Usefulness of 68Ga-DOTATOC PET/CT to localize the culprit tumor inducing osteomalacia

Tumor-induced osteomalacia (TIO) is an uncommon paraneoplastic syndrome presenting with sustained hypophosphatemia. Treatment of choice is removal of the tumor causing the TIO, but identification of the culprit tumor by routine imaging is challenging. This study aimed to assess the usefulness of somatostatin receptor imaging, called 68Ga-DOTATOC PET/CT, in the management of patients with TIO. Twelve patients who were suspected of having TIO underwent 68Ga-DOTATOC PET/CT. Lesion detectability and maximum standardized uptake value (SUVmax) were determined and retrospectively compared with the clinical/imaging surveillance and histopathologic diagnosis. The median duration of suspected TIO with hypophosphatemia was 7.8 years (range 2.1–21.0). Conventional radiologic and/or nuclear medicine images failed to identify the culprit tumors. However, 68Ga-DOTATOC PET/CT scans showed that 8 of the 12 patients had positive lesions, suggesting the presence of focal culprit tumors. The SUVmax of positive tumors was 1.9–45.7 (median: 11.5). Six skeletal lesions and two extra-skeletal lesions were identified. Seven of the lesions were pathologically confirmed as potential culprits of TIO. Hypophosphatemia was resolved in five patients who underwent lesion excision. The 68Ga-DOTATOC PET/CT is a useful whole-body imaging modality for the detection of causative tumors in patients with suspected TIO.

www.nature.com/scientificreports/ recent reports demonstrated improved localization of culprit tumors in TIO using these PET radiotracers 13,20,21 . Although minor changes in amino acid sequences result in a differential affinity toward the type 2 SSTR families, the three tracers have similar performances in clinical practice 22 . Several reports have demonstrated the performance of 68 Ga-DOTATOC PET/CT in these cohorts. In this context, we investigated the usefulness of 68 Ga-DOTATOC PET/CT for the management of patients with TIO from a single Korean tertiary hospital.

Study design and patient selection. The Institutional Review Board (IRB) in the Asan Medical Center
(IRB no. 2019-1290) approved this study protocol and waived informed consent from patients due to the retrospective nature of the study. The study was conducted in compliance with the Declaration of Helsinki regulation. From June 2017 to May 2019, we retrospectively recruited 12 patients with suspicion of TIO who underwent 68 Ga-DOTATOC PET/CT at the Asan Medical Center. Patient medical records were reviewed and documents regarding the commencement of clinical manifestations associated with TIO were retrieved. Additionally, the sequential biochemical laboratory results associated with bone metabolism and all the imaging studies, including radiology and nuclear medicine, were reviewed. When possible, we calculated the tubular reabsorption of phosphate (TRP) twice, according to the formula provided below, to evaluate the status of renal phosphate wasting 23 .
68 Ga-DOTATOC PET/CT imaging. The 68 Ga-DOTATOC was manually synthesized with a 68 Ge/ 68 Ga generator (iThemba LABS, South Africa) and GMP grade DOTATOC (ABX, Germany) in the cyclotron laboratory of our institution 24,25 . Quality control was performed according to European Pharmacopeia; the synthesized 68 Ga-DOTATOC satisfied all quality control criteria and the radiochemical purity of the 68 Ga-DOTATOC was 98.2 ± 1.0%, as measured by HPLC. We injected a median dose of 4.65 mCi (range 3.4-5.4) 68 Ga-DOTATOC, intravenously, in non-fasting patients. Sixty minutes after injection, PET/CT images were generated using the GE Discovery 690, 710, or 690 Elite systems. We acquired the emission PET scans at 3.5 min per bed. Nine patients underwent whole-body PET/CT from skull vertex to both feet. Three patients had torso PET/CT scan from the skull vertex to the proximal thigh. The lesion detectability and SUV max , normalized to body weight, were determined. Additionally, we compared the 68 Ga-DOTATOC PET/CT results with the outcomes of clinical/imaging surveillance and/or pathology. Table 1 summarizes the patient characteristics. We recruited 12 patients (four males and eight females) with a median age of 60 years (range 47-78). During the initial visit to our hospital, all patients had overt hypophosphatemia (serum phosphate ranging from 0.9 mg/dL to 2.4 mg/dL). Nine of ten patients had TRP levels below 85%, suggesting hypophosphatemia of renal origin. We measured FGF-23 in only two patients; both patients had abnormally increased FGF-23 values that were specifically suggestive of TIO.

