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Translational Cancer Mechanisms and Therapy

Biomarkers and Bone Imaging DynamicsAssociated with Clinical Outcomes of OralCabozantinib Therapy in Metastatic Castrate-Resistant Prostate CancerUlka N. Vaishampayan1, Izabela Podgorski2, Lance K. Heilbrun1,Jawana M. Lawhorn-Crews1, Kimberlee C. Dobson1, Julie Boerner1, Karri Stark1,Daryn W. Smith1, Elisabeth I. Heath1, Joseph A. Fontana1, and Anthony F. Shields1

Abstract

Purpose: Cabozantinib is a multitargeted tyrosine kinaseinhibitor that demonstrated remarkable responses on bonescan in metastatic prostate cancer. Randomized trials failed todemonstrate statistically significant overall survival (OS). Westudied the dynamics of biomarker changes with imaging andbiopsies pretherapy and posttherapy to explore factors that arelikely to be predictive of efficacy with cabozantinib.

Experimental Design: Eligibility included patients withmetastatic castrate-resistant prostate cancer with normal organfunction and performance status 0–2. Cabozantinib 60 mgorally was administered daily. Pretherapy and 2 weeks post,99mTc-labeled bone scans, positron emission tomographywith 18F-sodium fluoride (NaF-PET) and 18F-(1-(20-deoxy-20-fluoro-b-D-arabinofuranosyl) thymine (FMAU PET) scanswere conducted. Pretherapy and posttherapy tumor biopsieswere conducted, and serum and urine bone markers weremeasured.

Results: Twenty evaluable patients were treated. Eightpatients had a PSA decline, of which 2 had a decline of�50%. Median progression-free survival (PFS) and OS were4.1 and 11.2 months, respectively, and 3 patients were ontherapy for 8, 10, and 13 months. The NaF-PET demon-strated a median decline in SUVmax of �56% (range, �85to �5%, n ¼ 11) and �41% (range, �60 to �25%, n ¼ 9)for patients who were clinically stable and remained ontherapy for �4 or <4 cycles, respectively. The FMAU PETdemonstrated a median decline in SUVmax of�44% (�60 to�14%) and �42% (�63% to �23%) for these groups.The changes in bone markers and mesenchymal epithelialtransition/MET testing did not correlate with clinicalbenefit.

Conclusions: Early changes in imaging and tissue or serum/urine biomarkers did not demonstrate utility in predictingclinical benefit with cabozantinib therapy.

IntroductionProstate cancer is the most common cancer in males with an

estimated 164,690 new cases in 2018 in the United States withan anticipated mortality of 29,430 (1). Although most cases aretreated when localized, others present as disseminated diseaseor become metastatic after definitive treatment. In metastaticcastrate-resistant prostate cancer (mCRPC), various agents suchas sipuleucel T, abirateraone, enzalutamide, docetaxel, cabazi-taxel, and radium-223 have demonstrated survival benefit(2, 3). Despite demonstrating impressive efficacy in early trials,cabozantinib encountered a rocky road during the develop-

ment of an indication in metastatic prostate cancer. Initialphase I/II study of cabozantinib revealed tremendous promisewith an unprecedented normalization of bone scans that hadnever been observed even with the most effective treatment todate such as androgen deprivation therapy (4, 5). In addition,these effects were seen in a refractory pretreated patient pop-ulation, and measurable disease responses were noted. Thephase II trial results with this agent in prostate cancer led tothe conduct of 2 large registration trials. Unfortunately, the firsttrial of cabozantinib plus prednisone versus placebo plusprednisone showed no benefit in overall survival (OS), whichwas the primary endpoint (5). The second trial comparingcabozantinib and prednisone to mitoxantrone and prednisonewith predefined pain palliation endpoint was halted early dueto results demonstrating lack of benefit (6). These events ledto further drug development of cabozantinib being put onhold in prostate cancer. The mechanisms underlying the re-markable bone scan responses associated with significant clin-ical palliation have not been studied in depth. In addition, theevaluation of biomarker or imaging changes in correlation withclinical outcomes was not conducted.

