Is interim 18F-fluoride PET/CT a predictor of outcomes after radium-223 therapy?

Objective To determine whether an interim 18F-fluoride positron-emission tomography/computed tomography (PET/CT) study performed after the third cycle of radium-223 dichloride (223RaCl2) therapy is able to identify patients that will not respond to treatment. Materials and Methods We retrospectively reviewed 34 histologically confirmed cases of hormone-refractory prostate cancer with bone metastasis in patients submitted to 223RaCl2 therapy. All of the patients underwent baseline and interim 18F-fluoride PET/CT studies. The interim study was performed immediately prior to the fourth cycle of 223RaCl2. The skeletal tumor burden-expressed as the total lesion fluoride uptake above a maximum standardized uptake value of 10 (TLF10)-was calculated for the baseline and the interim studies. The percent change in TLF10 between the baseline and interim studies (%TFL10) was calculated as follows: %TFL10 = interim TLF10 - baseline TLF10 / baseline TLF10. End points were overall survival, progression-free survival, and skeletal-related events. Results The mean age of the patients was 72.4 ± 10.2 years (range, 43.3-88.8 years). The %TLF10 was not able to predict overall survival (p = 0.6320; hazard ratio [HR] = 0.753; 95% confidence interval [CI]: 0.236-2.401), progression-free survival (p = 0.5908; HR = 1.248; 95% CI: 0.557-2.797) nor time to a bone event (p = 0.5114; HR = 1.588; 95% CI: 0.399-6.312). Conclusion The skeletal tumor burden on an interim 18F-fluoride PET/CT, performed after three cycles of 223RaCl2, is not able to predict overall survival, progression-free survival, or time to bone event, and should not be performed to monitor response at this time.


INTRODUCTION
Baseline whole-body 18 F-fluoride PET/CT is ideal for staging and restaging prostate cancer and has been shown to be an independent prognostic imaging biomarker of patients undergoing radium-223 dichloride ( 223 RaCl 2 ) Radiol Bras. 2019 Jan/Fev;52(1): [33][34][35][36][37][38][39][40] After successful treatment of osteoblastic bone metastases, an osteoblastic reaction (flare) can occur, which increases bone uptake even in responsive cases. That can be confused with the osteoblastic reaction and inflammation that occur in response to tumor-associated growth factors during progression. This phenomenon has been well described in conventional bone scintigraphy, and that method is therefore not recommended for use as the sole means of determining the response to treatment (5,6) .
Although interim studies performed with 18 F-FDG PET/CT can change the management of patients with a variety of cancer types (7)(8)(9)(10) , the exact role of 18 F-fluoride PET/CT in evaluating the early response to therapy (interim study) is not well established. The importance of 18 F-fluoride PET/CT has extended beyond the diagnosis of metastases to the evaluation of optimal strategies for use in patients submitted to treatment with new therapeutic agents. Chemotherapy, hormone therapy, immunotherapy, and radionuclide therapies such as those involving 223 RaCl 2 (11) are costly approaches. Therefore, the ability to predict response, thereby avoiding overtreatment and reducing costs, will improve patient management. The purpose of this study was to determine whether an interim 18 F-fluoride PET/CT study is able to evaluate treatment responses in prostate cancer patients submitted to 223 RaCl 2 therapy.

MATERIALS AND METHODS
The local institutional review board approved this retrospective analysis (reference no. PA14-0848). We retrospectively reviewed histologically confirmed cases of hormone-refractory prostate cancer with bone metastasis in patients receiving 223 RaCl 2 therapy and undergoing two 18 F-fluoride PET/CT studies-a baseline study and an interim study (immediately prior to the fourth cycle of 223 RaCl 2 )-at our facility. All patients completed at least four cycles of 223 RaCl 2 (Xofigo; Bayer Pharma AG, Berlin, Germany), receiving intravenous infusions of 50 kBq/kg (1.4 µCi/kg) of 223 RaCl 2 at monthly intervals. 18 F-fluoride PET/CT images were acquired immediately prior to initiation of the 223 RaCl 2 therapy (baseline study) and immediately before the fourth cycle (interim study). True whole-body PET images were obtained 50-60 min after intravenous injection of 158-370 MBq of 18 Fsodium fluoride in dedicated PET/CT scanners (Discovery STe, RX, or VCT; 16 or 64 channel; GE Healthcare, Milwaukee, WI, USA, or Siemens mCT Flow; 64 channel; Siemens Healthcare, Knoxville, TN, USA), and whole-body noncontrast CT scans were used for attenuation correction.