Results
The median duration of suspected TIO was 7.8 years (range 2.1-21.0 years), reflecting the long-standing disease courses in most patients. During the period of suspected TIO, conventional radiologic (X-ray, CT, and MRI) and/or nuclear medicine images (bone scan and 18 F-FDG PET/CT) failed to pinpoint the culprit lesions. Even though some patients demonstrated several tumors in bones or soft tissues, those tumors were considered non-specific benign primary tumors by the interpreting radiologist or nuclear medicine physician. However, 8 of the 12 patients (66.6%) had focal positive uptake of 68 Ga-DOTATOC in areas other than the physiologic uptake in the pituitary gland, adrenal glands, and uncinate process of the pancreas, thereby suggesting the possibility of culprit tumors (Fig. 1). Regarding the quantitative parameters of the eight positive tumors, SUV max ranged from 1.9 to 45.7 (median: 11.5) and the longest tumor diameter was measured between 1.1 and 6.5 cm (median 1.95 cm). Six skeletal lesions were located in the second cervical vertebral body (C2), third lumbar vertebral body, pubic bone, femur, maxilla, and fibula and two extra-skeletal lesions were located in the groin and thigh. Among the eight patients with positive tumors, seven patients (87.5%) were pathologically confirmed and six patients finally underwent surgery to remove the tumor pinpointed by the 68 Ga-DOTATOC PET/CT. Phosphate levels completely recovered in five out of six patients after the surgery without requiring phosphate supplementation. One patient with a C2 vertebral mass underwent a second 68 Ga-DOTATOC PET/CT 6 months after the operation because of unresolved hypophosphatemia (Fig. 2). Compared to the basal 68 Ga-DOTATOC PET/CT, the second scan showed slightly decreased but residual 68 Ga-DOTATOC uptake by a persistent osteolytic lesion in the operative bed. Thus, the reason for the unresolved hypophosphatemia was a remnant tumor.
Two patients, who had focal positive uptake in 68 Ga-DOTATOC PET/CT, did not undergo surgery. A 48-yearold female patient (Number 7, Fig. 3), with bone biopsy results showing a phosphaturic mesenchymal tumor on her femur, refused curative surgery because of good compliance to phosphate supplement, albeit proportionally increased phosphate demand as the mass grows. A 70-year-old male patient (Number 8) with a focal pubic bone lesion also refused to undergo biopsy or surgery, despite sustained medication. Since the patient had previous surgeries of both femurs due to prior fragility fractures, the patient was concerned about post-operative complications.
Among the four patients with negative 68 Ga-DOTATOC PET/CT results, two patients seemed to have plausible clinical conditions, which excluded the possibility of TIO. One patient had a long history of the anti-viral medication, adefovir, due to chronic hepatitis B virus infection. This patient history implied an "adefovir-induced  www.nature.com/scientificreports/ hypophosphatemic osteomalacia. " Another patient had a history of celiac disease in which intestinal phosphate absorption is reduced. Concordantly, the patient's calculated TRP value was above 85%. The remaining two patients with negative 68 Ga-DOTATOC PET/CT results did not have clear reasons for persistent hypophosphatemia, and they were given phosphate supplements.