Cabozantinib is an inhibitor of tyrosine kinases, includingMET, AXL, and VEGFR, that results in abrupt clinical changes in

1Department of Oncology Karmanos Cancer Center/Wayne State University,Detroit, Michigan. 2Department of Pharmacology and Oncology Wayne StateUniversity, Detroit, Michigan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Ulka N. Vaishampayan, 4100 John R, Detroit, MI 48201.Phone: 313-576-8715; Fax: 313-576-8487; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-1473

�2018 American Association for Cancer Research.

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bone metabolism represented as an abrogation of 99mTc-MDPuptake on bone scan. This is likely due to inhibition of osteoclastfunction anddecrease in osteoblast activity.We hypothesized thatthe agent uniquely targets the cross-talk between C-MET andvascular endothelial growth factor receptor (VEGFR) axis andmodulates bone turnover via downstream cathepsin K–drivenpathways and activity of novel receptor tyrosine kinases, such asDDR-1 and DDR-2 (refs. 7, 8; Fig. 1). We conducted a pilot trialdesigned to study the pathophysiology and biomarker changes inbone metastases and correlate these with response and clinicaloutcome data in metastatic CRPC patients. The study alsoexplored any mechanistic clues for a subset within mCRPC thatmaybe worthy of targeting with cabozantinib therapy, given theclinical efficacy observed and reported by multiple investigatorsglobally. The MET receptor tyrosine kinase (RTK) for hepatocytegrowth factor (HGF) has been implicated as a mediator in manyimportant aspects of tumor pathobiology, including tumor sur-vival, growth, angiogenesis, invasion, and dissemination. (9)Several tyrosine kinase inhibitors of MET have been reported toshow antitumor activity in cell lines and animal models (10). TheVEGFR2 (vascular endothelial growth factor receptor) is a centralmediator of tumor angiogenesis, and several small-molecule andprotein therapeutics targeting this receptor are in clinical devel-opment. In addition to their individual roles in tumor pathobi-ology, preclinical data suggest that Met and VEGFR2 play syner-gistic roles in promoting tumor angiogenesis and subsequentdissemination (9).

Typically, anti-VEGF therapies have not been effective inmCRPC. Randomized trials with bevacizumab or ramucirumabdemonstrated lackof benefitwhen evaluated in combinationwithdocetaxel. Compounds that simultaneously inhibit VEGF andMET RTKs may be more effective anticancer agents than agentsthat target each of these receptors individually (10). Cabozantinibis a potent RTK inhibitor that targets primarily MET and VEGFR2and, by this mechanism, is likely to overcome resistance to anti-VEGF therapy. It has activity against other RTKs that have been

implicated in tumor pathobiology, including KIT, FMS-like tyro-sine kinase 3 (FLT3), and Tie-2. It is known to inhibit RET, an RTKknown to be causative for malignancy, such as in hereditarymedullary thyroid cancer (11, 12).

Imaging studiesThe conventional bone scan utilizes Technetium 99mTc meth-

ylene diphosphonate (MDP) and is a most widely used stan-dard-of-care method for evaluating skeletal metastases in pros-tate cancer. The 99mTc-MDP accumulates in new (woven) boneand is an indicator of changes in bone metabolism especiallyassociated with prostate cancer–induced osteoblastic response.However, 99mTc-MDP scan findings are nonspecific and areindirect markers of response to treatment (13). Positron emis-sion tomography with 18F-sodium fluoride (NaF-PET) scanshave improved anatomic detail over 99mTc-MDP scans, a higheraccuracy in detecting metastases and potentially allow quanti-fication of the extent of metastatic lesion (14). This imagingmodality may be superior to 18F-fluorodeoxyglucose (FDG)PET for prostate cancer, since the bone metastases in prostatecancer are primary osteoblastic. Osteoblastic metastases tend toexhibit a high rate of fluoride incorporation (13) and may havelow FDG uptake (14). Additionally, NaF-PET has improvedsensitivity, so earlier detection of changes is feasible and PETimaging offers the potential for rigorous quantification. Unlikeconventional bone scans that delineate the presence of a lesion(15), imaging techniques currently being evaluated in associ-ation with therapeutic trials in prostate cancer are designed todetect pharmacodynamic effects of novel agents. We utilizedthe 18F-fluoride PET in this study to trace the extent of absorp-tion of fluoride ion by bone tissue and to attempt quantifica-tion of response in bone metastases.

