The Role of F18-FLT PET / CT in Assessing Early Response to TARE and TACE in Patients with Primary and Metastatic Liver Tumors

Objective This study aims to identify F18-FLT PET / CT's role in the assessment of early response to intraarterial chemoembolization and radioembolization treatments in patients with unresectable primary and metastatic liver tumors. Methods This single-center study included 63 patients who underwent F18-FLT PET / CT for response evaluation after TACE and TARE treatments. After excluding 20 patients due to various reasons, 43 patients were analyzed. The compatibility of change in semi-quantitative values obtained from the F18-FLT PET / CT images with the treatment responses detected in F18-FDG PET / CT, CT, and MR images was evaluated. Results There was no correlation between early metabolic, morphological response, F18-FLT visual change, ΔSUVmax, ΔSUVmean, and ΔSUVpeak values. There was no signicant correlation between F18-FLT visual change, ΔSUVmax, ΔSUVmean, ΔSUVpeak, and OS, PFS for the target lobe PFS for the whole-body. The survival distributions for the patients with and without >30% change in ΔSUVmax and ΔSUVpeak values were statistically signicantly different (p < .009 and p < .024, respectively). primary metastatic tumors. signicant relationship between PFS for target lobe and change in SUVmax and SUVpeak values.


Introduction
Both metastases and primary malignancies, such as hepatocellular carcinoma (HCC) and cholangiocellular cancer, are common in the liver. Metastases are the most common liver malignancy, and leading tumors that metastasize to the liver are colorectal cancer, neuroendocrine tumors, other gastrointestinal cancers, and breast cancer. HCC is the sixth common cause of cancer and the third common cause of cancer-related deaths worldwide [1,2]. Since liver involvement is effective on survival, curative surgical applications are the rst-line therapy, either together with adjuvant chemotherapy or alone, providing the most signi cant survival advantage. However, surgery cannot be applied to most patients at diagnosis or tumor recurrence due to advanced-stage disease or inappropriate clinical status [2,3]. Local ablative applications such as radiofrequency (RFA), microwave (MWA) and cryo-ablation, irreversible electroporation (IRE), endovascular transarterial chemoembolization (TACE), and transarterial radioembolization (TARE) provide minimally invasive and safe treatment [4][5][6][7][8][9][10][11]. It has been reported that Page 3/16 TARE and TACE provide a long-term survival advantage and low toxicity, especially in patients with good performance and low tumor burden [7][8][9][10][11].
F18-FLT, an analog of thymidine, is phosphorylated with thymidine kinase-1 (TK1) and is converted to F18-FLT-monophosphate, which can not penetrate DNA and is trapped in the cytosol. F18-FLT is a TK1speci c substrate that increases in proliferating cells while not found in silent cells and correlates with a proliferation marker Ki-67 index [22,23]. Imaging with F18-FLT has advantages such as non-invasive quantitation of cell proliferation, three-dimensional tumor imaging, and evaluating the whole tumor proliferation heterogeneity in multiple tumor areas simultaneously. Studies show that tumor proliferation changes can be detected early with F18-FLT PET / CT after radiotherapy [22][23][24][25][26][27]. Knowing that TARE is an internal radiotherapy method, this study aims to describe F18-FLT PET / CT's role in assessing early response to TARE in patients with primary and metastatic liver tumors.

Materials And Methods
Ethics committee approval (I3-117-19) was obtained for this single-center study with prospective and retrospective components. The study was performed under the Helsinki Directive and Good Clinical Practices Guidelines. Informed consent was obtained from all volunteers included in the study.

Patients:
This study included the patients who underwent TACE and TARE treatment for histologically / cytologically or radiologically diagnosed primary (HCC, cholangiocellular carcinoma, etc.) or metastatic liver tumor; staged with CT, MR, and F18-FDG PET / CT or PET / MR imaging; has Eastern Cooperative Oncology Group (ECOG) performance score ≤ 2; was over 18 years old; were found suitable for TARE and TACE treatments; were followed for more than three months; had available data; gave informed consent to participate.
Patients with severe pain and claustrophobia, etc., preventing imaging; ECOG performance score> 2; patients who left the follow-up before the 3rd month; whose data are not available; who did not want to participate in the study were excluded.
There was no intervention in the treatment selection or management of the patients. The relevant specialist chose the treatment according to the standard evaluations.

