Diffusion-weighted Imaging as a Treatment Response Biomarker for Evaluating Bone Metastases in Prostate Cancer: A Pilot Study.

Purpose To determine the usefulness of whole-body diffusion-weighted imaging (DWI) to assess the response of bone metastases to treatment in patients with metastatic castration-resistant prostate cancer (mCRPC). Materials and Methods A phase II prospective clinical trial of the poly-(adenosine diphosphate-ribose) polymerase inhibitor olaparib in mCRPC included a prospective magnetic resonance (MR) imaging substudy; the study was approved by the institutional research board, and written informed consent was obtained. Whole-body DWI was performed at baseline and after 12 weeks of olaparib administration by using 1.5-T MR imaging. Areas of abnormal signal intensity on DWI images in keeping with bone metastases were delineated to derive total diffusion volume (tDV); five target lesions were also evaluated. Associations of changes in volume of bone metastases and median apparent diffusion coefficient (ADC) with response to treatment were assessed by using the Mann-Whitney test and logistic regression; correlation with prostate-specific antigen level and circulating tumor cell count were assessed by using Spearman correlation (r). Results Twenty-one patients were included. All six responders to olaparib showed a decrease in tDV, while no decrease was observed in all nonresponders; this difference between responders and nonresponders was significant (P = .001). Increases in median ADC were associated with increased odds of response (odds ratio, 1.08; 95% confidence interval [CI]: 1.00, 1.15; P = .04). A positive association was detected between changes in tDV and best percentage change in prostate-specific antigen level and circulating tumor cell count (r = 0.63 [95% CI: 0.27, 0.83] and r = 0.77 [95% CI: 0.51, 0.90], respectively). When assessing five target lesions, decreases in volume were associated with response (odds ratio for volume increase, 0.89; 95% CI: 0.80, 0.99; P = .037). Conclusion This pilot study showed that decreases in volume and increases in median ADC of bone metastases assessed with whole-body DWI can potentially be used as indicators of response to olaparib in mCRPC. Online supplemental material is available for this article.


Introduction
Prostate cancer is the second most commonly diagnosed cancer among men worldwide (1). Bone metastases are highly prevalent in patients with metastatic castration resistant prostate cancer (mCRPC), the late stage of prostate cancer, causing substantial disease-related morbidity and mortality in this population. Bone metastases occur in up to 84% of patients with mCRPC and frequently represent the only site of metastatic disease (2).
Standard imaging techniques, i.e. computed tomography (CT), and technetium-99m bone scintigraphy, fail to accurately evaluate the burden of bone metastases and detect changes in response to treatment (3). In fact, the widely used Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 (4), do not define response in bone metastases, considering these as non-measurable disease. The Prostate Cancer Working Group 2 criteria (PCWG2) define progression in bone metastases based on the appearance of new lesions on bone scintigraphy, but fail to state any criteria for response in bone metastases (5). Therefore, tumor responses in patients with bone only metastatic disease rely solely on prostate specific antigen (PSA) falls; the latter have not been proven to be a surrogate for improved survival (5)(6)(7). There is an urgent unmet need to identify, develop, and validate non-invasive response biomarkers for bone metastases in prostate cancer.
Diffusion weighted imaging (DWI) is a functional magnetic resonance imaging (MRI) technique that studies the motion of water molecules within a tissue. Apparent diffusion coefficient (ADC) is an objective measurement of this water diffusion, which has been demonstrated to inversely correlate with cellularity in different tumor types including bone marrow malignancies (8)(9)(10)(11)(12)(13). Changes in ADC values after treatment have been correlated with tumor responses in different tumor types including myeloma, ovarian carcinoma, primary peritoneal carcinoma and rhabdomyosarcoma (14)(15)(16). Additionally, the volume of bone metastases assessed with whole body (WB) DWI has prognostic value in patients with mCRPC (17). Limited data about the value of DWI in the assessment of response to bone metastases in mCRPC is currently available, coming from small series of patients (18)(19)(20)(21). In the setting of a prospective clinical trial, we aim to determine the usefulness of whole body diffusion weighted imaging (WB-DWI) to assess response of bone metastases to treatment in patients with metastatic castration resistant prostate cancer (mCRPC).

