Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8 TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8 TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8 TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39 CD8 TILs. Furthermore, frequencies of CD39 expression among CD8 TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.

The enumeration and characterization of circulating tumor cells (CTCs), found in the peripheral blood of cancer patients, provide a potentially accessible source for cancer diagnosis and prognosis. This work reports on a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. The technique utilizes the inherent Dean vortex flows present in curvilinear microchannels under continuous flow, along with inertial lift forces which focus larger CTCs against the inner wall. Using a trapezoidal cross-section as opposed to a traditional rectangular cross-section, the position of the Dean vortex core can be altered to achieve separation. Smaller hematologic components are trapped in the Dean vortices skewed towards the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. By using a single spiral microchannel with one inlet and two outlets, we have successfully isolated and recovered more than 80% of the tested cancer cell line cells (MCF-7, T24 and MDA-MB-231) spiked in 7.5 mL of blood within 8 min with extremely high purity (400-680 WBCs mL(-1); ~4 log depletion of WBCs). Putative CTCs were detected and isolated from 100% of the patient samples (n = 10) with advanced stage metastatic breast and lung cancer using standard biomarkers (CK, CD45 and DAPI) with the frequencies ranging from 3-125 CTCs mL(-1). We expect this simple and elegant approach can surmount the shortcomings of traditional affinity-based CTC isolation techniques as well as enable fundamental studies on CTCs to guide treatment and enhance patient care.

Purpose This multinational study evaluated the antitumor activity of nivolumab in nasopharyngeal carcinoma (NPC). Tumor and plasma-based biomarkers were investigated in an exploratory analysis. Patients and Methods Patients with multiply pretreated recurrent or metastatic NPC were treated with nivolumab until disease progression. The primary end point was objective response rate (ORR) and secondary end points included survival and toxicity. The expression of programmed death-ligand 1 (PD-L1) and human leukocyte antigens A and B in archived tumors and plasma clearance of Epstein-Barr virus DNA were correlated with ORR and survival. Results A total of 44 patients were evaluated and the overall ORR was 20.5% (complete response, n = 1; partial response, n = 8). Nine patients received nivolumab for > 12 months (20%). The 1-year overall survival rate was 59% (95% CI, 44.3% to 78.5%) and 1-year progression-free survival (PFS) rate was 19.3% (95% CI, 10.1% to 37.2%). There was no statistical correlation between ORR and the biomarkers; however, a descriptive analysis showed that the proportion of patients who responded was higher among those with PD-L1 positive tumors (> 1% expression) than those with PD-L1-negative tumors. The loss of expression of one or both human leukocyte antigen class 1 proteins was associated with better PFS than when both proteins were expressed (1-year PFS, 30.9% v 5.6%; log-rank P = .01). There was no association between survival and PD-L1 expression or plasma Epstein-Barr virus DNA clearance. There was no unexpected toxicity to nivolumab. Conclusion Nivolumab has promising activity in NPC and the 1-year overall survival rate compares favorably with historic data in similar populations. Additional evaluation in a randomized setting is warranted. The biomarker results were hypothesis generating and validation in larger cohorts is needed.

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