The role of interactive states of imnune checkpoint regulators in cancerdetermined by quantitative imaging

Abstract

Immune checkpoint blockade therapies have enhanced cancer therapy in lung and renal cancers aswell as melanoma. Most patients are stratified for anti-PD-1 or anti-CTLA-4 treatments based ontheir PD-L1 ligand expression that is currently assessed by traditional immunohistochemistry (IHC)approaches, which are subjective, lack quantitation and a dynamic range. We have developed a novelquantitative imaging platform, underpinned by time-resolved Förster resonance energy transfer(FRET) determined by frequency-domain fluorescence lifetime imaging microscopy (FLIM) tospatially quantitate these immune checkpoint interactions at a nanoscopic (<10nm) resolution. Thisassay is termed immune-FRET (iFRET). We validated the ability of iFRET to measure differentimmune checkpoints in patients with malignant melanoma and malignant non-small cell lungcarcinoma (NSCLC). Here, iFRET was predictive of patient outcome whereas IHC approaches werenot. This was then applied to a subset of colorectal cancer patients with bilateral lung metastases whofailed to respond to classical treatments and therefore had their metastases treated by radiofrequencyablation (RFA). iFRET could detect differential PD-1/PD-L1 and CTLA-4/CD80 interaction stateswithin patients and between patients. It was shown that neither CD80 nor PD-L1 expressioncorrelated with checkpoint interaction in these patients. Moreover, a negative correlation was seenbetween PD-1/PD-L1 interactive states and intratumoral CD3+ density. Taken together, these resultsindicate a novel technique by which to assess immune checkpoint engagement in patient samples.The results from this multi-disciplinary investigation and thus the implementation of iFRET to carryout quantitative immune surveyance may change the way patients are selected for immunotherapiesand may provide a mechanism by which to monitor their response to treatmen.