F18-FLT PET / CT imaging protocols and evaluation:
The presence or history of systemic or local ablative therapy, chronic disease, etc. that can affect the evaluation was questioned and noted. Oral hydration and bladder emptying before imaging was provided to reduce the total body radiation dose and increase the image quality. Approximately 60 minutes after the F18-FLT were given intravenously, the whole-body PET / CT images were taken. Following CT for attenuation correction, and anatomical correlation, whole-body PET images were obtained, in the supine position, from the vertex to the middle of the thigh, and 3 min per bed. PET /CT Discovery ST (GE Healthcare Waukesha, Wisconsin, USA) was used for PET hybrid imaging. After assessing maximum intensity projection (MIP), cross-sectional, and fusion images, areas with high (hyper-metabolic), heterogeneous (mixed), similar (iso-metabolic), and low (hypo-metabolic) uptake from adjacent liver parenchyma were noted.
F18-FLT PET / CT was performed twice: as a baseline before treatment and for response evaluation after treatment. Appearance and SUVmax, SUVmean, SUVpeak values of the target lesion-de ned as sole or the most extensive lesion in the target lobe were noted. Since the reference parenchymal SUV values of the patients showed a signi cant difference both between the patients and the pre-and post-treatment images of the same patient, the target background ratio (TBR) of the target lesions were calculated by proportioning the SUV values of the target lesion to reference values and were evaluated separately.
Patients were divided into groups with and without the change of SUV values calculated by the difference between the target lesion's post-treatment and pre-treatment SUV values, which were calculated and referred to as delta (Δ) SUV values.

Statistical analysis:
The changes between baseline and post-treatment F18-FLT PET / CT images were compared to the responses detected with F18-FDG PET / CT and CT/MRI, evaluated according to the PERCIST and RECIST 1.1 criteria, respectively, and progression-free survival and overall survival. All statistical analyses were performed using IBM SPSS for Windows version 15.0 (SPSS, Chicago, IL, USA). Shapiro-Wilks and Kolmogorov-Smirnov test was used to assess the assumption of normality. The continuous variables that do not have normal distribution were expressed as median (minimum-maximum). For non-normallydistributed continuous variables, differences between groups were tested using Mann Whitney U-test and Kruskal Wallis test. Lastly, Pearson chi-square analysis and Fisher's exact test determined associations between categorical variables, while Pearson and Spearman correlation analysis speci ed associations between continuous variables. The survival times of groups were obtained with Kaplan Meier analysis, and the difference between groups in survival times compared with the Log Rank test. A two-sided p-value<0.05 was considered statistically signi cant.