Material and methods
We conducted a phase II trial of the poly-(ADP)ribose polymerase (PARP) inhibitor olaparib (Lynparza, AstraZeneca) in mCRPC (TOPARP-A; CRUK/11/029); patients were enrolled from July 2012 to September 2014. A prospective MRI sub-study was conducted under institutional research board (IRB) approval at The Royal Marsden NHS Foundation Trust. Enrolment to this MRI sub-study was optional; written informed consent was obtained for MRI scan acquisition.

Study design
The primary endpoint of the TOPARP-A trial was response rate, with response defined as any of the following: a response of soft tissue/visceral disease according to Response Evaluation Criteria In Solid Tumors (RECIST) 1.1(4); a confirmed reduction in the prostate specific antigen (PSA) level of ≥50%; or a conversion in the circulating tumor cell (CTC) count, with a reduction in the number of CTC from ≥5/7.5 ml of blood at baseline to <5/7.5 ml of blood during treatment, with a confirmatory assessment at least 4 weeks later (22). Detailed information of the inclusion and exclusion criteria and the results of the TOPARP-A trial have been published showing a response rate to olaparib in mCRPC of 33% (95% confidence interval [CI], 20 to 48%) (23).
Participation in the optional MRI sub-study was offered to those patients without contraindication for MRI at The Royal Marsden NHS Foundation Trust. WB-MRI was performed at baseline (within 28-days prior to starting treatment) and at Cycle 4 Day 1 (corresponding to 12-weeks after starting treatment) and every 12-weeks subsequently. The primary endpoint of the MRI sub-study was to assess the association between changes in parameters derived from WB-DWI (volume of bone metastases and median ADC) and response to olaparib. For MRI sub-study purposes, patients were classified as responders if they met the definition of the primary endpoint of the TOPARP-A trial and if they had not experienced radiological progression by 12-weeks.

Patient population in the MRI sub-study
Patients were included in this study if: a) signed informed consent for the MRI sub-study in the setting of the TOPARP-A trial, b) bone metastases identified based on review of combined imaging modalities: MRI, CT and BS (in all the cases), c) a minimum of two paired WB-MRI studies performed at baseline and after 12-weeks of treatment. Patients with suboptimal quality WB-MRI or incomplete studies were considered un-evaluable for analysis and excluded from the study.

Clinical data collection
Data were collated into an anonymized database and analyzed by the Institute of Cancer Research Clinical Trials and Statistical Unit (ICR-CTSU; London, UK). PSA and CTC counts were collected at baseline and every 12-weeks while on treatment. CTC counts were also pursued at weeks 1, 2, 4 and 8. RECIST assessments were evaluated at baseline and every 12-weeks using CT.

Whole-body MRI parameters
WB-MRI was performed on a 1.5-T MRI scanner (Avanto Siemens Healthcare, Erlangen, Germany), using surface and body coils on patients positioned supine. Axial images were acquired using free breathing single-shot twice-refocused echo-planar DWI from the upper cervical spine to mid-thighs, sequentially across four imaging stations, each consisting of 50 slices respectively. In addition to WB-DWI, anatomical imaging was also acquired using breath-hold axial T1-weighted. The scan parameters are summarized in Appendix Table 1.

Imaging analysis
Images were processed and analyzed with open-access imaging assistant software (OsiriX v5.6). Evaluation of T1-weighted and DWI (b50, b900 and ADC maps) was performed in order to assess the presence of metastatic bone disease. Regions of interest (ROIs) including areas of signal abnormality on DWI b900, corresponding to high signal on DWI b900 and low signal on T1-weighted imaging, in keeping with metastatic bone disease were delineated. Different delineation techniques of the signal abnormality on DWI b900 corresponding to bone metastases were undertaken. Firstly, ROI analyses were performed including all areas of signal abnormality on DWI b900 and T1-weighted MRI corresponding to bone metastases observed in the axial skeleton (spine and pelvis, not including ribs) between C4 and mid-thighs, labeled as total diffusion volume (tDV). Secondly, a more limited analysis was performed using a RECIST approach of a maximum 5 target representative bone metastases chosen using the following criteria: maximum axial dimension >1cm, well-defined lesion border and representing different skeletal areas. For this analysis, ROIs including total volume of up to 5 target lesions and ROIs including the central slice of the same target lesions were chosen.
Additionally, a single radiologist (RPL) manually delineated the entire axial skeleton (spine and pelvis, not including ribs) enclosing normal and abnormal bone marrow from C4 to lesser trochanters. This delineation technique was included in view of its possible advantage for automated segmentation of the skeleton.
A semi-automated segmentation tool from the OsiriX software v.5.6 was used for delineating ROIs. All the delineation techniques in every WB-DWI were performed by a single radiologist (RPL) with 3 years of experience in WB-DWI; manual correction of the segmentation mask corresponding to the regions of interest (ROI) was performed by the radiologist where necessary ( Figure 1). The volume of metastases was calculated as the number of voxels for all ROIs multiplied by the voxel volume in each case. The ADC value of every pixel was recorded and histogram representations of the ADC values of bone metastases for each patient were generated using Microsoft Excel 2010.

