The feasibility of [18F]EF5-PET/CT to image hypoxia in ovarian tumors: a clinical study

Rationale Evaluation of the feasibility of [18F]EF5-PET/CT scan in identifying hypoxic lesions in ovarian tumors in prospective clinical setting. Methods Fifteen patients with a suspected malignant ovarian tumor were scanned with [18F]EF5 and [18F]FDG-PET/CT preoperatively. The distribution of [18F]EF5-uptake, total intraabdominal metabolic tumor volume (TMTV), and hypoxic subvolume (HSV) were assessed. Results [18F]EF5-PET/CT suggested hypoxia in 47% (7/15) patients. The median HSV was 87 cm3 (31% of TMTV). The [18F]EF5-uptake was detected in primary tumors and in four patients also in intra-abdominal metastases. The [18F]EF5-uptake in cancer tissue was low compared to physiological excretory pathways, complicating the interpretation of PET/CT images. Conclusions [18F]EF5-PET/CT is not feasible in ovarian cancer imaging in clinical setting due to physiological intra-abdominal [18F]EF5-accumulation. However, it may be useful when used complementarily to FDG-PET/CT.


Introduction
Ovarian cancer (OC) is the most lethal gynecological malignancy, and the majority of patients are diagnosed at an advanced stage [1]. Although OC is initially chemosensitive, most women experience multiple and finally chemoresistant relapses. The survival odds have not markedly improved despite extensive research, and completeness of surgery is still a major prognostic factor [2].
The presence of hypoxic regions in solid tumors is associated with a poor prognosis for many cancer types [3][4][5]. Hypoxia-mediated chemoresistance is also the greatest clinical challenge in OC [6,7].
There is a considerable need for non-invasive imaging of tumor hypoxia since it provides additional information, which could be integrated into strategies of treatment [8]. The method can improve therapeutic outcomes by predicting chemoresistance and selecting potentially treatment-resistant tumors for targeted surgery. 18 F-nitroimidazolpentafluoropropylacetamide ([ 18 F]EF5) is one of the extensively investigated and clinically tested tracers of tissue hypoxia [9,10]. 18 F]EF5 belongs to the nitroimidazole group and has considerable membrane permeability and capability to accumulate in viable hypoxic, though not in apoptotic or necrotic cells [11][12][13].
Since there is no systematic data evaluating the eligibility of hypoxia-imaging among patients with ovarian malignancy, we conducted the prospective clinical study to evaluate the feasibility of [ 18 F]EF5-PET/CT scan in identifying hypoxic lesions in ovarian tumors.

Study population
This prospective non-randomized study was conducted at Turku University Hospital, Finland, between November 2017 and June 2019. Patients between 38 and 79 years of age with ovarian tumor, who were not pregnant, nursing, or had a history of previous malignancies were included.
Ethical approval was obtained from the institutional review board (18.10.2016 §443), and all subjects signed an informed consent form, ClinicalTrials.gov identifier: NCT04001023.
A whole-body contrast-enhanced [ 18 F]FDG-PET/CT and [ 18 F]EF5-PET/CT of the abdomen were performed on separate days preoperatively. PET/CT images were then evaluated by a nuclear medicine specialist and gynecological oncologist to assess the distribution of the cancer and to determine regions of suspected hypoxia in the intraabdominal tumor load for targeted biopsies for future research.