F-fluoride PET/CT interpretation and quantification
Two board-certified nuclear medicine physicians evaluated baseline and interim 18 F-fluoride PET/CT images. Visual and quantitative analyses were performed.

Visual analysis
In the visual analysis, we compared the baseline and interim studies, classifying the responses as follows: • Complete response -Osteoblastic bone metastases identified in the baseline study no longer being present in the interim study.
• Partial response -Interim study showing decreased uptake in pre-existing bone metastases.
• Stable disease -No difference between the interim and baseline scans in terms of the uptake in pre-existing bone metastases.
• Progressive disease -Interim study showing an increase in the uptake or volume of a pre-existing bone metastases or new osteoblastic metastases.
The patients were followed for confirmation of these response classifications. The follow-up reference standards used in order to determine if the response classification was correct (i.e., to identify true-positive, true-negative, falsepositive, and false-negative responses) included clinical parameters-such as clinical worsening, disease progression, bone events, and death; biochemical parameters-such as the levels of alkaline phosphatase (ALP) and prostate-specific antigen (PSA); and imaging findings-such as those obtained with 18 F-fluoride PET/CT, 18 F-FDG PET/CT, bone scans, or CT scans. On the interim 18 F-fluoride PET/ CT study, images that demonstrated stable disease, a partial response, or a complete response were all considered to represent a true-positive response to therapy if the reference standards also indicated that the patient had responded to therapy (no clinical worsening, progression, or increase in the levels of the biochemical markers) or a false-positive response to therapy if those same standards demonstrated progressive disease (new areas of disease, clinical worsening, or death). In contrast, images that demonstrated progressive disease were considered to represent a true-negative response to therapy if the reference standards also indicated that the patient had not responded to therapy and it was confirmed during follow-up that there was no response to therapy or a false-negative response to therapy (flare response) if those same standards demonstrated a response (no clinical worsening, progression, or increase in the levels of the biochemical markers).

Quantitative analysis
Using quantitative analysis, we determined the wholebody skeletal tumor burden in the baseline and interim 18 Ffluoride PET/CT images. The skeletal tumor burden was determined after establishing the maximum standardized uptake value (SUV max ) threshold ≥ 10 to exclude normal bone (12) . To that end, we initially obtained the volume (in milliliters) of total fluoride activity, defined as the fluoride tumor volume above an SUV max of 10 (FTV 10 ), within the volume of interest (VOI). The FTV 10 calculation is equivalent to the metabolic tumor volume calculation used in 18 F-FDG PET/CT studies. The total fluoride lesion uptake above an SUV max of 10 (TLF 10 ) was then calculated as the product of mean SUV max × FTV 10 . The TLF 10 is equivalent to the total lesion glycolysis used in 18 F-FDG PET/CT studies. To evaluate the performance of interim 18 F-fluoride PET/CT, the percent change in the skeletal tumor burden between the baseline and interim studies was calculated as follows: %TLF 10 = interim TLF 10 − baseline TLF 10 / baseline TFL 10