Statistical analysis
Distribution of PSA, CTC counts, median ADC (mADC), tDV, volume and diameter of the target lesions at baseline and percentage change after 12-weeks on treatment are presented using descriptive statistics. Baseline distributions and median changes during treatment in mADC, tDV, volume and diameter of the target lesions are compared between responders and non-responders using non-parametric Mann-Whitney tests and their association with response to treatment using univariate and multivariate (adjusting for known prognostic factors of baseline PSA, lactate dehydrogenase [LDH] and alkaline phosphatase [ALP]) logistic regression models. The correlation between baseline and changes in tDV after 12weeks on treatment with baseline and best percentage change in PSA and CTC respectively were assessed using Spearman´s correlation coefficient (r), with 0.4≤|r|<0.6 indicating moderate correlation, 0.6≤|r|<0.8 strong correlation and |r|≥0.8 very strong correlation. A significance level of 0.05 and 95% CI have been used. No adjustment for reporting of multiple analyses has been performed; therefore significant results must be interpreted with caution. The analyses are based on a data snapshot taken on 24 th April 2015 and were performed with Stata version 13 (StataCorp).

Results
Thirty-two of the 42 patients (76.2%; 32/42) enrolled in the TOPARP-A trial at The Royal Marsden NHS Foundation Trust consented to the MRI sub-study. Six patients did not have baseline WB-MRI due to logistic or technical issues. All 26 patients with WB-MRI at baseline had bone metastases. Of the 26 patients with baseline WB-MRI, 5 patients did not have WB-MRI at 12-weeks due to poor performance status. None of the cases were excluded due to sub-optimal quality of the WB-MRI or incomplete studies. Therefore, 21 patients had evaluable WB-MRI at baseline and after 12-weeks of treatment ( Figure 2); all men, median age 68.2 years (minimum [min], maximum [max]: 40.8, 79.3 years). The population characteristics at baseline are summarized in Table 1. The baseline CT examinations were also reviewed using previously described terminology (24); 19 of the 21 patients had sclerotic bone metastases whereas 2 patients had mixed osteoblastic/osteolytic disease with predominantly lytic metastases. The other sites of metastatic disease outside the bone observed were in lymph nodes (57.1%; 12/21), liver (28.6%; 6/21) and lung (23.8%; 5/21). Seven patients had bone metastases only at baseline (33.3%;7/21). Six patients (29%; 6/21) were considered responders to olaparib as per the primary endpoint definition and had not progressed prior to 12-weeks.
The median time between the baseline WB-MRI and starting treatment was 6-days (1 st quartile [Q1], 3 rd quartile [Q3]: 2.5, 11 days). The absolute value of the tDV, sum of the 5 target lesions total volumes and of the central slice diameters and the mADC at baseline assessed by the different delineation techniques is summarized by response status in Table 2. The percentage change of these parameters after 12-weeks of treatment is summarized by response status in Table 3 and represented in box-plots in Appendix figure 1.