PETobtainedwith the thymidine analogue 18F-(1-(20-deoxy-20-fluoro-b-D-arabinofuranosyl) thymine (FMAU PET) scan is usedto image tumor metabolism and is based on incorporation of thetracer by mitochondrial thymidine kinase-2 (TK2; refs. 16, 17).The 3 modalities complement each other in distinguishingchanges in lesions on imaging. Changes in cellular metabolismeffected by therapy were hypothesized to have increased sensi-tivity in measurement of antitumor effects of cabozantinib thanthe more conventional approach of observing changes in size asseen on CT scans or just detecting areas of bone turnover or lackthereof, as seen with 99mTc-MDP conventional bone scans. Wehave previously reported on the use of FMAU scans for detectionof prostate cancer bone metastases from a study conducted at ourinstitution, validating the use of this imaging modality for bonemetastases (18).

Materials and MethodsStudy design

The primary objective of the study was to evaluate the timing,physiology, and magnitude of changes in tumor imaging, andpharmacodynamics (PDs) markers with cabozantinib treatmentin mCRPC. The secondary objectives were to evaluate the clinicalsafety, progression-free survival (PFS), andOSwith this agent andto correlate clinical outcomes with imaging and PD changesobserved. This was a single-arm, single-institution, pilot trial ofcabozantinib administered at a starting dose of 60mg orally dailyin patients with mCRPC. The study was approved by the WayneState University institutional review board and written informed

Translational Relevance

Radiologic response and progression represent key deci-sion-making endpoints in oncology therapeutics. Decisions tocontinue or change therapy hinge on changes noted in imag-ing. The cabozantinib experience in metastatic castrate-resistant prostate cancer (mCRPC) highlights the major chal-lenge of using imaging response as a surrogate for clinicaloutcomes in this disease. Due to the bone targeted, osteoclastinhibitory activity of cabozantinib, bone marker, and bonescan changes were misleading and did not correlate withclinical outcomes. Even novel imaging methods such as pos-itron emission tomography with 18F-sodium fluoride (NaF-PET) and 18F-(1-(20-deoxy-20-fluoro-b-D-arabinofuranosyl)thymine (FMAU PET) scans were unsuccessful in detectingresponses that would predict clinical benefit. Bonemarkers andtissue c-METexpression alsodid not yield predictionof efficacy.Cabozantinib represents a unique mechanism of action that isdistinct from currently approved therapies in mCRPC and isworthy of deeper investigation with genomic sequencing toevaluate predictive markers for patient selection and therapy.

Biomarkers Associated with Cabozantinib Therapy

www.aacrjournals.org Clin Cancer Res; 25(2) January 15, 2019 653




consent was obtained from all patients before registration. Thestudy was conducted in accordance with the ethical guidelines ofthe Declaration of Helsinki.

Patient selectionEligible patients were 18 years or older and had histologically

confirmed mCRPC and objective progression or risingPSA despite androgen deprivation therapy and antiandrogenwithdrawal. Patients with rising PSA had to demonstrate arising trend with 2 successive elevations at a minimum intervalof 1 week. A minimum PSA of 5 ng/mL or new area ofbony metastases on bone scan were required for patientswith no measurable disease. No minimum PSA was requiredfor patients with measurable disease. A maximum of 1prior chemotherapy regimen for mCRPC was allowed. Anyradiotherapy had to be completed at least 2 weeks prior tostarting study therapy. All patients had to be documented to becastrate with a testosterone level � 0.5 ng/mL. Luteinizinghormone releasing hormone agonist therapy was continued,

if required to maintain castrate levels of testosterone. Patientshad to be off antiandrogens for a minimum of 4 weeks forflutamide and 6 weeks for bicalutamide or nilutamide. Patientswith ECOG performance status �2 and life expectancy of 12weeks or more were eligible. Patients were required to haveadequate bone marrow, liver, and renal function. Other keyexclusion criteria included history of bowel perforation orfistula, uncontrolled brain or leptomeningeal metastases,uncontrolled hypertension or diabetes mellitus or history ofcongestive heart failure.

