Baseline and longitudinal variability of normal tissue uptake values of [F]-fluorothymidine-PET images

⁎ Corresponding author at: Department of Radiology an sity Medical Center, 7057, 1007MB Amsterdam, Netherlan E-mail address: m.cysouw@vumc.nl (M.C.F. Cysouw). http://dx.doi.org/10.1016/j.nucmedbio.2017.05.002 0969-8051/© 2017 The Authors. Published by Elsevier Inc Article history: Received 22 February 2017 Received in revised form 14 April 2017 Accepted 4 May 2017


Introduction
. Previous research investigating [ 18 F]-FLT uptake as biomarker of response to treatment has proven it to be a useful tracer for assessing or predicting progression-free and disease-free survival for several types of cancer [4].
In oncological PET(/CT) studies, normal tissue uptake can be used both qualitatively and quantitatively to assess malignancy of suspected lesions. Typically, a lesion is considered malignant if tracer uptake exceeds background uptake of surrounding tissue in qualitative visual analysis. In addition, uptake values of reference tissues such as blood or liver are used to normalize tumor uptake in semi-quantitative analysis, generating a tumor-to-background ratio (TBR). However, when variability of uptake in normal tissues within patients is high, TBR may become an unreliable parameter reflecting true tumor activity rather poorly and obscuring discriminative value of quantitative PET. Simplified measures, such as standardized uptake values (SUV) and TBR have proven useful for response assessment using [ 18 F]-FLT-PET/CT [5]. However, to use SUV reliably one needs to control for technical image flaws, such as mistakes made in injected activity [6]. Additionally, in order to use these simplified methods during treatment, biodistribution and pharmacokinetics may not change drastically over time. Hence, an image quality control method is necessary to allow for appropriate quantification of tumor activity.
Much research has been performed into normal tissue uptake and its intra-patient variability in [ 18 F]-FDG-PET [7][8][9][10]. However, as the biodistribution of [ 18 F]-FLT is inherently different to that of [ 18 F]-FDG, results from these studies cannot merely be assumed to be applicable to [ 18 F]-FLT-PET studies. As [ 18 F]-FLT is establishing itself as a valuable oncological PET-tracer, researchers and physicians should have knowledge of its normal tissue uptake variability within and between patients.
In this study we aimed at deriving normal [ 18 F]-FLT uptake values and their precisions for several tissues and/or organs and to explore if these could be used to assess image quality for individual scans and/or longitudinally. As [ 18 F]-FLT is often used in lung cancer patients, we assessed the effect of tyrosine-kinase inhibitor (TKI) treatment on normal tissue uptake values in lung cancer patients. Tissues investigated were lung, blood pool, liver, and bone marrow. Several SUV types were investigated, with several volumes of distribution normalizations.

Patients
Images of 90 patients scanned with [ 18 F]-FLT-PET(/CT) at baseline (i.e. while receiving no systemic treatment), were gathered from six single-center study cohorts and investigated in a retrospective analysis. One cohort of 28 patients with non-Hodgkin lymphoma (NHL) consisted only of baseline scans [11]. Twenty-three out of 90 patients received double baseline scans in order to assess intra-patient variability, with head and neck cancer (n = 6), non-small cell lung cancer (NSCLC; n = 9), and breast cancer (n = 8), [12,13]. In two cohorts of patients with NSCLC, a total of 39 patients were, in addition to the baseline scans, also scanned early (after approximately 1 week) and late (one cohort after 4 and one cohort after 6 weeks) during treatment with a TKI [5,14]. A description of the included study cohorts is provided in Table 1. All included studies were approved by the medical ethical committee of the respective research institutes and patients gave informed consent for participation.

Image acquisition
For extensive descriptions of image acquisition methods we refer to the included studies' primary publications. Reconstruction methods and scanning modes (i.e. dynamic and static) with acquisition times (post injection) are summarized in Table 1. Static scanning was performed approximately 60 min post-injection of [ 18 F]-FLT. Frames of dynamic scans were averaged over 45-60 min, 45-65 min, and 50-60 min, depending on available frame durations, to generate comparable static images allowing evaluation of effect of uptake intervals on SUVs.