Findings Patients
Sixty-three consecutive patients were included in the study between December 2018 and January 2020, who underwent pre-and post-treatment F18-FLT PET/CT to evaluate response to TARE and TACE treatments. Although all patients underwent baseline imaging, three of the TACE-receiving patients and sixteen of the TARE-receiving patients could not undergo F18-FLT PET/CT or other imaging for response evaluation either due to decreased performance status that hindered further procedure or death.
Three patients who received TACE did not undergo PET/CT or MR/CT imaging to evaluate response to treatment, and most of their data were missing. Only one TACE-receiving patient who underwent all imagings and survived more than six months had available data. This one TACE-receiving patient excluded from analysis, and 43 TARE-receiving patients were analyzed to have a homogenous population and statistical analysis.
30/43 patients were male, and the median age was 63 (min 38-max 79) years. Twenty-four patients had a primary liver tumor, and 14, 2, 2, 1 patient had liver metastasis from the colon, gastric, breast, and pancreas cancer, respectively. Detailed patient characteristics are listed in Table 1 of the hypermetabolic lesions remained, while 1/6 became mixed, 1/6 became iso-and 2/6 became hypo-metabolic. 1/5 of the target lesions with mixed appearance persisted; the rest majority became hypometabolic. 1/9 iso-metabolic lesion became hypo-metabolic while the remaining 8/9 lesions remained. All hypo-metabolic lesions persisted. PET/CT and contrast-enhanced liver MR images of the patient with persistent hypermetabolic lesions and progressive disease are presented in Figure 1.
There was no correlation between the diagnosis, longest diameter of the target lesion, volume and percent of tumor in the target lobe, age, the number of lesions in the target lobe, early metabolic, morphological response and F18-FLT visual change, ΔSUVmax, ΔSUVmean, ΔSUVpeak, ΔSUVmaxTBR, ΔSUVmeanTBR, and ΔSUVpeakTBR values. Calculated p values from statistical analyses are given in Table 3. There was no signi cant correlation between F18-FLT visual change, ΔSUVmax, ΔSUVmean, ΔSUVpeak, ΔSUVmaxTBR, ΔSUVmeanTBR, and ΔSUVpeakTBR and OS, PFS for target lobe and PFS for whole-body (Table 3). A log-rank test was run to determine if there were differences in the target lobe's progressionfree survival distribution for the ΔSUVmax and ΔSUVpeak groups when cut-off >30% change was applied. The survival distributions for the patients with and without a change in ΔSUVmax were statistically signi cantly different, χ2(2) = 6,774, p < .009. The survival distributions for the patients with and without >30% change in ΔSUVpeak were also statistically signi cantly different, χ2(2) = 5,095, p < .024. Statistical analysis could not provide a median value and estimated survival chance at 209. day was 0,549±0,129 STD for 17 patients with no change in SUVmax, while the estimated survival chance at 92. day was 0,500 ±0,098 STD in patients with >30% change in SUVmax. Estimated survival proportion at 209. day was 0,514 ±0,134 in 16 patients without change in SUVpeak value; while this proportion was 0,519 ±0,096 at 90.day (Figure 2, Table 3).

