University of Groningen Imaging hormone receptors in metastatic breast cancer patients Venema, Clasina Marieke

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Imaging hormone receptors in metastatic breast cancer patients

Venema, Clasina Marieke

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Publication date: 2018

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Venema, C. M. (2018). Imaging hormone receptors in metastatic breast cancer patients. Rijksuniversiteit Groningen.

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517613-L-sub01-bw-Venema 517613-L-sub01-bw-Venema 517613-L-sub01-bw-Venema 517613-L-sub01-bw-Venema Processed on: 7-3-2018 Processed on: 7-3-2018 Processed on: 7-3-2018

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Chapter 8

Visualizing the effects of bicalutamide on the androgen receptor

availability in metastatic breast cancer patients

C.M. Venema1, A.W.J.M. Glaudemans2, E.F.J. de Vries2, H. Boersma³ G.A.P. Hospers1, B. Rikhof4, C. Zielstra-Dorbitz5, C.P. Schröder1

1

From the departments of Medical Oncology, 2 Nuclear Medicine and Molecular Imaging, 3 Pharmacy, and 5Radiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. 4

Department of Medical Oncology, Leeuwarden Medical Center, Friesland, The Netherlands.

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BACKGROUND: The androgen receptor (AR) is expressed in 70-80% of all

breast cancer patients and offers a potential new target for treatment. Bicalutamide is an AR antagonist which showed a clinical benefit rate at 6 months in 19% of the metastatic breast cancer patients. Optimal selection of breast cancer patients benefiting from AR-targeting treatment is needed. With 16β-18F-fluoro-5α-dihydrotestosterone (18F-FDHT) PET, the density of available AR can be visualized in vivo. The reduction in 18F-FDHT uptake during treatment with bicalutamide may be predictive for response. We assessed the feasibility of 18F-FDHT PET to detect a change in available AR binding sites during bicalutamide treatment in metastatic breast cancer patients.

METHODS: Post-menopausal women with AR positive metastatic breast

cancer were eligible for this pilot study. All patients were staged with bone scintigraphy and contrast enhanced CT. At baseline and during bicalutamide treatment 18F-FDHT PET was performed. The primary endpoint was the reduction in FDHT uptake in tumor lesions after 4-6 weeks of bicalutamide monotherapy, relative to baseline. Relative changes in uptake were calculated per lesion. RESULTS: Ten patients were included in the trial. All patients had at least one 18F-FDHT positive lesion at baseline. A scan during treatment was available in eight patients. The median SUVmax of all lesions of all patients during treatment was significantly lower than at baseline (per lesion p < 0.001 and per patient p = 0.016). On a patient basis, the percentage change in FDHT uptake varied from -100% to +14%. The percentage change in 18F-FDHT uptake was highly variable between lesions within individual patients. CONCLUSION: It is feasible to detect changes in 18F-FDHT uptake in patients treated with bicalutamide. This may be the basis for future larger prospective studies to explore whether analyzing the positive predictive value of serial 18F-FDHT PET scanning can be used to predict treatment response.

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INTRODUCTION

Hormone receptors, such as the estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR), play a vital role in the development and progression of several malignancies (1). In breast cancer, 75% of the patients express the ER, which makes these patients eligible for ER-targeted therapy (2). While ER and PR are routinely determined in breast cancer patients, this is not (yet) the case for the AR (3). The AR is expressed in tumors of 70-80% of all breast cancer patients and its expression can be found in all breast cancer subgroups (4-6). Even in the so-called triple negative tumors, i.e. ER-, PR-, and human epidermal growth factor receptor 2 (HER2)-negative tumors, AR expression was observed in 25-30% of the cases (4). Preclinical studies indicate that this offers a new potential treatment strategy for this poor-prognosis subcategory that can nowadays only be treated with chemotherapy (7,8). Not only for triple-negative breast cancers, but also for other subtypes, the AR may be a relevant drug target. ER-positive breast cancer can adopt a phenotype similar to prostate cancer in which AR exerts oncogenic rather than tumor suppressor activity (9). Early clinical trials with AR intervention strategies in metastatic breast cancer indeed are showing clinical benefit rates up to 35 percent and an acceptable toxicity profile (10,11).