Quantitative image analysis
Volumes of interest (VOI) of fixed size and shape were placed in lung, liver, bone marrow, and ascending aorta. Within the lung, uptake may be a function of variation in perfusion, air density, liver spill-in, and diaphragmatic motion. Therefore, we placed 3 cm diameter spheres apically as well as basally. For blood pool, a VOI 2 or 4 voxels (depending on voxel size) in diameter with an axial length of 2 cm was placed in the ascending aorta, which was identified on low-dose CT or on dynamic PET-images acquired shortly after injection of 18 F-FLT. In six patients with head and neck cancer the aorta was outside the field of view, in which case the left carotid artery was used. In liver, a 3 cm diameter sphere was placed in the upper right lobe. Vertebrae were used to quantify bone marrow uptake; both single and multiple (3) lower thoracic vertebrae were delineated using circular VOIs (5 or 9 voxels, depending on voxel size), avoiding the vertebral cortex. Tissues with known or apparent disease were avoided.
From each VOI, SUVmean (mean SUV within the VOI), SUVmax (maximum SUV value within the VOI), and SUVpeak (mean SUV within a 12 mm diameter sphere positioned within the VOI to yield the highest value) were extracted. SUV was calculated according to the following equation: where AC = activity concentration (Bq/mL), ID = injected dose at time of scanning (Bq). Normalization factors were bodyweight (BW; kilogram), lean body mass (LBM; kilogram), and body surface area (BSA; m 2 ). The latter two were calculated according to the following equations: Normalization factors were indicated as suffix to SUV (e.g. SUVpeak-bw, being the peak SUV normalized to bodyweight).

Statistical analysis
For baseline SUVs, mean with standard deviation (SD) was calculated for (averaged) dynamic and static data, to illustrate SUVs as a function of acquisition time. In addition, coefficients of variation (COV%) were calculated to describe variability of uptake values. Shapiro-Wilks test was used to assess normality of data. For data with skewed distribution, data were log-transformed for statistical analysis. To assess differences between dynamic and static data for each SUV type, first Levene's test was used to assess equality of variances. Next, an independent samples t-test was applied to investigate differences between the two samples. Additionally, we calculate reference ranges using 5th-95th percentiles. Extreme outliers (b[Q 1 -3*IQR] or N[Q 3 + 3*IQR]) were censored for calculation of reference ranges. The effect of age and gender on baseline uptake values were analyzed using multivariate linear regression.
To investigate repeatability (i.e. intra-patient variability), repeatability coefficients (RC) were calculated. Relative differences and RCs were calculated as follows: where the SD is taken from the relative differences.
To illustrate the effect of treatment on normal tissue uptake, relative change of SUV was calculated using the following formula: Relative changes were described using mean ± SD. All statistical analyses were performed using IBM SPSS v22.0 (Armonk, USA).

Baseline uptake values
Baseline scans were categorized into two groups to investigate tissue uptake as function of uptake interval; one group consisted of dynamic images (frames averaged 45/50 to 60/65 min p.i.) and one group of static images (60 min p.i.).
The effects of age and gender on uptake values were analyzed in multivariate analysis, while correcting for acquisition type (static or dynamic). Age was only a significant modifier for lung SUVmax-bw (p = 0.035-0.046). Gender was a significant modifier for all SUV types of bone marrow (single vertebra), liver, and blood pool when using LBM for normalization (p = 0.003-0.046). For bone marrow (multiple vertebrae), gender was a modifier for SUVmean-LBM (p = 0.029). For lung, gender was a modifier for SUVmean-bsa and SUVpeak-bsa (p = 0.013-0.026).
Henceforth, the remainder of the paper will focus on results of SUVpeak normalized to bodyweight. All other data can be found in the supplemental data. Table 2 contains average SUVpeak-bw, of all investigated tissues (Supplemental Table 1 for SUVmean and SUVmax). No extreme outliers were identified. Blood pool and apical lung SUVpeak were significantly different between averaged dynamic and static images (p = 0.001 and p = 0.041, respectively). In contrast, SUVpeak of basal lung, liver, and bone marrow were not significantly different between dynamic and static images, for all normalization factors (p = 0.604, p = 0.126, and p = 0.444-0.592, respectively). For bone marrow using multiple vertebrae, only SUVmax was significantly different between dynamic and static images, regardless of normalization factor (p = 0.027-0.049).
The distribution of SUVpeak (COV%) is illustrated in Fig. 1 as a function of dynamic and static images. COVs of blood pool and bone marrow between static and dynamic images were similar within 1%. For apical and basal lung, COVs between dynamic and static images were −8.8% and 1.7% different, respectively. For liver, COVs between dynamic and static images were −10.8% different.
To further illustrate the differences in SUV between the averaged dynamic and static images, time-activity curves of mean uptake in the investigated tissues are displayed in Fig. 2 for a dynamic scan of a typical patient. A gradual decrease in activity concentrations can be seen for blood pool and lung. Notably, the time-activity curve of lung tissue has a very similar shape to the blood pool curve, suggesting that it is mainly blood pool which is measured in lung. A decrease in liver activity is noted over time, which corresponds to the data in Table 2. Bone marrow seems to be increasing at 55 min p.i., but it could have reached a plateau at 60 min p.i. since there were no significant differences in bone marrow SUVmean between averaged dynamic and static images.
Before defining reference ranges for SUVs of whole-body static scans (60 min p.i.), differences in SUVs between the static PET and PET/CT cohort were investigated. For liver and bone marrow, significant differences were found for SUVmean and SUVpeak (all normalizations; p = 0.000-0.024), but not for SUVmax. For lung, significant differences were found for SUVmax (all normalizations; p = 0.000-0.005), but not for SUVmean and SUVpeak. For blood pool, only PET/CT data were available for the static scans. As differences in SUV between PET and PET/CT static whole-body scans were observed, reference ranges were calculated for the hybrid PET/CT cohort only. Reference ranges for SUVpeak-bw (Supplemental Table 2 for other SUVs) of whole body static PET/CT scans 60 min post-injection were 0.44-1.04 for blood pool, 0.34-0.83 for lung (apical), 3.46-7.46 for liver, and 4.86-11.36 for bone marrow (multiple vertebrae).