Analysis of axial skeleton DWI b900 signal abnormality
When delineating all the areas of DWI signal abnormality in keeping with bone metastases in the axial skeleton, the median tDV in this population was 0.45 L (min, max: 0.01, 1.31 L) and mADC was 782 x10 -6 mm 2 /s (min, max: 684, 1121 x10 -6 mm 2 /s). These parameters grouped by responders and non-responders are summarized in Table 2; there were no statistically significant differences between the baseline distribution of tDV and mADC between the two groups (p=0.243 and p=0.312 respectively).
The two patients with mixed osteoblastic/osteolytic pattern with predominantly lytic bone metastases were non-responders who had +55.5 % and +24.6 % increase of tDV and +3.40 %, + 15.6 % increase of mADC after 12 weeks on treatment respectively.
The correlation between PSA levels, CTC counts and DWI parameters was also explored; baseline PSA levels and CTC counts showed strong and moderate positive association with baseline tDV (r=0.64, 95% CI 0.29-0.84; and r=0.59, 95% CI 0.22-0.82 respectively) and there was a strong positive association between changes in tDV and best post-treatment percentage change in PSA and CTC (r=0.63, 95% CI 0.27-0.83; and r=0.77 95% CI 0.51, 0.90 respectively), indicating that changes in tDV correlate with response to therapy.
Of the six responding patients, four had a further evaluable WB-MRI at the time of radiological and/or PSA progression. In all four cases, we observed a decrease in tDV while responding to olaparib, followed by a later increase in tDV at the time of radiological and/or PSA progression. Three of these four responding patients also had an increase in mADC while responding to treatment, followed by a decrease in mADC at the time of radiological and/or PSA progression. The fourth patient experienced minimal mADC change at PSA nadir and disease progression. (Appendix figures 3 and 4).

Analysis of five target lesions (total volume and central slice)
With the aim of evaluating more limited radiological analyses, to decrease workload, we correlated changes in up to 5 target lesions per patient with treatment response. We evaluated 5 target lesions in 19 of the 21 patients (90%); the remaining two patients had only one and three evaluable bone lesions respectively. The median sum of total volumes corresponding to the target lesions in the population at baseline was 0.05 L (min, max: 0.01, 0.52 L), and the mADC when delineating total volume of the target lesions was 814 x10 -6 mm 2 /s (min, max: 606, 1712x10 -6 mm 2 /s). In patients with non-widespread bone disease (N=9) we also assessed the diameter of the target lesions in the central slice. When assessing only the central slice of the same target lesions, the median of the sum of diameters at baseline was 12.6mm (min, max: 2.8, 20.2 mm) and the mADC was 835 x10 -6 mm 2 /s (min, max: 554.5, 1263 x10 -6 mm/s). These parameters grouped by responders and nonresponders are summarized in Table 2; there were no statistically significant differences between the baseline distribution of volume, diameter and mADC (central slice and volume) of the target lesions between the two groups (p=0.876, p=0.143, p=0.312 and, p=0.073 respectively).
Then, we assessed the same target lesions for each patient in their follow-up WB-MRI after 12-weeks of treatment; the percentage change of these parameters after 12-weeks of treatment is summarized by response status in Table 3, Figure 2. Changes in entire lesion volume of the target bone metastases also inversely associated with response (OR: 0.89, 95%CI 0.80, 0.99, p=0.037) ( Table 4). The mADC change at 12-weeks, when analyzing the target bone metastases (total volume and central slice), also associated with response, although these associations did not reach statistical significance (p=0.056 and p=0.082 respectively) ( Table 4). Results from the multivariate logistic regression analyses showed similar trends ( Table 4).

Analysis of the axial skeleton (enclosing normal and abnormal bone marrow)
The baseline median of the mADC in our population when delineating the entire axial skeleton including both normal and abnormal bone marrow was 805 x10 -6 mm 2 /s (min, max: 614, 1182 x10 -6 mm 2 /s). mADC at baseline grouped by responders and non-responders are summarized in Table 2; there were no statistically significant differences between the baseline distributions of mADC between the two groups (p=0.94). The percentage change of mADC after 12-weeks of treatment is summarized by response status in Table 3, Figure 2. When comparing the mADC of the entire axial skeleton (normal and abnormal bone marrow) pre-and-post treatment with olaparib, changes in mADC did not associate with response to treatment (p=0.518). (Table 4)

