Al[18F]F-NOTA-Octreotide Is Comparable to [68Ga]Ga-DOTA-TATE for PET/CT Imaging of Neuroendocrine Tumours in the Latin-American Population

Simple Summary In the present work we investigated the clinical utility of Al[18F]F-NOTA-Octreotide (Al[18F]F-OC) in comparison to [68Ga]Ga-DOTA-TATE in patients diagnosed with neuroendocrine tumours. Our aim was to verify the recently published, promising results for Al[18F]F-NOTA-Octreotide in the Latin-American population. Al[18F]F-NOTA-Octreotide provided excellent image quality, detected NET lesions with high sensitivity and represents a highly promising, clinical alternative to [68Ga]Ga-DOTA-TATE. Abstract PET imaging of neuroendocrine tumours (NET) is well established for staging and therapy follow-up. The short half-life, increasing costs, and regulatory issues significantly limit the availability of approved imaging agents, such as [68Ga]Ga-DOTA-TATE. Al[18F]F-NOTA-Octreotide provides a similar biodistribution and tumour uptake, can be produced on a large scale and may improve access to precision imaging. Here we prospectively compared the clinical utility of [68Ga]Ga-DOTA-TATE and Al[18F]F-NOTA-Octreotide in the Latin-American population. Our results showed that in patients with stage IV NETs [68Ga]Ga-DOTA-TATE presents higher physiological uptake than Al[18F]F-NOTA-Octreotide in the liver, hypophysis, salivary glands, adrenal glands (all p < 0.001), pancreatic uncinated process, kidneys, and small intestine (all p < 0.05). Nevertheless, despite the lower background uptake of Al[18F]F-NOTA-Octreotide, comparative analysis of tumour-to-liver (TLR) and tumour-to-spleen (TSR) showed no statistically significant difference for lesions in the liver, bone, lymph nodes, and other tissues. Only three discordant lesions in highly-metastases livers were detected by [68Ga]Ga-DOTA-TATE but not by Al[18F]F-NOTA-Octreotide and only one discordant lesion was detected by Al[18F]F-NOTA-Octreotide but not by [68Ga]Ga-DOTA-TATE. Non-inferiority analysis showed that Al[18F]F-NOTA-Octreotide is comparable to [68Ga]Ga-DOTA-TATE. Hence, our results demonstrate that Al[18F]F-NOTA-Octreotide provided excellent image quality, visualized NET lesions with high sensitivity and represents a highly promising, clinical alternative to [68Ga]Ga-DOTA-TATE.


Introduction
Neuroendocrine tumours (NETs) are highly heterogeneous neoplasms that arise from neuroendocrine cells and affect the diffuse neuroendocrine system, intestinal tract and bronchia [1]. The slow progression of these tumours and their unspecific symptoms lead to a high prevalence and late diagnoses [2][3][4]. Unfortunately, late diagnoses favour the development of distant metastasis resulting in high mortality [5]. To improve the clinical

Image Analysis
Volumes of interest (VOIs) were drawn around tumour lesions, visually distinguished as regions of increased radiotracer uptake relative to background uptake and expected physiological radiotracer uptake. To perform semi-quantitative analysis, mean, peak and maximum standard uptake values (SUV bw ) were calculated using Siemens SyngoVia software. Two nuclear medicine experts who were not blinded to clinical data, independently analysed the PET images. The biodistribution profiles in normal organs were compared for both tracers by analysing SUV mean and SUV max values. Likewise, SUV max and SUV peak values were used to compare the uptake of Al[ 18 F]F-NOTA-Octreotide and [ 68 Ga]Ga-DOTA-TATE in NET lesions. Tumour-to-liver (TLR) and tumour-to-spleen ratios (TSR) were calculated by dividing the SUV max of different tumour lesions by the SUV mean of the liver and spleen, respectively.

Statistical Analysis
Continuous variables were evaluated for normal distribution with histograms and Q-Q plots. Nonparametric quantitative data were compared using a two-sided Wilcoxon signed-rank test to analyse and compare SUV, TLR, and TSR values between scans with p-values < 0.05 considered as statistically significant. To test the non-inferiority of Al[ 18 F]F- NOTA-Octreotide compared to [ 68 Ga]Ga-DOTA-TATE, malignant lesions detected in each patient were registered and counted. In the case of an excessive number of lesions (≥50) an arbitrary number of 50 lesions was used. Since cancerous lesions within a subject are likely to be more correlated than cancerous lesions between subjects, a linear mixed-effects model of non-inferiority for repeated measures was employed [24]. The study had a clinically significant non-inferiority margin of 5% to show a non-inferiority of Al[ 18 F]F-NOTA-Octreotide compared to [ 68 Ga]Ga-DOTA-TATE in tumoral lesion detection, with 80% power and an alpha of 2.5% (one-sided). R version 4.2.0 (22 April 2022) was used for all statistical analyses [25].

