240 pts were required to show a 20% increase in sensitivity (power 80% and α=0.05) to detect mediastinal nodal disease with a prevalence of 50%. Results 118 patients were randomised to Arm A and 123 to Arm B. The sensitivity for detection of mediastinal metastases by surgical staging in Arm A was 80% (95% CI, 68 to 89) vs 94% (95% CI, 85 to 98) for endoscopic (± surgical) staging in Arm B (p=0.04). Nodal metastases were found in 41 (35%) of surgically staged patients in Arm A and 62 patients (50%) (56 by EBUS/EUS + 6 by subsequent surgical staging) in Arm B (p=0.019). Overall, the prevalence of mediastinal disease in each arm was similar (p=0.24). Thoracotomy was considered futile in 21 (18%) in those staged in Arm A vs 8 patients (7%) in Arm B (p=0.009). Complication rate was similar in both arms (6 vs 7 patients, p=0.8); however, 12 of 13 complications were due to surgical staging procedures. Conclusions Mediastinal staging for NSCLC should commence with combined EBUS/EUS (followed by surgical staging if endoscopic findings are negative for malignancy) as this improves the detection of nodal metastases and reduces futile thoracotomies compared to surgical staging alone.

The Tumor Inflammation Signature (TIS) is an investigational use only (IUO) 18-gene signature that measures a pre-existing but suppressed adaptive immune response within tumors. The TIS has been shown to enrich for patients who respond to the anti-PD1 agent pembrolizumab. To explore this immune phenotype within and across tumor types, we applied the TIS algorithm to over 9000 tumor gene expression profiles downloaded from The Cancer Genome Atlas (TCGA). As expected based on prior evidence, tumors with known clinical sensitivity to anti-programmed cell death protein 1 (PD-1) blockade had higher average TIS scores. Furthermore, TIS scores were more variable within than between tumor types, and within each tumor type a subset of patients with elevated scores was identifiable although with different prevalence associated with each tumor type, the latter consistent with the observed clinical responsiveness to anti PD-1 blockade. Notably, TIS scores only minimally correlated with mutation load in most tumors and ranking tumors by median TIS score showed differing association to clinical sensitivity to PD-1/PD-1 ligand 1 (PD-L1) blockade than ranking of the same tumors by mutation load. The expression patterns of the TIS algorithm genes were conserved across tumor types yet appeared to be minimally prognostic in most cancers, consistent with the TIS score serving as a pan-cancer measurement of the inflamed tumor phenotype. Characterization of the prevalence and variability of TIS will lead to increased understanding of the immune status of untreated tumors and may lead to improved indication selection for testing immunotherapy agents.

Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular diversity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse samples and protein profiling of 100-plex for human samples. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD ⁺ ), which reduced T cell proliferation and function. Because NAD ⁺ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD ⁺ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD ⁺ -mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Background How the immune microenvironment changes during neoadjuvant chemotherapy of primary breast cancer is not well understood. Methods We analyzed pre- and post-treatment samples from 60 patients using the NanoString PanCancer IO360™ assay to measure the expression of 750 immune-related genes corresponding to 14 immune cell types and various immune functions, and assessed TIL counts and PD-L1 protein expression by immunohistochemistry. Treatment associated changes in gene expression levels were compared using t-test with Bonferroni correction. TIL count, PD-L1 protein and immune metagenes were compared using Wilcoxon test. Baseline immune markers were correlated with pathologic complete response (pCR) using estrogen receptor and treatment arm adjusted logistic regression. Results At baseline, high TIL counts and high expression of chemoattractant cytokines (CCL21, CCL19) and cytotoxic T cell markers were associated with higher pCR rate. High expression of stromal genes (VEGFB, TGFB3, PDGFB, FGFR1, IGFR1), mast and myeloid inflammatory cell metagenes, stem cell related genes (CD90, WNT11, CTNNB1) and CX3CR1, and IL11RA were associated with residual disease (RD). After treatment, in cases with pCR, TIL counts and most immune genes decreased significantly. Among RD cases, TIL counts and PD-L1 expression did not change but cellular stress and hypoxia associated genes (DUSP1, EGR1), and IL6, CD36, CXCL2, CD69 and the IL8/VEGF metagene increased. Conclusions Activated T cells in the tumor microenvironment are associated with pCR whereas stromal functions are associated with residual disease. Most immune functions decrease during neoadjuvant chemotherapy but several immunotherapy targets (PD-L1, IL6, IL8) remain expressed in RD suggesting potential therapeutic strategies.

The recent successes in the use of immunotherapy to treat cancer have led to a multiplicity of new compounds in development. Novel clinical-grade biomarkers are needed to guide the choice of these agents to obtain the maximal likelihood of patient benefit. Predictive biomarkers for immunotherapy differ from the traditional biomarkers used for targeted therapies: the complexity of the immune response and tumour biology requires a more holistic approach than the use of a single analyte biomarker. This paper reviews novel biomarker approaches for the effective development of immune-oncology therapies, highlighting the promise of the advances in next-generation gene expression profiling that allow biologic information to be efficiently organized and interpreted for a maximum predictive value at the individual patient level.