Statistical analyses
Categorical variables were expressed as absolute and relative frequencies, whereas continuous variables were expressed as mean ± standard deviation when presenting normal distribution and as median (minimum-maximum) when presenting non-normal distribution. All outcome measures were correlated with the %TFL 10 values obtained. The primary end point was overall survival (OS), which was calculated from the first 223 RaCl 2 cycle to the date of death or last follow-up. Secondary end points were progression-free survival (PFS), time to a bone event (TTBE), and bone marrow failure (BMF). PFS was calculated from the first 223 RaCl 2 cycle to the date of progression, death, or last follow-up. The TTBE was calculated as the time from the date of the first 223 RaCl 2 cycle to the next bone event. Lastly, BMF was defined as the development of hematologic toxicity (World Health Organization grade 3 or 4), together with no recovery after six weeks or death due to BMF after the last 223 RaCl 2 cycle.
Kaplan-Meier survival curves were generated, and Cox proportional hazards regression was used in order to analyze predictors of survival. Backward stepwise selection was performed for multivariate Cox models. Logistic regression was used in order to model the odds of a bone event as a function of all of the PET variables. We used Spearman's correlation coefficient to assess the level of agreement between the PET variables. For the statistical analyses, we used the Statistical Analysis System, version 9.3 for Windows (SAS Institute Inc., Cary, NC, USA).

RESULTS
We analyzed the cases of 34 patients, with a mean age of 72.4 ± 10.2 years (median, 72.5 years; range, 43.3-88.8 years) (Table 1), who had had prostate cancer for a mean of 6 ± 4 years (range, 2-20 years). The mean Gleason score was 7 ± 3. Prior to the initiation of 223 RaCl 2 therapy, 26.9% of the patients had received chemotherapy, 5% had received radiotherapy, 59% had received hormone therapy, and 9% had received blood transfusion. At the first 223 RaCl 2 cycle, the mean ALP was 193.9 IU/L and the mean PSA was 103.2 ng/mL. The median time of follow-up after the interim study was 28.1 months (range, 11-52 months). The 34 patients were submitted to a collective total of 179 223 RaCl 2 cycles: 55.9% of the patients received six cycles of 223 RaCl 2 ; 14.7% received five cycles; and 29.4% received four cycles. The principal causes of treatment interruption were progression (in 44.4%), hematologic toxicity (in 17.8%), a significant decline of the Eastern Cooperative Oncology Group performance status (in 13.3%), and a bone event (in 2.2%).

Visual analysis of interim 18 F-fluoride PET/CT
A complete response was not perceived in any of the interim 18 F-fluoride PET/CT studies or on the basis of the follow-up reference standards. A partial response was identified in 16 (47%) of the patients in the interim 18 F-fluoride PET/CT studies (Figure 1), and the reference standards demonstrated that a partial response had indeed been  A patient with hormone-refractory prostate cancer, accompanied by bone metastasis, who showed a partial response to 223 RaCl 2 , and the interim 18 F-fluoride PET/CT study demonstrating a true-positive response. A: The baseline 18 F-fluoride PET/CT study revealing widespread osteoblastic metastases. B: The interim 18 F-fluoride PET/CT study, performed after the third 223 RaCl 2 cycle, showing a reduction in osteoblastic metastases, especially in the rib cage, pelvis, and right femur, consistent with a partial response to 223 RaCl 2 . There was a 70% reduction in the %TLF 10 . During follow-up, the ALP levels dropped and no new bone lesions appeared. After the last 223 RaCl 2 cycle, the patient resumed enzalutamide to control lymph node metastases that had been present prior to the first 223 RaCl 2 cycle.