PET/CT scanning procedure
The PET/CT studies were performed with a digital PET/ CT scanner: Discovery MI (General Electric Medical Systems, Milwaukee, WI, USA). It has combined PET/ CT-scanners with a 128-slice CT and a 3D PET imaging capability. The PET imaging field of view (FOV) was 70 cm in diameter and 20 cm in axial length. To obtain attenuation correction for 511 keV photon distribution, the transmission scan was performed using a low-dose (noise index 30, automatic 3D current modulation, 10-120 mAs, and 120 kVp) CT protocol.
The patients received an intravenous injection of 370 MBq of 18 F-EF5. A static emission scan was acquired 180 min from the tracer injection to cover the entire abdomen (3 bed positions, 7.5 min/bed). The patients voided prior to the scan. The sinogram data was corrected for deadtime, decay, and photon attenuation and reconstructed in a 256 × 256 matrix. Image reconstruction followed the  The scans were performed in a random order depending on the availability of the [ 18 F]EF5 and camera. The mean interval between scans was 2 (range 1-7) days. A hypoxic voxel was defined using a threshold tumor to gluteus maximus muscle ratio (TMR) for [ 18 F]EF5uptake of 1.5, based on earlier experience [14]. The hypoxic subvolume (HSV) was defined as the sum volume of all lesions with TMR over 1.5.

Statistical analyses
Statistical analyses were performed using JMP Pro 13 software from SAS. Continuous variables were compared using a Wilcoxon rank-sum test. A non-parametric Spearman rank correlation test was used to evaluate the association between SUVmax values. Two-tailed P values < 0.05 were considered statistically significant.
Patients' clinical and imaging characteristics are presented in Table 1   Our study included two patients with non-malign tumors (patient nr 8 and 11), which presented no The physiological uptake of [ 18 F]EF5 in the gall bladder/bile, small intestine, and urinary bladder was notably higher than in the tumors (Fig. 3).
A demonstrative EF5-and FDG-PET/CT images of a patient with advanced ovarian cancer are presented in Fig. 4.

Discussion
Hypoxia is a common phenomenon in cancer with 50-60% of solid tumors containing hypoxic regions [15]. While hypoxia has a well-established role in promoting hematogenous metastases of cancer cells [16], the hematogenous spread is rare in OC at the time of diagnosis [17]. It should also be noted that the role of hypoxia in the transcoelomic spread to the peritoneum and omentum (common to OC) has not been widely investigated. On the basis of our study, [ 18 F]EF5-PET/CT suggested hypoxia in half of the patients and the distribution of [ 18 F]EF5-uptake was variable. [ 18 F]EF5-uptake was detected mainly inside the ovarian tumor and less often in metastases. One preclinical study suggested a hypoxic environment to induce omental/ peritoneal metastases [18]. Another study [19] which included two OC patients detected EF5-uptake and severe hypoxia in a peritoneal carcinosis biopsied laparoscopically promptly after the injection of EF5. Our cases with widespread peritoneal carcinosis typically had several [ 18 F]FDG-avid areas but only one patient had [ 18 F]EF5-avid peritoneal lesion.
The previous hypoxia imaging studies are conducted mostly on solid and locally advanced tumors [3,5,14,20,21] [22], where cancer cells rely widely on glycolysis and reduce their respiration regardless of tissue oxygenation level. Unlike OC, head and neck and lung cancer are isolated tumors that are not surrounded by physiologically EF5-affine tissues. Our study is prospective, and two tumors eventually appeared to be benign. Nevertheless, we consider it important to present them especially as they showed no EF5 uptake.
Previously, two excretory paths of highly lipophilic [ 18 F]EF5-tracer have been demonstrated [23,24]. In the latter, it was assumed that due to slow tracer biliary excretion, only small amounts of activity would be seen in the small intestine. However, our study revealed an excessive [ 18 F]EF5-uptake in the bile and small intestine. In contrast to the supradiaphragmatic tumors, this phenomenon imposes limitations on assessing tumors presenting weak [ 18 F]EF5-uptake. Especially when located near to the intestine, metastases may be easily be mistaken to physiological uptake and remain unnoted.

Conclusion
Non-invasive hypoxia imaging with [ 18 F]EF5-PET/CT is possible, but its clinical use is restrained by the weak tumor uptake of the tracer compared to the non-specific uptake in excretory organs. The potential usefulness of with [ 18 F]EF5-PET/CT in OC could be complementary to FDG-PET/CT with the intent to determine high-risk patients. The role of hypoxia in OC is intensively studied and [ 18 F]EF5-PET/CT forms an attractive tool for patient stratification.