Bicalutamide is an oral, nonsteroidal, AR antagonist, approved by the United States Food and Drug Administration, and the European Medicines Agency for the treatment of metastatic prostate cancer. In a phase II study in patients with metastatic breast cancer, a clinical benefit rate at 6 months was seen in 19% of the patients treated with bicalutamide (11). The median progression free survival was 12 weeks. Although this rate is comparable to other treatment options in triple negative metastatic breast cancer patients, bicalutamide may be an attractive option, as it exerts less toxic side effects than chemotherapy.

The modest clinical benefit rate suggests that optimal selection of breast cancer patients benefiting from AR-targeting treatment is needed. It is likely that only those patients with tumors expressing the AR, can benefit from AR-targeted therapies such as bicalutamide. To date however, it is not part of standard care to test for AR expression in breast cancer samples. Standard histological staining of AR on the primary tumor is inexpensive and easy to

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apply, but no cutoff value for AR positivity in breast cancer is established yet and several problems could arise in the setting of metastatic breast cancer. Receptor status can change over time, and can vary across metastases (inter-tumor heterogeneity) (12-15). Obtaining biopsies to assess AR expression, is not always feasible and a single biopsy from a metastatic lesion is not necessarily representative for the AR status throughout the body.

Molecular imaging of AR using positron emission tomography (PET) is a non-invasive way to assess AR expression throughout the whole body. 16β-18 F-fluoro-5α-dihydrotestosterone (18F-FDHT) is a radiopharmaceutical designed for imaging the density of available AR in vivo (16,17). 18F-FDHT PET has been shown to visualize the AR expression in metastatic breast cancer and prostate cancer lesions. 18F-FDHT uptake was shown to be blocked by competition with the AR antagonists flutamide and enzalutamide in prostate cancer patients (14,17,18). The reduction in FDHT uptake during treatment with bicalutamide can be regarded as a measure for the occupancy of the receptor by the drug and may therefore be predictive for response, similar to change in 18F-FES binding to the ER during selective estrogen receptor degrader therapy (19-21). We performed the present study in metastatic breast cancer patients to assess the feasibility of 18F-FDHT PET to detect the binding of bicalutamide to AR binding sites, as reflected by a difference in tracer uptake in the tumor between baseline and during bicalutamide treatment. This could be the basis for prospective studies to analyze the positive predictive value of serial 18 F-FDHT PET scanning for treatment response.

METHODS AND MATERIALS

This prospective, single center feasibility study was conducted at the University Medical Center Groningen, the Netherlands, in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki. The institutional Review Board approved the protocol, and patients provided written informed consent before participation. The study was registered in clinicaltrials.gov as NCT02697032.

Patients

Patients with histologically proven AR-positive, HER2-negative metastatic breast cancer were eligible for the study, independent of their ER and PR status. Inclusion criteria were the presence of measurable disease according

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to the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1) or evaluable disease, e.g. non-irradiated bone metastases (22). Patients had to be post-menopausal. Exclusion criteria included symptomatic metastases in the central nervous system, or a history of cardiac disease within 6 months prior to screening. Also the concomitant use of CYP3A4 inhibitors was not allowed as bicalutamide is metabolized via CYP3A4 and strong inhibitors could increase the serum levels of bicalutamide.

Study Design

All patients were staged with contrast enhanced CT of the thorax and abdomen, and with bone scintigraphy. Before the start of treatment, patients underwent an 18F-FDHT PET scan. Thereafter all patients were treated with bicalutamide 150 mg daily. All patients were treated until progressive disease or unacceptable toxicity was encountered. After start of treatment patients visited the out-patient clinic for biweekly safety checks and a second 18F-FDHT PET scan was performed after 6 weeks of treatment. Due to rapid progression of disease in the first 3 patients, the protocol was amended to perform the second 18_{F-FDHT PET scan after 4 weeks. Responses were evaluated with} repeated contrast enhanced CT according to RECIST 1.1 after 8 weeks and every 3 months thereafter in case of measurable disease. In case of non-measurable, bone only disease, imaging was performed according to the physicians’ choice and a deterioration of symptoms (such as pain or increased fatigue) was considered to be clinical progression (figure 1). Primary objective was the feasibility to detect changes in available AR binding sites, observed as a difference between baseline and on-treatment 18F-FDHT uptake in metastatic breast cancer patients during bicalutamide treatment.