Biodistribution of [ 68 Ga]Ga-DOTA-TATE Compared to Al[ 18 F]F-NOTA-Octreotide
In this prospective study, 20 patients with biopsy-proven NET were enrolled to compare the biodistribution and clinical utility of [ 68 Ga]Ga-DOTA-TATE and Al[ 18 F]F-NOTA-Octreotide. No adverse events, adverse drug reactions or significant changes in vital signs were observed during the study. Both tracers showed similar physiological uptake in spleen, vascular pool and bone. However, [ 68 Ga] Ga-DOTA-TATE exhibited significantly higher uptake in liver (p < 0.01), hypophysis (p < 0.01), salivary glands (p < 0.01), uncinate process (p < 0.05), adrenal glands (p < 0.01), kidneys (p < 0.05) and small intestine (p < 0.05). The highest uptake of Al[ 18 F]F-NOTA-Octreotide was observed in the spleen, adrenal glands and kidneys, whereas a low uptake was observed for vascular pool, salivary glands and bone, a pattern similar to that seen with [ 68 Ga]Ga-DOTA-TATE ( Figure 1).

Biodistribution of [ 68 Ga]Ga-DOTA-TATE Compared to Al[ 18 F]F-NOTA-Octreotide
In this prospective study, 20 patients with biopsy-proven NET were enrolled to compare the biodistribution and clinical utility of [ 68 Ga]Ga-DOTA-TATE and Al[ 18 F]F-NOTA-Octreotide. No adverse events, adverse drug reactions or significant changes in vital signs were observed during the study. Both tracers showed similar physiological uptake in spleen, vascular pool and bone. However, [ 68 Ga] Ga-DOTA-TATE exhibited significantly higher uptake in liver (p < 0.01), hypophysis (p < 0.01), salivary glands (p < 0.01), uncinate process (p < 0.05), adrenal glands (p < 0.01), kidneys (p < 0.05) and small intestine (p < 0.05). The highest uptake of Al[ 18 F]F-NOTA-Octreotide was observed in the spleen, adrenal glands and kidneys, whereas a low uptake was observed for vascular pool, salivary glands and bone, a pattern similar to that seen with [ 68 Ga]Ga-DOTA-TATE ( Figure 1).     Figure 1, Table 2). We obtained similar results when calculating tumour-to-background ratios with spleen as reference organ. However, differences in TLR and TSR values were not statistically significant ( Figure 2).  Figure 1, Table 2). Ga]Ga-DOTA-TATE. We obtained similar results when calculating tumour-to-background ratios with spleen as reference organ. However, differences in TLR and TSR values were not statistically significant ( Figure 2).       Hence, to determine a non-inferior detection of neoplastic lesions of Al[ 18 F]F-NOTA-Octreotide PET compared to [ 68 Ga]Ga-DOTA-TATE PET we employed a multilevel model since the results indicated evidence of clustering, confirmed by the correlation coefficient (0.68) and with a significant ANOVA test (p < 0.05). The results showed a mean difference, test-reference, of 0.074% (95% confidence interval: −3.874-4.022%) with a lower margin higher than the pre-specified boundary for non-inferiority (-5%), indicating that Al[ 18 F]F-NOTA-Octreotide PET is non-inferior to [ 68 Ga]Ga-DOTA-TATE PET (Figure 4).  non-inferiority chart 2-sided 90% confidence interval (red) and 95% confidence interval (light blue). UCL: upper clinically significant margin limit 5%. LCL: lower clinically significant margin limit of 5%. The mean difference is indicated. Outliers are represented as circles.  non-inferiority chart 2-sided 90% confidence interval (red) and 95% confidence interval (light blue). UCL: upper clinically significant margin limit 5%. LCL: lower clinically significant margin limit of 5%. The mean difference is indicated. Outliers are represented as circles.