Treatment planThe study consisted of open-label daily, oral administration

of cabozantinib at a starting dose of 60 mg to eligible patients.This was administered with a full glass of water (minimum of 8oz/240 mL) after fasting (with exception of water) for a min-imum of 2 hours before and at least 1 hour after ingestion.Subjects were advised to record dosing time and doses taken ina study drug dosing diary while on study treatment. The

Figure 1.

Proposed mechanism of action of XL 184 in prostate cancer. XL184 predominantly targets RTKs involved in tumor-induced bone resorption. Inhibition of osteoclastactivity by XL184 results in reduced levels of the key osteoclast collagenase CTSK and overall inhibition of bone turnover. Inhibition of DDR-1 and -2 directly byXL184 and indirectly by reduced availability of resorbed collagen abrogates collagen-induced osteoblast differentiation and woven bone deposition.Abbreviations: RANKL, receptor activator of NF-kB ligand; VEGF, vascular endothelial growth factor; VEGFR1-, 2-, VEGF receptors; HGF, hepatocyte growth factor;cMet, HGF receptor; MCSF, macrophage colony–stimulating factor; c-fms, MCSF receptor; SCF, stem cell factor; c-kit, SCF receptor; FLt3, FMS-like tyrosine kinase 3;DDR-1,2, discoidin domain receptor; PIGF, placental growth factor; CTSK, cathepsin K; TRAcP, tartrate-resistant acid phosphatase; NTx, N-telopeptide;CTx, C-telopeptide; ET-1, endothelin-1.

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original schedule of assessments continued even if doses werewithheld. The subjects were instructed to not make up anymissed or vomited doses and to adhere to the planned dosingschedule. The study allowed a maximum of 2 dose reductionsof cabozantinib to 40 mg and 20 mg orally daily, respectively. Ifgrade 3 or 4 toxicities or grade 2 toxicity lasting for 7 days orgreater was noted, then medication was held until the toxicityresolved to grade 1 or pretherapy baseline. Dose reductioncould be considered when resuming therapy. If toxicity per-sisted after 2 dose reductions and optimal supportive care, thenstudy therapy had to be discontinued.

Correlative studiesImagingmethods. This study utilized standard-of-care 99mTc-MDPbone scan using a gamma camera, NaF-PET to measure effects ofcabozantinib treatment on bone tissue, and FMAU PET to mea-sure changes in tumor metabolism in response to therapy. Imag-ing was performed using a gamma camera and PET/CT scanners.

We proposed evaluating patients with NaF-PET pre- and postcabozantinib therapy to determine the optimal timing whenbone scan normalization occurs. So approximately 5 patientswere evaluated with NaF-PET imaging pretherapy and 2 to 3weeks posttherapy. The optimal time point to perform imagingwith FMAU-PET scans to evaluate for antitumor effect wasdetermined to be at 2 weeks and subsequent patients had scansperformed during that timeline. PET imaging was performedusing a GE Discovery STE PET/CT system (GE Medical Sys-tems), located at the PET Center, Children's Hospital of Michi-gan. Patients were positioned on their back on a PET/CTscanner in a high-sensitivity mode. Vital signs were monitoredat the beginning and end of each scan. Patients were injectedwith FMAU PET using an intravenous catheter with dosesstandardized to body weight (mean, 360 MBq; range, 196–407 MBq) with a specific activity of at least 18,500 MBq/microM and a purity greater than 98%. Dynamic images wereacquired at 6 to 11 minutes with 1 bed position over the areaof interest of 2 frames: (1 � 5 minutes and 1 � 6 minutes).Thereafter, a whole-body image was obtained using a 2D/3Dmodality, of 3 bed positions. For NaF-PET scans, patientsreceived an intravenous injection with a mean of 340 MBq(range, 259–407 MBq) and imaging began 45–60 minuteslater. Patients were positioned with their arms down andscanned from the vertex to upper thigh and then repositionedto scan the patient's legs. Whole-body acquisition time con-sisted of 3 minutes per bed position. Reconstructed imageswere viewed and analyzed using Osirix Imaging Software.Tumor SUVmax values were obtained at baseline and follow-up scans by drawing a 1-cm diameter region of interest over 5 ofthe most active boney lesions on the NaF-PET scans. Weselected no more than 2 active lesions per bone region, includ-ing the skull, thorax, spine, pelvis, and extremities. The samelesions were selected on the FMAU PET scans. A decrease inmean SUV by 20%was considered a PET response and was usedas the threshold to detect changes.