A B
achieved in eight of those patients (true-positive cases), whereas the other eight patients had progressed (false-positive cases), as shown in Figure 2. Stable disease was noted in five (15%) of the patients in the interim 18 F-fluoride PET/CT studies, although only three of those patients were categorized as true-positive cases (showing stable disease or a partial response), whereas the two remaining patients progressed. Progressive disease was identified in 13 (38%) of the patients in the interim 18 F-fluoride PET/CT studies, 12 (35.3%) of whom were categorized as true-negative cases (Figure 3), the remaining patient (3.0%) being categorized as a false-negative case because the increased uptake noted on the interim 18 F-fluoride PET/CT (when compared with that observed in the baseline study) was actually due to a flare phenomenon ( Figure 4). Therefore, the responses were categorized as true positive in 11 cases (32.4%), false positive in 10 (29.4%), true negative in 12 (35.3%), and false negative in 1 (2.9%). The interim 18 F-fluoride PET/CT study was found to have a sensitivity of 91.6%, a specificity of 54.5%, a positive predictive value of 52.4%, a negative predictive value of 92.3%, and an accuracy of 67.6% ( Figure 5). For distinguishing between responders and nonresponders, a reduction in the ALP level had a sensitivity of 38% and a specificity of 85% when the follow-up parameters were taken as the reference. Figure 6 illustrates the quantitative method employed to obtain the TLF 10 and FTV 10 values. Spearman's correlation coefficient showed that the %TLF 10 and %FTV 10 values correlated strongly with each other (rho = 0.95). Therefore, Although that pattern is consistent with progression (with a %TLF 10 increase of 65%), the PSA and ALP dropped remarkably, after which the patient responded and was stable at 12 months after the last 223 RaCl 2 cycle. Therefore, the images were clearly due to a flare (false-negative) response. Figure 6. Example of determination of TLF 10 and FTV 10 . A: A semi-automatic VOI (orange rectangle) is placed within the whole-body maximum intensity projection image. A threshold SUV max of 10 is then established as the cut-off to separate normal bone from abnormal bone. Consequently, the software will automatically delineate only SUV max regions above the set threshold of 10, defining the VOI with an isocontour threshold set at 41% of the SUV max . After all regions have been defined, a careful inspection should be performed to exclude all non-tumor-related VOIs. The sum of all the VOIs outlined with the SUV max of 10 provides the FTV 10 .

Quantitative analysis of interim 18 F-fluoride PET/CT
To obtain the TLF 10 , the FTV 10 is multiplied by the SUVmean 10 (VOI 10 × mean 10 ), which is also measured in milliliters. B: In this particular example, the patient had only one lesion with an SUV max higher than 10, which corresponded to a rib metastasis with an SUV max of 25. The TLF 10 was 65.8, and the FTV 10 was 4.2.

