Fine needle aspirates characterise the hepatocellular carcinoma immune niche to predict immune checkpoint inhibitor outcomes.

BACKGROUND & AIMS: Antitumour immunity involves a complex balance of immune effectors and regulators, many of which are adapted or compartmentalised within the local microenvironment. How this influences responses to immunotherapy in hepatocellular carcinoma (HCC) is poorly understood because of limited access to the tumour immune landscape. We postulated that fine needle aspirates (FNA) could allow for minimally invasive, in-depth tumour immune profiling in patients with advanced HCC. METHODS: Patients with radiological evidence of advanced HCC were prospectively enrolled to provide matched blood, FNA (n = 29 baseline, 2 also on treatment) and biopsy (n = 20 baseline only) for ex vivo spectral flow cytometric characterisation of multiple immune populations. RESULTS: FNA yielded more viable leukocytes than biopsies (mean 800,000 vs. 250,000 cells) allowing reproducible characterisation of a broad range of viable immune effectors and regulators enriched within the tumour. Tissue-resident memory CD8(+)T cells (CD8(+)T(RM)), a subset critical to cancer control that are excluded from blood, could also be aspirated from HCC for phenotypic/functional assessment (mean 10% vs. 0.1%, p <0.0001). PD-1 and alternative checkpoints (TIM-3/LAG-3/2B4/CD39) were strikingly enriched on CD8(+)T(RM) and CD4(+)T(RM), which were also more likely to co-express multiple checkpoints than their circulating counterparts (CD8(+)T(RM): 21.6% vs. 7.4%, p = 0.0003, CD4(+)T(RM): 16.4% vs. 5.6%, p = 0.011). Expression of checkpoints on circulating T cells was discordant with levels on the fraction compartmentalised within tumours. FNA revealed an intratumoral expansion of neutrophils with an immunosuppressive phenotype that were increased in non-responders to immunotherapy (mean 50.2% vs. 25.5% in responders, p = 0.015) and correlated inversely with CD8(+)T(RM) and CD4(+)T(RM) frequencies (r = 0.6, p = 0.001). CONCLUSION: FNA are suitable for rapid, comprehensive sampling of HCC prior to and during immunotherapy, revealing features of the tissue-resident tumour immune niche that cannot be predicted from blood. These features have the capacity to predict clinical outcomes. IMPACT AND IMPLICATIONS: This study addresses the need for simple and minimally invasive assessment of the tumour immune microenvironment in hepatocellular carcinoma, revealing key compartmentalised immunotherapy targets that are not predictable from blood. Findings are important for researchers, clinicians and patients to guide personalised immune checkpoint inhibitor selection and the development of novel approaches to block immunosuppressive neutrophils in order to improve on limited responses to current hepatocellular carcinoma therapies. We demonstrate the potential of rapid fine needle aspirate-based immune profiling to be integrated into larger studies, including clinical trials, to guide personalised selection of patients for existing and future immune checkpoint inhibitors, provide insights into mechanisms of primary and secondary resistance, and inform the development of novel immunotherapy targets.