Discussion
68 Ga-labelled tracers for SSTR are the gold standard for imaging NET patients. However, countries with vast territorial areas and limited 68 Ge/ 68 Ga generators face an enormous logistical challenge. Al[ 18 F]F-NOTA-Octreotide has emerged as an exciting alternative to 68 Ga-tracers, especially due to the longer half-life of fluorine-18 compared to gallium-68 and production yield, facilitating distribution to distant clinical facilities. Moreover, fluorine-18 presents a shorter positron range resulting in an improved spatial resolution compared to gallium-68 [17]. F]F-NOTA-Octreotide showed significantly less accumulation in the liver, hypophysis, salivary glands, uncinate process, adrenal gland, kidney, and small intestine compared to [ 68 Ga]Ga-DOTA-TATE. The differences in uptake were most pronounced in salivary glands which is in line with previous studies reporting four to sixfold higher uptake [21]. Collectively our results revealed a high background uptake for [ 68 Ga]Ga-DOTA-TATE compared to Al[ 18 F]F-NOTA-Octreotide, which is consistent with previously published data [14]. However, while writing the present article, a new multicentric prospective study including a cohort of 75 NET patients histologically confirmed was published showing no significant differences in mean SUV max in most organs [22]. Contrary to this report, we observed a higher mean SUV max for [ 68 Ga]Ga-DOTA-TATE compared to Al[ 18 F]F-NOTA-Octreotide. This discrepancy may be due to the smaller group sample included in our study (20 patients). When the mean of the tumour-to-background ratio was analysed (using the liver, TLR, and spleen, TSR, as background tissue), non-significant differences were observed (Fig 2), demonstrating a lower liver and spleen background with Al[ 18 [22].
Our study included patients with NETs (mainly G1/G2), which, in some cases, obstructed the lesions counting process. Thus,patients No. 7,No. 11,13,18, and 20 presented countless liver metastasis, which was registered as ≥50 lesions. This was also true for bone metastasis in the case of patients 11 and 13 (  (Figure 3). Interestingly, this patient presented a G3 NET tumour. This particular lesion was small, suggesting that 18 F presents a superior capacity than [ 68 Ga]Ga-DOTA-TATE to detect small lesions. This hypothesis is supported by the better spatial resolution of fluorine-18 compared to gallium-68 and by previous results with comparison using both tracers to detect < 5mm peritoneal metastasis [14,26].
The differences between both tracers in detecting liver lesions neither change the therapeutic approach nor the disease prognosis. In fact, none of these differences was statistically significant. In other metastatic lesions such as primary tumour, bone, lymph nodes, soft tissue, ovary lung or peritoneal carcinomatosis, [ 68 Ga]Ga-DOTA-TATE and Al[ 18 F]F-NOTA-Octreotide had comparable effectiveness, detecting the same number of lesions.
Our results confirm that Al[ 18 F]F-NOTA-Octreotide is not inferior to [ 68 Ga]Ga-DOTA-TATE PET to detect lesions in NET patients since the lower margin of the 95% of the confidence interval was higher than the lower pre-specified boundary of −5% for noninferiority ( Figure 4). Thus, our results confirmed previous results [14,22] in Latin-American NET patients in which the distribution and production of radiotracers is a crucial challenge, especially considering the geography and prevalence of cancer [27].
Our study found non-significant differences in detection rates between Al[ 18 F]F-NOTA-Octreotide and [ 68 Ga]Ga-DOTA-TATE PET. However, one limitation of our study is the small subgroup of patients included and missing complementary data such as other imaging modalities, such as magnetic resonance imaging or 18 F-FDG PET/CT imaging. Ongoing research at our centre is now focused on evaluating the relationship between the tumoral grade and lesion uptake in larger groups of patients.

Conclusions
Our study is the first, to our knowledge, to be performed on  Figure S1: Chemical structures of DOTA-TATE and NOTA-Octreotide with differences highlighted in colored areas; Figure S2: Synthera ® + Synthesizer with loaded IFP and reagents (left), IFP with reagents and cartridges; Figure S3: Representative HPLC-Chromatogram for Al[ 18 F]F-NOTA-Octreotide; UV signal (upper row) and radioactivity (lower row). Product elutes at 12.4 min (isomer 1) and 13.0 min (isomer 2) and main impurity is [ 18 F]F-eluting at 1.2 min. The UV peak at t = 0.8 corresponds to ascorbate; Figure S4: Zoom of HPLC-Chromatogram in figure S2. for Al[ 18 F]F-NOTA-Octreotide; UV signal (upper row) and radioactivity (lower row). Product elutes at 12.4 min (isomer 1) and 13.0 min (isomer 2) and main impurity is [ 18 F]F-eluting at 1.2 min. The UV peak at t = 0.8 corresponds to ascorbate; Figure   Institutional Review Board Statement: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the principles of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study was approved by the regional ethics committee board (CEC SSM Oriente, permit number 11082020) and written informed consent has been obtained from all participants.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to restrictions regarding privacy of patients and ethical reasons.