DISCUSSION
We have demonstrated that an interim 18 F-fluoride PET/CT study is unable to predict outcomes after 223 RaCl 2 therapy. Novel therapies for osteoblastic metastases, including 223 RaCl 2 therapy, are costly, and it is therefore important to establish a diagnostic test to predict responses to these new, expensive treatments. In one study evaluating treatment responses after six cycles of 223 RaCl 2 in ten patients (13) , conventional bone scintigraphy demonstrated that increased areas of uptake were due not only to treatment response but also to reparative bone changes after therapy (a flare response).
Previous studies have shown that a baseline 18 F-fluoride PET/CT study plays a prognostic role in patients with breast or prostate cancer treated with 223 RaCl 2   (1,14) . However, 18 F-fluoride PET/CT is not traditionally used in evaluating the response to any therapy, because the process of bone healing involves an osteoblastic reaction than can increase 18 F-fluoride uptake, as in conventional bone scintigraphy (15) . Because of comparable pharmacokinetics between 223 RaCl 2 (2) and 18 F-fluoride (16) , we hypothesized    that 18 F-fluoride would be able to evaluate osteoblastic metastases before, during, and after 223 RaCl 2 therapy. In our study sample, the interim study demonstrated that a decrease in uptake was generally due to a response (partial or stable disease) to therapy. However, we find it interesting that, in six (17.6%) of the patients, the decreased uptake was caused by extensive tumor infiltration of the bone marrow, ultimately leading to BMF. To our knowledge, there have been no previous studies describing the latter imaging pattern (caused by BMF) in interim studies. In contrast, although the interim study was able to demonstrate that increased uptake was due to progression, that pattern of uptake was in fact a flare phenomenon in one case. This increased uptake most likely occurred because of the bone healing process after successful 223 RaCl 2 treatment, which involves an osteoblastic reaction. In the subset of patients in which the flare phenomenon occurred, the CT portion of the study revealed reparative changes with increased extent of the sclerotic lesions. However, even on CT, it was not possible to determine which patients were progressing and which were responding. Although we hypothesized that bone levels of ALP could help evaluate patient outcomes, it demonstrated higher specificity and lower sensitivity than did the interim 18 F-fluoride PET/CT study.
Quantitative analyses of 18 F-fluoride PET/CT images have been conducted to assess its role in predicting outcomes, by determining the peak SUV max values of bone metastases. Apolo et al. (17) performed 18 F-fluoride PET/ CT after 6 and 12 months of standard therapy in prostate cancer patients, reporting that progression was associated with SUV increases of more than 57%, as well as that a greater increase in SUV was associated with worse survival. Yu et al. (18) evaluated responses to therapy with dasatinib using SUV max in five target lesions on 18 F-fluoride PET/CT and detected only a borderline correlation with PFS; the changes also correlated with the ALP level. Another study, involving only five patients, showed a reduction in SUV max at 6 and 12 weeks after the use of 223 RaCl 2 (19) . In our population, the SUV max did not correlate with OS. Although the above mentioned studies performed 18 F-fluoride PET/CT for therapeutic evaluation, its precise role in determining the early response to therapy has yet to be extensively studied, especially in terms of assessing survival as an end point.
The reported frequency of the flare phenomenon in prostate cancer patients undergoing conventional bone scintigraphy ranges from 6% to 25% (20,21) . Although the flare phenomenon has also been described in patients undergoing 18 F-fluoride PET/CT (15) , there have been no reports of its frequency in patients treated with 223 RaCl 2 and undergoing 18 F-fluoride PET/CT. Although we identified the flare phenomenon on 18 F-fluoride PET/CT in only a small proportion of our patient sample (3%), that proportion is probably higher than in conventional bone scintigraphy, given the greater sensitivity of PET/CT. The most likely explanation for the fact that the frequency of the flare phenomenon was not higher is that our study sample was composed of patients with extensive disease, in whom the likelihood of progression is greater than is that of a response to therapy. In addition, the number of patients might have been insufficient to detect this phenomenon.
In our patient sample, the interim 18 F-fluoride PET/ CT (%TLF 10 ) after three cycles of 223 RaCl 2 was not able to predict OS, PFS, TTBE, or BMF. These findings are quite similar to those of a previous study, involving ten prostate cancer patients treated with 223 RaCl 2 (22) , although the interim 18 F-fluoride PET/CT studies were performed at different time points: at baseline; after one (or two) cycles of 223 RaCl 2 ; and at the end of treatment. A correlation with outcome was only noted between baseline and end-oftreatment 18 F-fluoride PET/CT results were found to correlate with outcomes, as previously reported (1) .
One major limitation of our study was the relatively small number of patients. We believe that the interim 18 Ffluoride PET/CT could have potential for the prediction of BMF, given that 6 of the 9 patients who evolved to BMF showed a reduction in uptake. However, due to the small sample size, those results were not significant. Another limitation was the fact that it was not possible to obtain histological confirmation in the patients who showed progression. Although the 18 F-fluoride PET/CT images were acquired in different scanners, the same software was employed in all quantifications, guaranteeing uniformity in the metrics.
To our knowledge, this is the first study to evaluate the role of interim 18 F-fluoride PET/CT in predicting the response to 223 RaCl 2 therapy, using quantitative methods to determine the skeletal tumor burden. It would be interesting to know whether these findings could be replicated in other populations, such as that of breast cancer patients treated with 223 RaCl 2 .

CONCLUSION
In prostate cancer patients undergoing 223 RaCl 2 therapy, interim 18 F-fluoride PET/CT performed after three cycles of 223 RaCl 2 does not seem able to predict outcomes. It also appears to be unable to differentiate a flare response from progressive disease, and we therefore discourage the use of interim 18 F-fluoride PET/CT to evaluate the response to 223 RaCl 2 therapy in prostate cancer patients. There is a need for studies involving a larger number of patients and patients with other types of cancer, in order to verify our findings.