Serum and urine markers. Serum bone markers were assessed pre-and posttherapy. These included serum bone-specific alkalinephosphatase (BSAP) and N-terminal telopeptide of collagentype I (NTx). High levels of these markers (>146 u/L for BSAPand >100 nmol/mmol for NTx) have been reported to be signif-icantly predictive of higher incidence of skeletal complications

(relative risk of 3.32, P < 0.001), prostate cancer progression (RR2.02, P < 0.001), and death (RR of 4.59, P < 0.001; ref. 19).

A number of other bone turnover markers such as osteocalcin,pyridinoline, and deoxypyridinoline have been implicated to bepredictive of therapeutic response. A study evaluating the efficacyofmatrixmetalloproteinase inhibitors in prostate cancer reportedthat decline of the bone resorption markers, including NTx,procollagen I NH2-terminal propeptide, osteocalcin, and deox-ypyridinoline, correlated with improved PFS and OS outcome(19–21). This led to the hypothesis that detectable changes inbone markers could act as surrogates of therapeutic effect inprostate cancer bone metastases.

We selected the serumNTx andBSAP and urineNTx as the boneturnover markers due to the validation of these markers in priorlarge studies utilizing zoledronate therapy (22). Decline in thelevelswas predictive of lower incidence of skeletal events aswell asPFS and OS. Hence, the measurement of these markers (NTx andBSAP) pre- and posttherapy was correlated with PET scan findingsand clinical outcomes.

Serum and urine (24-hour urine collection sample) N-telopep-tide were assessed using the Vitros ECI Immunodiagnostic Systemcompetitive assay (Johnson & Johnson Ortho-Clinical Diagnos-tics). SerumBSAP levels were assessed using a chemical inhibitionand differential inactivation assay.

The levels of bone resorption markers in serumwere assayed atbaseline and at 8, 15, and 28 days after treatment with cabozan-tinib. Tartrate-resistant acid phosphatase (TRAcP) levels weremeasured using Human TRAcP ELISA (RayBiotech). For theevaluation of osteocalcin levels, Quantikine Colorimetric Sand-wich ELISA assays (R&D Systems) were used. All samples wereassayed in triplicate according to the manufacturer's instructions,and cytokine levels were quantified by colorimetric detection at450 nm against appropriate standards.

Circulating tumor cell count. This wasmeasured by the Cell Searchmethod pretherapy, and 2 and 4 weeks after therapy and atprogression.

IHC for MET testing. Paraffin sections were deparraffinized in axylene–ethanol series. Endogenous peroxides were removed by amethanol/1.2% hydrogen peroxide incubation at room temper-ature for 30 minutes. HIER antigen retrieval with a pH9 EDTAbuffer and the BIOCARE Decloacking Chamber. A 40-minuteblocking step with Super Block Blocking buffer (Thermo Scien-tific) was performed prior to adding the primary antibody. Metantibody from Abcam (ab51067) was used at a dilution of 1:100.Detectionwas obtained usingHRP/DAB chromogen and counter-stained with Mayer's Hematoxylin. Sections were dehydratedthrough a series of ethanol-to-xylene washes and cover slippedwith Permount. The staining was evaluated, categorized as 0, 1þ,2þ, and 3þ by a qualified pathologist whowas blinded to clinicaldata, and reported.

Statistical methodsThe trial was a prospective pilot adaptive design study to

obtain preliminary data. Each patient would undergo up to 4PET scans, using different radiotracers. It was desired to esti-mate the mean SUV at any time point to approximately one-third of a standard deviation (SD) with 80% confidence. With15 patients, the mean SUV could be estimated to within 0.347SD units of the true mean with 80% confidence. These






preliminary estimates would be of sufficient precision for usein designing a subsequent larger study. Because not allpatients would undergo all PET scans, the mean SUV may beless precisely estimated at some time points.