The primary endpoint was the percentage change in FDHT uptake in tumor lesions after 4-6 weeks of bicalutamide monotherapy, relative to baseline. Secondary endpoints were 1) the difference in the change in 18F-FDHT uptake during treatment between those patients with an objective response according RECIST criteria and those with no objective response, and 2) the correlation between 18F-FDHT tracer uptake and AR tumor expression on (previously resected) tumor tissue.

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Figure 1 Study procedure overview. Patients were staged with a bone scan and

contrast enhanced CT scan, and a biopsy was performed when feasible. Before start of treatment with bicalutamide 150 mg daily and after 4-6 weeks treatment patients were scanned with an 18F-FDHT PET scan.

Tumor histology

All patients underwent a biopsy of a metastasis before the start of treatment, when this was considered safe, except for those patients who were biopsied within six months prior to the baseline 18F-FDHT PET scan. Biopsies were formalin fixed and paraffin embedded. ER (CONFIRM anti-Estrogen Receptor (SP1) Rabbit Monoclonal Primary Antibody, Ventana, Illkirch, France) and AR (anti-Androgen Receptor (SP107) Rabbit Monoclonal Primary Antibody, Ventana, Illkirch, France) expression were analyzed by immunohistochemistry using a Ventana Benchmark automated stainer (Ventana, Illkirch, France). Antibodies were prediluted by the supplier. ER was scored according to the American Society of Clinical Oncologypathologists guideline (23). A threshold of >10% positive nuclear staining was used as a discriminator for AR positivity, based on current use in the literature (4,5,10).

Molecular Imaging

A whole body (head to mid-thigh) 18F-FDHT PET scan was performed at baseline and after 4 to 6 weeks (see section “Study design”). 18F-FDHT was produced as previously described (24). Approximately 200 MBq of 18F-FDHT was injected intravenously. Sixty minutes post injection the patients were scanned using a Siemens Biograph 40 or 64-slice mCT (Siemens Medical Systems, Knoxville, TN) with a 2-mm reconstructed spatial resolution and emission acquisition time of 3 minutes per bed position. Low dose CT (for attenuation and scatter correction) and PET imaging were performed within one procedure. If contrast enhanced CT was not performed prior to the PET

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scan, it was performed immediately after the PET procedure on the same scanner. Reconstructions of the scans and analysis of the images were performed according to the European Association of Nuclear Medicine (EANM) Research Limited (EARL) approved protocols for 18F imaging (25).

Imaging Analysis

CT scans were evaluated according to RECIST v1.1 guidelines by the same dedicated radiologist (CZ) (22). All tumor lesions visible on CT (>1 cm) and bone scans were documented.

The 18F-FDHT-PET scan was first assessed qualitatively. A nuclear medicine physician (AG) visually identified lesions with tracer uptake above the background signal, which could not be attributed to physiological uptake or an artifact. Next, 18F-FDHT uptake was quantified for all individual tumor lesions and recorded as the maximum standardized uptake value (SUVmax). We also measured the SUVmean using a 50% isocontour of the hottest pixelin the lesion to assess the average SUV in the volume of interest. The SUVpeak was used to calculate uptake in a 1 cm³ spherical volume of interest surrounding the voxel with the highest activity. 18_{F-FDHT tumor uptake was corrected for} physiological uptake by drawing a region of interest at the contralateral side for symmetric structures or in adjacent tissue of the same origin for asymmetric structures. The SUV from healthy tissue was subtracted from the SUV of the tumor (i.e. lesion SUVmax/mean/peak minus background SUVmax/mean/peak), resulting in a background corrected SUVmax/mean/peak, corSUVmax/mean/peak. Absolute and background-corrected SUV were calculated for individual lesions. Relative changes in uptake were calculated per lesion and the median (changes in) SUVs were calculated for all lesions that showed 18

F-FDHT uptake above background for patient-based analysis.

Statistical Analysis

This feasibility study was aimed to assess whether a change in tracer uptake after 4-6 weeks of bicalutamide monotherapy could be visualized in individual tumor lesions. Considering the feasibility setting of the trial, only descriptive statistics were performed. Statistical analyses of the differences between baseline and residual uptake during treatment were performed using the paired t-test. Correlations between baseline uptake, or residual uptake during treatment and outcome were calculated using the Pearson correlation coefficient. A p-value below 0.05 was considered statistically significant.

