E-Poster Presentation 34th Lorne Cancer Conference 2022

Multi-omic and spatial dissection of immunotherapy response groups in non-small cell lung cancer (NSCLC) (#337)

Arutha Kulasinghe 1 , James Monkman 2 , Honesty Kim 3 , Aaron Mayer 4 , Ahmed Mehdi 1 , Nicholas Matigian 1 , Rahul Ladwa 1 , Scott Mueller 5 , Mark Adams 2 , Ken O'Byrne 2
  1. University of Queensland, Woolloongabba, QLD, Australia
  2. Queensland University of Technology, Kelvin Grove, QUEENSLAND, Australia
  3. Enable Medicine, California
  4. Enable Medicine, Californa
  5. University of Melbourne, Melbourne

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 2nd line retrospective NSCLC ICI-treated cohort (n=41 patients; n=25 responders, n=16 non-responders).

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. 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, 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 in a retrospective cohort and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.