Repeatability
Intra-patient variability of normal tissue SUVs between double baseline scans was investigated using RCs. There were no significant dissimilarities between the normalizations of each SUV, with differences in RCs between normalizations ranging 0.03-0.80%. For all SUV types, liver and bone marrow (multiple vertebrae) provided the lowest RCs.

Longitudinal variability
Two cohorts of NSCLC patients were scanned at baseline, and early (approximately 1 week) and late (4 or 6 weeks, respectively) after starting treatment with TKIs. Since between the SUVs of late scans of the two cohorts (at 4 and 6 weeks) no significant differences in relative changes from baseline were found, these data were pooled together. For one cohort blood pool could not be evaluated due to absence of coregistered CT and early dynamic PET images [14]. In the other cohort liver could not be evaluated due to the chest only scan trajectory [5]. Table 3 contains the mean relative changes of SUVpeak-bw during systemic treatment (Supplemental Table 3 for SUVmean and SUVmax). Early during treatment the smallest relative changes were observed for blood pool (−1.24 ± 15.71%). In contrast, blood pool had the largest relative changes late during treatment (7.89 ± 17.63%). It should be For further illustration, Fig. 3 exhibits relative changes of SUVs, respectively, from baseline for all individual patients, in which the large ranges of changes from baseline are clearly visible (Supplemental Fig. 2 for absolute changes). For each tissue, the SUV with optimal intra-and inter-patient variability is displayed. Fig. 4 illustrates images of two patients with aberrant increases in uptake (early  during treatment) in lung (+209%) and bone marrow (+67%), respectively. Remarkably, these patients only demonstrated these aberrant increases in one tissue type.