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Figure 2 Consort diagram of included patients RESULTS

At the data cut-off on July 1st 2017, eleven patients were screened for study entry. Ten patients were included in the trial and started treatment (Figure 2). One patient was excluded based on the presence of brain metastases during screening. All patients had received prior palliative therapy. Median age was 73 years (range 51 - 89). In total 40% of the patients had measurable disease according to RECIST 1.1 (lung n = 1, liver n = 2, lymph node n = 1). Detailed patient characteristics at study entry are provided in Table 1.

According to RECIST 1.1 criteria, stable disease was observed in five patients for more than 10 weeks. One of these patients had measurable liver lesions at baseline, which cannot be evaluated by 18F-FDHT PET. Four patients had radiologic progressive disease within 8 weeks, and one patient had clinical progressive disease within 8 weeks. The median follow-up was 9 weeks. Of the 10 patients, 9 had discontinued bicalutamide at the data cut-off date, with a median progression free survival of 8 weeks. The remaining patient is still on treatment and has received bicalutamide for more than 3 months.

Bicalutamide was relatively well tolerated by most patients. Two serious adverse events occurred in two patients during the trial, both related to progressive disease (i.e. pleural effusion and cholangitis based on tumour obstruction), rather than to the drug. In total six adverse events were related to the use of bicalutamide (see Table 2).

Eligible (n = 11)

No second scan (n = 2)

Second scan after 6 weeks (n = 2)

Second scan after 4 weeks (n = 6) Excluded (n = 1) Included (n = 10)

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Table 1. Patient characteristics

Characteristics Number of patients

Age mean years (range) 71 (51 - 89)

BMI, kg/m² median (SD) 27.2 (4.8)

Primary tumor characteristics IHC

ER+/AR+ ER-/AR+

7 3

Metastatic tumor characteristics IHC

ER+/AR+ ER+/AR- ER-/AR+ ER-/AR- Unknown 3 0 3 1 3 Previous treatment

Hormone therapy (median lines) Chemotherapy (median lines)

7 (3) 6 (2)

Metastatic locations

Bone metastases only Lung and bone metastases Liver metastases and bone Lymph nodes and bone

6 1 2 1

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F-FDHT PET Results at Baseline

All patients had at least one 18F-FDHT positive lesion at baseline. In total 154 lesions were found with conventional imaging (CT: n = 24, bone scintigraphy: n = 130). Liver lesions (n = 2) were not analyzed due to high physiological 18 F-FDHT uptake in the liver. In total, 141 lesions were visible on the18F-FDHT PET scan. Two lymph node metastases were non-evaluable due to close proximity of large veins which showed high physiological FDHT uptake. In total, 139 lesions were included for FDHT PET analysis. The majority of the lesions were bone lesions (n = 119). The remaining lesions were lymph nodes (n = 7) and visceral lesions (n = 13). The CT scans revealed 5 FDHT negative metastases, and the bone scans revealed 31 FDHT negative lesions, of which 26 were found within one patient.

Baseline SUVmax of 18F-FDHT varied greatly among lesions (median 4.08; range 0.8 – 20.2) and between patients (median SUVmax: 3.3; range 2.1 – 4.8) (see also Table 3 and Figure 4 for per patient analysis). The median SUVmax of bone lesions was 4.0 (range 0.8 - 8.3), of lymph nodes 6.1 (4.7 - 20.2) and of visceral

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metastases 2.9 (1.8 - 12.2). The baseline SUVpeak, SUVmean and corSUVmax/peak/mean are described in Supplemental Table 1.

Change In Tracer Uptake During Treatment

An on-treatment scan was available in eight patients of which six patients received the second scan after 4 weeks of treatment (Figure 2) and two after 6 weeks. Two patients did not receive the on-treatment scan due to early progression. An example of the maximum intensity projection of the baseline and during treatment FDHT PET scan is depicted in Figure 3. On treatment, the median residual SUVmax of all lesions (n = 109) of all patients was 2.13 (range 0.6 – 4.4) and a median residual SUVmax per patient 2.1 (range 1.9 – 2.8), which was significantly lower than at baseline (per lesion p < 0.001 and per patient p = 0.016). Residual SUVpeak, SUVmean and background corrected SUVmax/peak/mean per organ are described in Supplemental Table 2.