Discussion
We hypothesized that changes in volume of bone marrow metastases assessed by DWI and changes in mADC are indicators of response of bone metastases to treatment in patients with mCRPC. In our study we explored different delineation techniques for assessing bone metastases quantitatively and qualitatively with WB-DWI. One technique included all the areas of DWI signal abnormality in keeping with all bone metastases in the axial skeleton (tDV); the other focused on two simpler techniques assessing five target lesions, based on the widely used RECIST 1.1 (4), to determine whether a simplified approach may be viable in clinical practice. Finally, we explored if changes in mADC delineating the entire spine and pelvis (including areas of normal and abnormal bone marrow), which may facilitate automated delineation, was associated to response.
We have shown that when delineating all the areas of DWI signal abnormality in keeping with bone metastases in the axial skeleton (from C4 to mid-thigh), the changes detected in tDV and mADC after 12-weeks on treatment allow the identification of responders in mCRPC with bone metastases. Decreases in tDV correlated with decreases in PSA levels and CTC count falls, and also with overall response as defined as a composite endpoint in the TOPARP-A clinical trial (23). Consistent with the fact that tumor cell death results in increased water diffusivity manifested as higher ADC values, patients who responded to olaparib also showed a greater increase in mADC compared to non-responders. In our population, the results of simpler ways of assessing bone metastases on WB-DWI in 5 selected target lesions (total volume or central slice) support further evaluation of this faster and more practical approach in future studies; as decreases in volume and diameter of the five target lesions after 12-weeks on treatment associated with response. There was also a trend of significance when associating mADC increases of the target lesions at 12-weeks and response. Therefore, overall, these data indicate that WB-DWI may have a role in bone metastases response assessment in mCRPC, without need of ionizing radiation or intravenous contrast, potentially allowing the detection of differential responses in visceral or nodal metastases and bone metastases. Clinical qualification of WB-DWI as response biomarker in bone metastases would improve assessment of response to treatment in mCRPC, allowing for optimization of patients care, treatment decision and drug development in this common disease. Conversely, when delineating spine and pelvis, including all areas of normal and abnormal bone marrow, increases in mADC after 12-weeks on treatment did not associate to response; probably due to the fact that changes in mADC in bone metastases are diluted by the absence of changes in mADC in normal bone marrow.
We acknowledge the potential limitations of our study; firstly, due to the small size of this pilot study, only limited exploration of the impact of adjustment for other clinical factors on the association of changes in tDV and mADC is possible. Analyzing larger populations in multi-center studies is now needed for future validation of these results and to allow multivariate analyses. Secondly, all our patients were treated with one drug, the PARP inhibitor olaparib; however, previous studies have identified similar changes in DWI in bone metastases responding to hormonal therapy and cytotoxics (18)(19)(20)(21). Prospective studies to replicate our results with established treatments for mCRPC are now needed. Thirdly, it should be noted that the ROI delineation depends on the quality of the acquired DWI data, the semi-automatic segmentation tool and radiologist expertise. Prior studies reported high intra-reader reproducibility of DWI analysis using similar bone metastases delineation methodology (17,25), although this needs to be validated in larger, properly powered studies. Finally, we acknowledge that the majority of our population had sclerotic bone metastases, only 2 patients had predominantly lytic bone metastases; therefore, it was not feasible to perform comparison between the sclerotic vs lytic nature of the bone metastases. Despite these limitations, our study represents the largest prospective series to date in a trial of a novel therapeutic assessing response to drug treatment in bone metastases in patients with mCRPC using WB-DWI. The data presented here highlight the potential of DWI for bone metastases response assessment and warrants further evaluation of WB-DWI in this disease.
In conclusion, we have shown that assessment of bone metastases with WB-DWI during anticancer treatment is feasible, with changes in bone metastases volume and mADC being indicators of response to treatment in mCRPC in our pilot study. Moreover, the more efficient study of five target lesions has substantial practical merit for disease evaluation, which can be more easily adopted into clinical practice. These results support further evaluation of DWI as a response biomarker in prospective mCRPC patient cohorts, ideally embedded into clinical trials (26).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

1.
Clinical qualification of whole body diffusion weighted imaging (WB-DWI) as response biomarker in bone metastases would improve assessment of response to treatment in mCRPC, allowing for optimization of patients care, treatment decision and drug development in this common disease.

Summary statement
Assessment of bone metastases with whole body diffusion-weighted imaging during cancer treatment is feasible, with changes in bone metastases volume and median apparent diffusion coefficient being indicators of response to treatment in metastatic castration resistant prostate cancer in our pilot study. Images show the different delineation techniques in two-dimensional coronal or axial views for illustrative purpose. Areas of signal abnormality corresponding to high signal intensity on DWI (b = 900 mm/s 2 ) and low signal intensity on T1-weighted image, in keeping with bone metastases observed between C4 and the mid-thigh were delineated on DWI (b= 900mm/s 2 ) (a). In order to explore a more limited approach, total volume (b) and central axial slice (c) of up to 5 target lesions were delineated on DWI (b= 900mm/s 2 ). (d) Finally,    Table 4 Associations of total diffusion volume (tDV), volume and diameter of the target lesions and median ADC (mADC) changes between baseline and 12 weeks with binary response to treatment were assessed using logistic regression. OR: Odds ratio