Our pilot study is the first study on humans to systematically compare 18F-FDG and 68Ga-NODAGA-RGD uptake in a HNSCC patient population. It shows that: (1) every primary HNSCC tumour and lymph nodes were visually detectable with both tracers, but with different uptake patterns; (2) 68Ga-NODAGA-RGD uptake was heterogeneous with a low target-to-background ratio while 18F-FDG uptake is mostly homogeneous with higher target-to-background ratio; and (3) 68Ga-NODAGA-RGD uptake was not related to tumor grade, p16 or HPV status.
18F-FDG PET-CT has a high clinical value in the initial workup and follow-up of patients with HNSCC tumors [4–7]. It however only allows evaluation of tumor cells metabolism but not neoangiogenesis. To this purpose we conducted a one-to-one comparison of tracers to assess the clinical potential of 68Ga-NODAGA-RGD. All HNSCC primary tumors, lymph nodes and metastases detected on 18F-FDG PET/CT images were also seen with the angiogenesis radiotracer. Only few studies have been conducted in humans; while Haubner et al.  demonstrated that 68Ga-NODAGA-RGD uptake was not sufficient to be used in patients with hepatocellular carcinoma, other authors reported sufficient uptake for diagnostic purpose in human xenografts of esophageal carcinoma, melanoma and glioblastoma [18, 21]. As both 68Ga-NODAGA-RGD and 18F-Galacto-RGD demonstrated similar preclinical results , our results are in line with the previous work by Beer et al. , who concluded that thanks to its significant uptake, 18F-Galacto-RGD might be used for the assessment of angiogenesis and for planning and response evaluation of αvβ3 targeted therapies in HNSCC.
However, it is worth to mention that tracer uptake patterns were very different between 18F-FDG and 68Ga-NODAGA-RGD. Indeed, TATV were larger with 68Ga-NODAGA-RGD, with heterogeneous uptake within the primary tumor and lymph nodes, and relative low target-to-background ratio compared with 18F-FDG. While this seems to preclude the use of 68Ga-NODAGA-RGD as a single tracer for tumor staging, we assume that it brings complementary information about the tumor itself. Part of volume difference can be due to difference in positron energy between the fluorine-18 and gallium-68. Also, the threshold used for TATV delineation is subject to discussion. We used a 42% SUVmax fixed threshold similarly to MTV delineation, which may have resulted in larger TATV due to lower SUVmax values with 68Ga-NODAGA-RGD. Threshold adaptation for 68Ga-NODAGA-RGD could be performed defined based on tumor margins if defined on whole tumor histopathological specimen, which was out of the scope of our study, as not all tumors and lymph nodes were resected in toto. Nevertheless, we believe that difference in uptake patterns and volume are mainly attributable to difference in the tracer targeting. Although we did not perform the immunohistochemistry staining of human HNSCC tissue microarray to properly correlate uptake with angiogenesis , it is known that 68Ga-NODAGA-RGD improves imaging of αvβ3 expression . Beer et al.  demonstrated that the uptake of 18F-Galacto-RGD mostly represented αvβ3 expression in the neovasculature, but not in the HNSCC tumor cells themselves. This was also confirmed with other RGD-based tracers on HNSCC tumor xenografts . 68Ga-NODAGA-RGD uptake beyond 18F-FDG avid areas could thus reflect the presence of the formation of neovessels. Isal et al.  demonstrated that tumor areas with high 68Ga-NODAGA-RGD uptake also exhibited the highest rates of cell proliferation and integrins expressions irrespective of cell density in engrafted glioblastomas. This seems to be different in HNSCC, as we did not find any significant association between tracers’ uptake and HNSCC grade. Despite different uptake patterns, we found a significant correlation between 18F-FDG and 68Ga-NODAGA-RGD SUVmean values, which overall might indicate the coexistence of interrelated pathophysiological phenomenon within the tumor, i.e. cell proliferation and neoangiogenesis. Finally, no significant difference in 68Ga-NODAGA-RGD activity was found regarding HPV or p16 protein expression (p ≥ 0.22). Although HPV and p16 have demonstrated significant prognostic value in HNSCC tumors [1, 26], this may not preclude the use of 68Ga-NODAGA-RGD as a prognostic biomarker. Indeed, taking into account that tumor neovessels are of paramount importance for tumor oxygenation, the prognostic value of 68Ga-NODAGA-RGD could be assessed in HNSCC patients undergoing chemoradiotherapy. Recent preclinical  and clinical pilot studies  hence reported that 111In-RGD2 and 18F-RGD-K5, two tracers targeting integrin αvβ3, having the potential to monitor response to therapy and to identify patients with incomplete responses to concurrent chemoradiotherapy. This point has to be explored in a larger prospective study.
We acknowledge several limitations inherent to a pilot study. First, we evaluated a small sample of HNSCC patients, which limits discriminating power, especially regarding correlation with histology. Second, immunostaining was not performed to confirm regional αvβ3 expression, but rather characterize whole tumor distribution. Third, as already mentioned, we used a fixed threshold for TATV definition in a first approximation, which might have overestimated tumor volume; threshold optimization based on spatial comparison with αvβ3 immunostaining on whole-tumor histological slices would need to be performed for more precision. Finally, larger, longitudinal studies would need to be performed to determine the prognostic value of 68Ga-NODAGA-RGD.