NK cell activity and methylated HOXA9 circulating tumor DNA as prognostic biomarkers in patients with non-small cell lung cancer treated with PD-1/PD-L1 inhibitors.
Authors: Witting Christensen Wen Sara , Nederby Line , Andersen Rikke Fredslund , Hansen Torben Schjoedt , Haugaard Christa , Hilberg Ole , Jakobsen Anders , Hansen Torben 
Published: June 2022

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Background: PD-1/PD-L1 inhibitors have improved survival for patients with non-small cell lung cancer (NSCLC), but better prognostic biomarkers are needed. Methods: We prospectively collected plasma from 82 patients with NSCLC before initiating treatment with PD-1/PD-L1 inhibitors and before treatment cycles 2-4. We used the NK Vue assay to measure interferon gamma (IFNγ) as a surrogate for natural killer cell activity (NKA) with a cutoff of 250 pg/mL. Circulating tumor DNA (ctDNA) in the form of methylated HOXA9 was measured by droplet digital PCR. ctDNA status was classified as detectable or undetectable ctDNA. Results: Patients were classified into three groups according to IFNγ levels at the available time points. The NKA-low group had a persistently low level of IFNγ or dropped to and remained at a low level after baseline (<250 pg/mL, n=29), the NKA-mixed group experienced either an increase from low to normal levels or vice versa (n=34), while the NKA-high group maintained a normal level of IFNγ (≥250 pg/mL, n=13). The median PFS was 64 days (95% confidence interval (CI) 48-115 days), 228 days (95% CI 146-353 days), and 214 days (95% CI 101-693 days), respectively, for NKA-low, NKA-mixed, and NKA-high (p=0.003). The median OS was 170 days (95% CI 110-285 days), 487 days (95% CI 361-761 days), and 1,131 days (95% CI 235 days to not reached), respectively (p<0.001). Patients were divided according to detectable (ctDNA+, n=41) or undetectable (ctDNA-, n=32) ctDNA after one treatment cycle. Median PFS was 97 days (95% CI 58-192 days) and 228 days (95% CI 146-353 days), respectively, for ctDNA+ and ctDNA- (p=0.018). Median OS was 235 days (95% CI 170-525 days) and 544 days (95% CI 361-1158 days), respectively (p=0.007). A score combining NKA and ctDNA both measured after the first treatment cycle had a strong prognostic impact. Group 1 had a low level of IFNγ (<250 pg/mL) and detectable ctDNA (n=27), group 2 had either low levels of IFNγ and undetectable ctDNA or vice versa (n=29), and group 3 had normal levels of IFNγ (≥250 pg/mL) and undetectable ctDNA (n=15). Median PFS was 69 days (95% CI 48-213 days), 183 days (95% CI 102-235 days), and 307 days (95% CI 140-693 days), respectively, for group 1, 2, and 3 (p=0.022). Median OS was 221 days (95% CI 121-539 days), 419 days (95% CI 235-650 days), and 1,158 days (95% CI 250 days to not reached), respectively (p=0.002). Biomarker score 1 was a marker of poor prognosis for OS with a hazard ratio (HR) of 3.971 (95% CI 1.763-8.943, p=0.001) compared to biomarker score 3. It remained statistically significant with a HR of 5.560 (95% CI 2.359-13.101, n=71, p<0.001) when adjusting for PD-L1 status, histology, and performance status. Conclusions: A biomarker score combining the levels of NKA and ctDNA status after the first cycle of treatment may be used to stratify the prognosis in patients with NSCLC treated with PD-1/PD-L1 inhibitors.