Discussion
In this study, we detected the culprit tumors that induced osteomalacia in seven out of nine patients whose calculated TRP was suggestive of renal hypophosphatemia 26 29 . Among the nine patients who had suspicions for culprit lesion according to the PET/CT, seven were confirmed to have those lesions. In addition, the Singh study focused on unraveling the other uptake induced by fracture or degenerative changes beyond the strength of detecting the culprit lesion. We also observed such uptake, usually induced by fracture (Blue arrows in Fig. 2B highlight the rib fractures). Those lesions exhibited a mild uptake with symmetric distributions, which contrasted with the unilateral manifestation of most of the culprit lesions. Therefore, close examination is recommended for interpretation of the 68 Ga-DOTA-based PET radiotracers in patients who have concomitant fractures.
Sincere efforts have been made to reveal the culprit tumors in patients with TIO using various imaging modalities other than specific SSTR scan, including simple radiography, bone scan, CT, MRI, and 18 F-FDG PET/ CT [14][15][16]30 . However, almost all conventional modalities showed low performance due to the non-pathognomonic findings or small tumor size. This could be the reason for inappropriately delaying the diagnosis of TIO. Indeed, we also had patients whose culprit tumors were observed on CT, MRI, or 18 F-FDG PET/CT a few years before the 68 Ga-DOTATOC PET/CT. However, the presence of culprit tumors was not specifically demonstrated at that time and the opportunity for early detection was lost. Figure 3 presents one such representative case.
In this context, the specificity for SSTR with "one-shot" whole-body functional imaging available by 111 Indium octreotide scintigraphy is considered as a mainstay in assessing patients with TIO 18,31 . However, superior spatial www.nature.com/scientificreports/ resolution inherent in the PET/CT system, better pharmacokinetics, and a higher affinity toward SSTR by shorter half-life PET radiotracers with a cyclic chelator render 68 Ga-DOTA-based PET radiotracers the imaging of choice not only for patients with neuroendocrine tumors but also for patients with TIO 13,17 . The evaluation of treatment response or detection of the recurred/remnant tumor using post-treatment 18 F-FDG PET/CT is a popular approach in the field of oncology 32 . To the best of our knowledge, we have shown, for the first time, the additional value of follow-up with 68 Ga-DOTATOC PET/CT after the basal scan to detect residual tumors whose hypophosphatemia persists even after surgery. Our results provide another option in the management of patients with TIO using 68 Ga-DOTA-based PET/CT beyond the pivotal role of detecting the primary occult tumor. Previously, a case that also showed the usefulness of detecting the residual culprit tumor surgery using 68 Ga-DOTANOC PET/CT was reported, but an initial PET/CT for comparison was not available 33 .
There are a few limitations to this study. First, even though FGF-23 is a key hormone and a sensitive indicator of TIO, testing for FGF-23 is not available in clinical practice in our country, preventing us from getting FGF-23 results. For this reason, we had just two patients who had the serum FGF-23 test. Easy accessibility to FGF-23 assays would be helpful for the early detection of TIO. Nevertheless, the results of 68 Ga-DOTATOC PET/CT in this study were not affected by the results of the FGF-23 assay. Second, not all patients underwent whole-body PET/CT imaging, including that of the lower extremity, due to our inexperience. Given the positive lesion detected in the fibula of one patient, whole-body PET/CT imaging rather than torso imaging is recommended in these patients. Third, in the patients who had negative 68 Ga-DOTATOC PET/CT without definite plausible reasons, we did not perform any gene mutation tests. Hypophosphatemia-related genetic diseases, such as X-linked hypophosphatemic rickets or autosomal dominant hereditary hypophosphatemic rickets [34][35][36] , are very rare and but cannot be excluded. Finally, the total number of patients is quite low because of the rarity of the disease and limited accessibility of 68 Ga-labeled radiopharmaceuticals. Due to the small number of patients included in this study, we omitted the statistical analysis regarding the diagnostic performance of 68 Ga-DOTATOC PET/CT. www.nature.com/scientificreports/ In summary, 68 Ga-DOTATOC PET/CT appears to be a very robust and innovative imaging modality for detecting causative tumors in patients with suspected TIO and for detecting remnant or recurred tumors after surgery.