Figure 3

Example of a maximum intensity projection of the 18F-FDHT PET at baseline (A) and during treatment (E). Physiological uptake is seen in the large veins, porth-a-cath, and liver as well as the gastrointestinal and urinary tract. High AR expression in the tumor lesions are visible in thoracic lymph nodes (B), primary tumor (C), and inguinal lymph node (D), which is decreased after 6 weeks of treatment (F, G, H).

The median change in 18F-FDHT uptake corrected for background (corSUVmax) was -79% (range -100 to +183) when all lesions were taken into account. Bone lesions showed a median corSUVmax change of -83% (range -100% to +183), lymph nodes -78% (range -78% - -77%) and visceral lesions -36% (range -80 to +50%). Per patient analysis showed a median change in corSUVmax of -64.5 %

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(range -100% to +14%) (Figure 5). Heterogeneity in the reduction in 18F-FDHT uptake was seen among lesions within individual patients (Table 3). In 11 lesions of five patients an increase in 18F-FDHT uptake was observed during treatment.

Uptake of healthy liver and in the descending thoracic artery did not significantly differ between baseline and during treatment.

Table 2. Adverse events during treatment, and their relation to bicalutamide.

Relation of AE AE or SAE Max. grade of AE Total

1 2 3 4 Unknown AE Hyponatriemia 1 1 Increased ASAT 1 1 Loss of appetite 1 1 Changing stools 1 1 SAE 0 Total 4 Related to Bicalutamide AE Flush 1 1 Flatulence 2 2 Fatigue 2 1 3 SAE 0 Total 6 Related to progressive disease AE Anemia 1 1 Pain shoulder 1 1

SAE Dyspnea due to pleural effusion 1 1 Sepsis based on cholangitis due to obstruction 1 1 Total 4 Total AE 8 3 1 0 12 Total SAE 2 2 Total 14

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Figure 4 Distribution of baseline 18F-FDHT uptake, expressed as the maximum

standardized uptake value (SUVmax), in individual patients.

Figure 5 Baseline and on-treatment background-corrected median FDHT maximum

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Correlation Between Treatment Outcome and FDHT PET Results

In this small study, no correlation was found between response or treatment duration, and median SUVmax at baseline, or SUVmax change between baseline and during treatment (Figure 6).

Figure 6 Waterfall plot showing the relative change in median FDHT standardized uptake value corrected for background (corSUVmax) in individual patients. Patients with

a clinical benefit for more than 10 weeks are depicted in blue, versus no clinical benefit in red.

DISCUSSION

This is the first study to visualize the effect of bicalutamide on AR availability with 18F-FDHT PET in patients with metastatic breast cancer. We show that it is feasible to detect a decline in 18F-FDHT uptake during treatment with androgen receptor blockade.

A reduced uptake on 18F-FDHT PET during androgen blockade with enzalutamide, was previously shown in 22 prostate cancer patients (18). In that study, the reduction in 18F-FDHT uptake ranged from 20%-100%. Similar to our study, this reduction was not related to response. From these data and our study, it might be suggested that sufficient receptor occupation by AR blockers may be a prerequisite, but no guarantee for treatment response.

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However, these are small studies, which should be regarded as hypothesis generating. Larger studies are required to establish whether a (change in) 18 F-FDHT uptake can ultimately serve as a marker for response.

Heterogeneity of tumor characteristics is increasingly considered as a crucial feature affecting outcome and response to treatment (26). In our previous molecular imaging work, profound heterogeneity was observed with regard to ER- and HER2 expression between and within patients and lesions (27,28). In the present study, heterogeneous uptake of 18F-FDHT in tumor lesions was seen in seven out of ten patients, indicating the presence of both receptor positive- and negative lesions in those patients. Eleven bone lesions that were visible on the bone scan did not show uptake above background on the baseline 18F-FDHT PET. It is possible that the bone scan was positive due to other reasons (degenerative bone lesions e.g.), but it could also indicate that these lesions have converted to AR-negative lesions. An AR positive metastasis biopsy does not exclude this possibility.