Discussion
Both metastases and primary malignancies are commonly seen in the liver. As liver involvement is effective on survival, early detection of response has a crucial role in patient management (2)(3)(4)(5). After LRTs such as TARE and TACE, there is no standard response evaluation and follow-up protocol; valuation is performed at different times with PET / CT, CT, or MR, depending on the center's practice and patient management. Since response assessment with CT and MRI takes a longer time and have their limitations, PET, a functional and molecular imaging technique, is used for early response evaluation with agents that re ect tumor-speci c metabolism. Providing metabolic and anatomical information, PET / CT, and PET / MR have become the leading imaging techniques in cancer patient management [12][13][14][15][16][17][18][19][20][21][22].
F18-FDG PET / CT is the most common metabolic imaging method in diagnosing, staging, restaging and evaluating the treatment response due to increased glucose metabolism in many types of cancer. F18-FDG PET-CT can be used to assess treatment response in poorly differentiated and high-grade tumors. However, since small and well-differentiated tumors (such as HCC, NET) shows low or no FDG uptake due to low glucose metabolism and cellularity, imaging with new tumor-speci c agents is needed [17][18][19][20][21][22][25][26][27]. PET / CT imaging with F18-FLT re ects cells' proliferation, is a non-invasive method in a personalized treatment approach [25][26][27].
In addition to complex and competing factors in the FLT uptake mechanism, there are notable differences between patient preparation, imaging time after injection, protocol, amount of injected activity, reconstruction method, timing before and after treatment, and patient numbers in studies withF18-FLT PET / CT. The power of studies is weak, as the number of patients achieving all the treatments and an ideal comparison in different disease groups is limited. It is noteworthy that data and various analysis techniques, such as semi-quantitative or visual evaluation are used [22][23][24][25][26][27][28][29][30]. Considering all the factors mentioned above are in uential in the response evaluation, and there is no standard protocol, it is di cult to compare the studies. colorectal cancer liver metastases. They reported that 26/33 metastases were visible after kinetic spatial ltering (FLT-PETKSF). FLT-PET SUVave or SUVmax and FLT-PETKSF showed a signi cant decrease in responders two weeks after the rst-line chemotherapy. It has been reported that the change in FLT can distinguish those who responded to the treatment with 83% sensitivity and 78% speci city from nonresponders [30]. In this current study, F18-FDG and F18-FLT PET / CT were evaluated for the treatment response after TARE, an LRT, not a systemic treatment. There was no signi cant correlation between ΔSUVmax, ΔSUVmean, and ΔSUVpeak values among the patients with and without response; therefore, sensitivity and speci city could not be calculated.
The critical point in the early evaluation of the treatment response is to distinguish non-responder to discontinue unnecessary treatment, thus avoid toxicity and cost. It is essential to determine the resectable disease from those that require more aggressive treatment. Patients with shrinkage of tumors up to 30% are considered to have stable disease, according to RECIST 1.1, and unresponsive to treatment [12][13][14][15][16][17]. In this study, tumor sizes of patients with stable disease decreased, re ecting the bene cial effect of treatment. However, since this decrease in size remained below the RECIST 1.1 criteria, it was accepted as a stable disease and unresponsive to treatment. It should be recognized that patients with stable disease, especially with colon cancer, are admitted as responders and continue to receive systemic therapy in clinical practice [32]. Generally, chemotherapy-refractory liver metastases are referred for LRTs such as TARE. Thus, even de ned stable disease can provide more prolonged survival and can be accepted as responsive. If patients with stable disease are admitted as responders to therapy, statistical analysis can be found signi cant in long-term follow-up results. Since there was no increase in the F18-FLT uptake after treatment in any of the patients, it could not be evaluated whether this was related to progressive disease. Since the tumors were hypometabolic in most patients, the changes in the F18-FLT SUVmax, SUVmean, and SUVpeak values were not signi cantly different in responder and non-responder patients.
Since liver resection was not performed on any of the patients after embolization, except for the transplantation patient, post-treatment histopathological tumor changes and correlation with F18-FLT values could not be evaluated.
The most signi cant limitation of this study is the small sample size, consequent heterogeneous patient population, and the small number of patients who responded to the therapy. Therefore, in statistical analysis, results reaching a signi cant degree could not be obtained in SUV parameters. TARE candidate patients have many different clinical scenarios such as highly variable liver lesion number and size, disease stage, history of single or multi-step systemic treatment, liver resection, transplant, and LRT's.
Also, since there is a clear difference in disease etiologies, current clinical and radiological status, it was impossible to standardize the patient group. Further studies with larger and standardized patient populations are needed.
It can be argued that the timing of the F18-FLT was not right. TARE is an internal radiotherapy procedure, and response to radiotherapy is generally evaluated later than chemotherapy/ selective systemic therapies [25,26]. F18-FDG PET/CT and F18-FLT PET/CT imaging were done ≥4 weeks after the procedure. Studies evaluating radiotherapy response revealed a signi cant relationship between F18-FLT PET/CT and response or survival in patients with head-neck, esophageal, breast, lung, rectal, etc. cancer [26]. This study aimed to distinguish real responders from non-responders who were grouped based on post-radiotherapy response assessment techniques F18-FDG PET/CT and CT or MR. No correlation was found between the semi-quantitative values such as ΔSUVmax, ΔSUVmean, ΔSUVpeak, SUVmaxTBR, SUVmeanTBR, and SUVpeakTBR values calculated from F18-FLT PET / CT images. There was a signi cant relationship between OS and ΔSUVmax, and ΔSUVpeak values only when >30% change accepted as signi cant.

Conclusion
It has not been shown a signi cant relationship between the changes in F18-FLT PET/CT SUVmax, SUVmean, SUVpeak, SUVmaxTBR, SUVmeanTBR, and SUVpeak values with neither with the early metabolic response in F18-FDG PET/CT nor with the morphological response in contrast-enhanced CT or MR in patients with primary and metastatic unresectable liver tumor undergoing embolization therapy.
There was a signi cant relationship between OS and ΔSUVmax, and ΔSUVpeak values when >30% change accepted as signi cant. Reproducible and re-applicable clinical data from a larger and standardized patient population is required to assess the role of F18-FLT PET-CT in the evaluation of response to TARE treatment. Although the clinical use of F18-FLT PET / CT is limited, it can be used as an alternative/complementary to F18-FDG PET / CT in the early evaluation of the treatment response in patients undergoing TARE for primary or secondary liver tumor.

Declarations
Funding: Author's own work.