Introduction. Despite the complexity of the immune response to cancer and its systemic nature, the study of tumor immunology has predominantly focused on the immune milieu within the tumor microenvironment. However, historical knowledge [1] and recent findings [2, 3], highlight the critical significance of processes on the periphery, and urge a more expansive attention on the immune system’s role both intra-tumoraly and peripherally. In this study we use [¹⁸F]F-AraG,  a mitochondrial metabolic tracer that tracks activated T cells [4], to assess the systemic immune status in patients with non-small cell lung cancer (NSCLC) prior to starting immunotherapy.

Methods. This study included 15 patients enrolled in two clinical trials, both aimed at imaging T cell activation in patients with NSCLC (NCT04524195 and NCT04726215). The cohort included 8 males and 7 females, 11 patients with stage IV, 3 patients with stage III, and one patient with stage II disease. Participants were enrolled at three institutions: Palo Alto Veterans Affairs (five patients), Sutter Medical Center in Sacramento (six patients) and Johns Hopkins Kimmel Cancer Center (four patients). [¹⁸F]F-AraG PET/CT imaging was conducted at the institution where participants were enrolled following the same imaging protocol. Standard of care, diagnostic [¹⁸F]FDG PET/CT scans were also collected.  All individual [¹⁸F]FDG and [¹⁸F]F-AraG PET/CT scans were analyzed using TRAQinform IQ software (AIQ Solutions, Madison, WI). From every [¹⁸F]F-AraG PET/CT image, TRAQinform IQ software extracted SUV_max, SUV_mean, Volume and SUV_total for all lesions/hotspots as well as for the spine, spleen, liver, thyroid, bowel and heart. To comprehensively evaluate systemic immunity, we performed discriminant analysis using [¹⁸F]F-AraG SUV values extracted from these organs along with those from all tumor lesions and active lymph nodes.

Results. Differences between [¹⁸F]FDG and [¹⁸F]F-AraG uptake were noted across lesions  within the same patient as well as within the same lesion, indicating that these two tracers capture distinct aspects of the tumor microenvironment. Similar to what was observed in the preclinical studies [5], [¹⁸F]F-AraG’s intralesional distribution patterns resembled discrete cancer-immune phenotypes: inflamed, immune excluded and immune desert (Persuasive data, Figure 1). The observed differences between [¹⁸F]FDG and [¹⁸F]F-AraG, as well as the [¹⁸F]F-AraG’s pattern of lesional uptake indicate that within tumors [¹⁸F]F-AraG does not accumulate in cancer cells. In the subgroup of patients with distant metastases, none of the [¹⁸F]FDG biomarkers associated with tumor burden correlated with overall survival. In contrast, [¹⁸F]F-AraG SUV_total  in the lumbar bone marrow significantly differed between patients who experienced disease progression during immunotherapy and those who did not. In addition, the patients with high lumbar [¹⁸F]F-AraG SUV_total exhibited significantly lower overall survival compared to those with low vertebral signal (Persuasive data, Figure 2).

Conclusion. The results presented here underscore the significance of evaluating systemic immunity and offer initial evidence to support the assessment of [¹⁸F]F-AraG bone marrow signal as a predictive imaging biomarker for patient stratification and treatment guidance in patients with NSCLC.

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Figure 1. [¹⁸F]F-AraG uptake in the lumbar bone marrow and overall survival in immunotherapy treated patients with NSCLC. A. Kaplan Meier analysis of the patient cohort with advanced disease revealed significantly lower probability of survival in patients with high accumulation of [¹⁸F]F-AraG in the lumbar bone marrow. B. Tumor burden in two patients with different clinical outcome. Patient 1 (TLG = 1800) had progressive disease and died less than 5 months after starting therapy. Patient 2 (TLG = 2162) had a complete response and was alive at the time of analysis, more than 12 months after the start of the therapy. C. Lung lesion in the two patients showed comparable [¹⁸F]F-AraG accumulation (top row). [¹⁸F]F-AraG accumulation in the bone marrow L4 vertebrae of the patient with shorter survival was considerably higher than the uptake in the patient with longer survival. 

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