In addition to heterogeneity of uptake at baseline, we also observed a large variation in receptor occupancy during treatment in this study. An increase in uptake in 11 lesions among five patients was even shown. This suggests that bicalutamide did not bind to the AR in these lesions, which could be due to inadequate accumulation of the drug in these lesions, or to mutations or conformational changes that prevent binding of the drug to the receptor. Also, the relatively low binding affinity of bicalutamide to the AR may play a role. Bicalutamide is a first generation AR blocker. New, second generation, AR blockers such as enzalutamide have a higher binding affinity to AR compared to bicalutamide (29). Furthermore, we used SUV to analyze the change in 18F-FDHT uptake. This is the most common used value to analyze uptake, however this value is subject to different factors that could change over time. The perfusion of the tumor and metabolism of the tracer can change during treatment. We therefore corrected for background uptake, but it is possible that we missed changes in metabolism of the tracer. Therefore, it is conceivable that 18F-FDHT uptake during treatment was affected by multiple factors, including target heterogeneity, treatment binding efficacy, as well as tracer perfusion and metabolism. Future studies may increase insight in these factors, and how they should be taken into account when interpreting the PET results.

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More clinical studies exploring the efficacy of AR-targeted therapies in metastatic breast cancer patients are ongoing. This study shows that it is feasible to visualize the effect of androgen blockade by means of 18F-FDHT PET in this patient population. Previously, molecular imaging of the effects of ER modulators with PET scans provided information about ER occupancy and guided dose selection for phase II trials (19,21). Likewise, a (change in) 18 F-FDHT uptake on serial 18F-FDHT PET scans may ultimately serve as a marker for response. Therefore, the present study can provide the basis for future, larger studies to explore this.

ACKNOWLEDGMENTS

Financial support: Healthy ageing pilots CD015.004/275 and JK de Cock Stichting

Protocol design: 16th workshop on methods in clinical cancer research, Flims, Switzerland 2014

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Ta b le 3 18 F-FDHT PET result s in in dividu al patients Patient Tumor sites FD H T negativ e le sio n s FD H T posi tiv e le sio n s Basel ine SUV ma x Basel ine corS UV ma x Resi dual SUV ma x Resi dual corS UV ma x % chang e SUVc o r P value Δ basel ine corS UV ma x and resi dual corS UV ma x

Weeks until prog

1 Bone 0 20 4.19 (2.72 – 5.27) 2.35 (0.8 8 – 3.43 2.81 (1.4 2 – 4.24) 0.55 (0 – 1.98) -79 ( -10 0 -+38) P < 0.0 0 1 2 Lu ng, bone 1 4 2.21 (1.81 – 2.89) 1.34 (0.7 6 – 1.60) 0 2.11 (1.6 9 – 2.29) 1.00 (0. 5 5 – 1.58) -18 (-6 2 - +55) P = 0.475 3 Bone, lym ph node s, sk in 1 10 5.09 (2.75 – 20.24) 3.92 (0.8 8 – 19.05 ) 2.94 (1.8 2 – 4.41) 1.16 (0. 3 3 – 3.04) -65 (-8 0 - +150) P = 0.0 4 3 4 Bone, skin 2 14 2.61 (1.70 – 3.10) 1.48 (0.6 5 – 2.68) 1.90 (1.2 4 – 2.58) 0.56 (0 – 1.40) -64 (100 -20) P < 0.0 0 1 5 Bone 0 23 4.60 (2.95 – 8.33 2.84 (1.1 9 6.57) 1.99 (1.3 9 – 2.65) 0.61 (0. 0 1 – 1.27) -82 (100 -40) P < 0.0 0 1 6 Bone 0 24 4.02 (2.60 – 5.97) 1.64 (0.2 2 – 3.59) 1.94 (1.4 4 – 2.56) 0 (0 – 0.3 7 ) -100 ( 100 -93) P < 0.0 0 1

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7 Bone, lung 4 11 2.51 (0.83 – 4.97) 1.35 (0.1 0 – 3.81) 2.37 (0.6 4 – 3.16) 1.59 (0 – 2.38) +14 ( -10 0 -+183) P = 0.731 8 Bone 0 2 2.35 (2.29 - 2.40) 0.36 (0.3 1 – 0.42) 1.95 (1.6 7 – 2.22) 0.24 (0 – 0.48) -25 ( -10 0 -+50) P = 0.745 9 Bone 26 4 3.92 (3.32 - 4.22) 2.25 (1.6 5 – 2.55 - - NA 10 Bone 2 21 4.79 (3.79 – 8.52) 3.39 (2.5 1 – 5.23) - - NA

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