Discussion
[ 18 F]-FLT is a PET tracer increasingly used in oncological response assessment PET(/CT)-studies, providing a biomarker of tumor proliferation for several types of cancer, such as NSCLC, head and neck squamous cell carcinoma, and breast cancer [4]. The aims of this paper on normal reference tissue uptake values of [ 18 F]-FLT-PET(/CT) images were to evaluate their inter-and intra-patient variability, and to assess the longitudinal variability of these values during systemic treatment with TKIs. In addition to the most commonly used reference tissues, being liver and blood pool, we also investigated lung and bone marrow uptake.
Knowledge of reference values of normal tissue is critical for qualitative image analysis, where malignancy is suspected if lesion uptake exceeds background uptake. Furthermore, in quantitative analysis a TBR may be used to distinguish between benign and malignant lesions and for measuring response to treatment. Additionally, reference tissue uptake may be used to calculate a threshold for malignancy, as done for [ 18 F]-FDG-PET in the PERCIST criteria using mean liver uptake [15]. Also, reliability of (semi-)quantitative analysis of tumor tracer uptake is affected by the technical quality with which a PET study is performed [16,17]. Hence, a reliable reference value with low inter-patient variability may serve as an image-based quality control method.
SUVmean, SUVmax, and SUVpeak have different characteristics regarding sensitivity to noise and inter-and intra-observer variability caused by VOI placement [18,19]. Most commonly, in clinical practice and clinical studies, SUVmax is used due to its low dependency on accurate and reliable VOI placement and hence ease of applicability. However, its sensitivity to noise level may render it an unreliable parameter of normal tissue tracer uptake, especially in multi-center studies. SUVpeak seems an appropriate alternative in this context, due to its intrinsically lower sensitivity to noise fluctuations and similarly low dependency on VOI placement. Lodge et al. found that for quantification of primary brain tumors SUVpeak indeed provided best repeatability at 60 min post-injection, making it an attractive SUV for tumor quantification [20]. In this study, however, while intra-patient variability was different between SUV types, conclusions regarding the most reliable tissues (liver and bone marrow) were similar regardless of SUV type.
Considering the intra-patient variability of normal tissue uptake in this study, liver seems to be the most stable reference tissue (Fig. 3). Remarkably, SUVmax demonstrated the lowest intra-and inter-patient variability for this tissue. However, SUVpeak had very Results are relative changes (mean ± SD) from baseline, in percentages. . Each line represents an individual patient. Blue and red lines indicate relative changes below and above repeatability coefficients (%), respectively, for the SUV with optimal repeatability determined per tissue type (Fig. 3). For a patient in (B) relative change in lung SUVmean of 209% exceeded the y-axis. similar repeatability and is preferred due to its lower sensitivity to image noise. The second lowest RCs were observed for bone marrow SUVpeak when derived from multiple vertebrae. Additionally, SUVpeak was not different between averaged dynamic and static images for both liver and bone marrow ( Table 2). Blood pool and lung SUVs seemed less stable than liver and bone marrow, and these tissues should not be used as reference.
During treatment (with TKIs) we observed changes in biodistribution larger than test-retest limits (Fig. 3), which could be a consequence of altered tracer kinetics and/or metabolism during systemic treatments. Drugs affecting glucuronidation of FLT in liver or renal clearance of FLT may result in altered area-under-the-curves of the arterial input functions. Also, drugs affecting tissue proliferation (specifically bone marrow in this study) may explain these changes in FLT uptake. The observed treatment-induced alterations in FLT kinetics can explain why in previous research tumor-to-blood ratios seemed most appropriate for assessment of treatment response in locally advanced breast cancer and NSCLC, when correlated with full kinetic modeling [5,21]. A rationale for this is that tumor SUVs fluctuate with blood pool activity, and use of tumor-to-blood ratios may compensate for variations in arterial input functions. Overall, this emphasizes the importance of dynamic PET/CT validation studies before SUVs can be used for tumor quantification during treatment.
Taken together, we recommend liver and bone marrow to be used as normal tissue references for [ 18 F]-FLT-PET images. SUVpeak of these tissues demonstrated good repeatability and was not affected by differences in uptake intervals. Liver uptake can be easily quantified by placing a 3 cm diameter spherical VOI in the right upper liver lobe and measuring SUVpeak. For bone marrow quantification we suggest delineating three adjacent vertebrae, avoiding the vertebral cortex, and measuring SUVpeak. We observed no significant advantage of using either bodyweight, lean body mass, or body surface area for SUV normalization in this context. However, LBM and BSA may be affected by gender type, thus using bodyweight is preferred.
As both liver and bone marrow uptake can change during systematic treatment a conservative approach could be to assess liver and bone marrow and verify if these uptakes are within expected ranges and remain constant during treatment. If this would not be the case it does not necessarily imply poor image quality and/or poor execution of the PET/CT study, but it could alert the observer to perform additional quality control on the images such as verification of injected activities, uptake times and acquisition/reconstruction settings. Also it should alert the observer that there are changes in tracer uptake biodistribution that may affect lesion uptake interpretation as well. The changed biodistribution could namely also result from a change in arterial input function as was shown before for tumor quantification for [ 18 F]-FLT and [ 18 F]-FDG [5,22].
This study has several limitations, among which the heterogeneity between studies due to the retrospective nature. However, Kuhnert et al. found that while different reconstructions provide different SUVs for tumors, this was not the case for liver tissue [23]. This is probably due to the fact that differences in noise and contrast between reconstructions do not affect SUV of homogeneous tissue when using a mean uptake value, such as SUVmean or SUVpeak. Despite heterogeneity between cohorts we were able to identify liver and bone marrow as the most reliable reference tissues, with similar RCs as seen for tumor SUVs [13]. In addition, perhaps a smaller reference range for liver and bone marrow uptake can be determined in future studies performing commonly used PET acquisition modes for 18 F-FLT-PET images (i.e. static whole body scans performed 60 min after injection) with larger sample sizes.

Conclusion
We investigated blood pool, lung, liver, and bone marrow uptake of [ 18 F]-FLT -PET(/CT) images in oncological patients. Liver and bone marrow seemed most appropriate for image-based quality control due to high repeatability. However, clinicians should be aware of the variable susceptibility of normal tissue SUVs to physiologic treatment effects in patients undergoing systemic treatments. Simultaneous evaluation of liver and bone marrow uptake in longitudinal response studies may be used to assess image quality, where changes in SUVs outside repeatability limits should trigger investigators to perform additional quality control on individual PET images.

Compliance with ethical standards
Part of the research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www. imi.europa.eu; grant agreement number 115151), whose resources are composed of a financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and an in-kind contribution from the companies of the European Federation of Pharmaceutical Industries and Associations. Authors Cysouw, Kramer, de Langen, Frings, Wondergem, Aboagye, Kenny, Kobe, Hoekstra, Boellaard declare no conflicts of interest. Author Wolf is on advisory boards (compensated) of, and receives lecture fees from AstraZeneca, BMS, Boehringer-Ingelheim, MSD, Clovis, Novartis, Pfizer, and Roche, and has received research support from Novartis, Pfizer, BMS, and MSD. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.