Arutha Kulasinghe, PhD
NHMRC Research Fellow
The University of Queensland
Multiomic spatial delineation of immunotherapy response groups in non-small cell lung cancer (NSCLC)
Introduction: Immunotherapies, such as immune checkpoint inhibitors (ICI) have shown durable benefit in a subset of non-small cell lung cancer (NSCLC) patients. The composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becomingly increasingly recognised as a driving factor in treatment-refractory disease.
Methods: Here, we employed multiplex IHC (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments of pre-treatment samples from a retrospective 2nd line NSCLC ICI-treated cohort (n=41 patients; n=25 responders, n=16 non-responders). In parallel, the immune proteome of a standard of care (SOC), platinum treated NSCLC cohort (n=47) was assessed to evaluate treatment specific associations.
Results: We demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in ICI refractory tumours (p=0.012). Patients sensitive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p=0.028) within the tumour compartments, which corresponded with the increased expression of IL2 mRNA (p=0.001) within their stroma, indicative of key conditions for ICI efficacy prior to treatment. IL2 mRNA levels within the stroma positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p=2e-5) and BAD (p=5.5e-4) and negatively with levels of memory T cells (CD45RO) (p=7e-4). Immuno-inhibitory markers CTLA-4 (p=0.021) and IDO-1 (p=0.023) were supressed in ICI-responsive patients. Tumour CD44 (p=0.02) was depleted in the response group and corresponded inversely with significantly higher stromal expression of one of its ligands, SPP1 (osteopontin, p=0.008). Orthogonal validation of CD44 by multiplex immunofluorescence confirmed both its association with response and localisation to tumour cells rather than immune cell infiltrate. Analysis of dysregulated transcripts indicated the potential inhibition of stromal interferon-gamma (IFNγ) activity and estrogen-receptor and Wnt-1 signalling activity within the tumour cells of ICI responsive patients. Cox survival analysis indicated tumour CD44 expression was associated with poorer prognosis (HR=1.61, p=0.01), consistent with its depletion in ICI sensitive patients. Similarly, stromal CTLA-4 (HR=1.78, p=0.003) and MDSC/M2 macrophage marker ARG1 (HR=2.37, p=0.01) were associated with poorer outcome while BAD (HR=0.5, p=0.01) appeared protective. The SOC cohort paralleled similar roles for immune checkpoints and pro-apoptotic markers, with LAG3 (HR=3.81, p=0.04) indicating poorer outcome, and BIM (HR=0.16, p=0.014) with improved outcome. Interestingly, stromal mRNA for E-selectin (HR=652, p=0.001), CCL17 (HR=70, p=0.006) and MTOR (HR=1065, p=0.008) were highly associated with poorer outcome in ICI treated patients, indicating pro-tumourigenic features in the tumour microenvironment that may facilitate ICI resistance.
Conclusions: Through multi-modal approaches, we have dissected the characteristics of NSCLC treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy. Whilst further validation of putative markers is needed, our findings provide early insights into predictive biomarkers associated with response to therapy in NSCLC.
Dr. Arutha Kulasinghe is a Peter Doherty NHMRC Research Fellow, and leads the Clinical-oMx Lab at the University of Queensland. Dr Kulasinghe has pioneered spatial transcriptomics using digital spatial profiling approaches in the Asia-Pacific region, contributing to world-first studies for lung cancer, head and neck cancer, and COVID-19. His research aims to understand the underlying pathobiology by using an integrative multi-omics approach. Dr Kulasinghe has published his research in 60 manuscripts and is supported by the NHMRC, Australian Academy of Sciences, Cancer Australia, Cure Cancer, MRFF and numerous philanthropic and hospital foundations.