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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The tumour-initiating cell (TIC) model accounts for phenotypic and functional heterogeneity among tumour cells. MicroRNAs (miRNAs) are regulatory molecules frequently aberrantly expressed in cancers, and may contribute towards tumour heterogeneity and TIC behaviour. More recent efforts have focused on miRNAs as diagnostic or therapeutic targets. Here, we identified the TIC-specific miRNAs, miR-1246 and miR-1290, as crucial drivers for tumour initiation and cancer progression in human non-small cell lung cancer. The loss of either miRNA impacted the tumour-initiating potential of TICs and their ability to metastasize. Longitudinal analyses of serum miR-1246 and miR-1290 levels across time correlate their circulating levels to the clinical response of lung cancer patients who were receiving ongoing anti-neoplastic therapies. Functionally, direct inhibition of either miRNA with locked nucleic acid administered systemically, can arrest the growth of established patient-derived xenograft tumours, thus indicating that these miRNAs are clinically useful as biomarkers for tracking disease progression and as therapeutic targets.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Purpose Mesenchymal epithelial transition factor ( MET) activation has been implicated as an oncogenic driver in epidermal growth factor receptor ( EGFR)–mutant non–small-cell lung cancer (NSCLC) and can mediate primary and secondary resistance to EGFR tyrosine kinase inhibitors (TKI). High copy number thresholds have been suggested to enrich for response to MET inhibitors. We examined the clinical relevance of MET copy number gain (CNG) in the setting of treatment-naive metastatic EGFR-mutant–positive NSCLC. Patients and Methods MET fluorescence in situ hybridization was performed in 200 consecutive patients identified as metastatic treatment-naïve EGFR-mutant–positive. We defined MET-high as CNG greater than or equal to 5, with an additional criterion of MET/centromeric portion of chromosome 7 ratiο greater than or equal to 2 for amplification. Time-to-treatment failure (TTF) to EGFR TKI in patients identified as MET-high and -low was estimated by Kaplan-Meier method and compared using log-rank test. Multiregion single-nucleotide polymorphism array analysis was performed on 13 early-stage resected EGFR-mutant–positive NSCLC across 59 sectors to investigate intratumoral heterogeneity of MET CNG. Results Fifty-two (26%) of 200 patients in the metastatic cohort were MET-high at diagnosis; 46 (23%) had polysomy and six (3%) had amplification. Median TTF was 12.2 months (95% CI, 5.7 to 22.6 months) versus 13.1 months (95% CI, 10.6 to 15.0 months) for MET-high and -low, respectively ( P = .566), with no significant difference in response rate regardless of copy number thresholds. Loss of MET was observed in three of six patients identified as MET-high who underwent postprogression biopsies, which is consistent with marked intratumoral heterogeneity in MET CNG observed in early-stage tumors. Suboptimal response (TTF, 1.0 to 6.4 months) to EGFR TKI was observed in patients with coexisting MET amplification (five [3.2%] of 154). Conclusion Although up to 26% of TKI-naïve EGFR-mutant–positive NSCLC harbor high MET CNG by fluorescence in situ hybridization, this did not significantly affect response to TKI, except in patients identified as MET-amplified. Our data underscore the limitations of adopting arbitrary copy number thresholds and the need for cross-assay validation to define therapeutically tractable MET pathway dysregulation in EGFR-mutant–positive NSCLC.

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