The Prostate Cancer Clinical Trials Working Group (PCWG2)criteria were used to determine a response (23). For measurabledisease response, RECIST criteria 1.1 were used (24). For thepatients imaged on a common schedule, the continuous end-points (e.g., SUV, bone scan measurements, and all continuouslydistributed correlatives) were summarized with standard descrip-tive statistics, separately at each measurement time point. Wehypothesized that biomarker changes in bone metastases andimaging correlated with response and clinical outcome data inmetastatic CRPC patients treated with cabozantinib. The categor-ical endpoints such as toxicities, the clinical response, and IHCexpression levels were summarized via their frequency distribu-tion, point estimate of the proportion, and the Wilson type 90%confidence interval (CI).

The distributions of percent change in SUV by FMAU PET andpercent change in each bone marker were quite nonnormal,despite various transformations applied. Accordingly, the rela-tionship between pre/posttherapy changes in imaging (percentchange in SUV by FMAU PET and by NaF) and changes in each ofthe 3 bone marker levels (percent change from day 1 to week 4)were first assessed using the Spearman rank correlation coefficient(rho, and its 90%CI). Fisher Z-transformation of rhowas requiredin order to calculate the confidence limits for the rank correlationcoefficient. To explore the relationships of change inbonemarkerswith change in imaging, we used a nonparametric regressionapproach.Wefit a locally estimated scatterplot smoother (LOESS)curve using the LOESS procedure in SAS 9.4 software. A LOESScurvewasfit to 2 selected bivariate relationships of change inbone

marker and change in imaging. For exploratory analysis purposes,the selections were the only 2 relationships having rho > 0.20.The default smoothing parameter (percentage of the totalobservations used in each smoothing neighborhood) was usedin the LOESS procedure. These nonparametric LOESS curvesbetter described the nonlinear character of those 2relationships.

The distribution of censored PFS was summarized via theKaplan–Meier (K-M) survivorship estimate. Summary statistics(e.g., median, 6-month, and 12-month progression-free rates)were calculated from the K-M life table. Similar analyses will beperformed for OS as well.

PFS was measured from treatment start date to the first dateof documented progression, whether by PSA or by imaging, ordeath from any cause, whichever occurred first. Patients notexperiencing progression were censored for PFS as of the date oftheir last PSA or imaging result. OS was measured from treat-ment start date to the date of death from any cause. Patientswere censored for OS as of the last date on which they wereconfirmed to be still alive.

ResultsPatient characteristics, toxicity, and efficacy

Twenty-six patients were consented, of which 20 were eligibleand enrolled; 1 withdrew from the study and 5 were screenfailures. The median age was 69 years (range, 56–76 years;Supplementary Table S1). Thirteen patients had bone pain at thetime of enrollment. Sixteen patients discontinued therapy dueto progression and 4 discontinued due to toxicities which con-sisted of hand–foot syndrome, fatigue, urinary infection, and ele-vated creatinine each in 1 patient. No unexpected toxicities or

Figure 2.

Waterfall plot of PSA levels.

Vaishampayan et al.





treatment-related prolonged morbidity or mortality were noted.Each cycle consisted of 28 days of therapy. Themedian number ofcycles received was 4 (range, 1–17 cycles) and 6 (30%) patientsreceived 8ormore cycles of therapy. Three patients did not requiredose reductions and 11 and 6 required 1 and 2 dose reductions,respectively.

EfficacyResponsewas assessed in all 20 patients based on PSA aswell as

measurable disease per RECIST1.1. Six of 9 patients with mea-surable disease showed tumor shrinkage per RECIST 1.1 criteria(median �20%, range, �10 to �38%). Eight of 20 patientsdemonstrated a PSA decline with a median of 21% (Fig. 2) with

Figure 3.

Illustrations of imaging changes seen with cabozantinibtherapy in mCRPC patients treated on study. A,NaF-PET scan obtained in a 64-year-old male pre- andposttreatment. B and C were obtained from a 71-year-oldmale using 18F-NaF PET and 18F-FMAU PET, respectively.






absolute change of 22.3 ng/mL (range, 6.4% to 70.8%, absolutedecline range 0.9–282.9 ng/mL). Two patients had a 50% orgreater reduction in PSA levels. The PSA decline did not correlatewith PFS. Ten patients received therapy for �4 cycles and 6patients continued on therapy for �8 cycles (maximum of 17cycles). All patients were evaluable for PFS and OS. Median PFSwas 4.1 months (90% CI, 2.3–5.3 months), and median OS was11.2 months (90% CI, 15.0–29.7 months).

Imaging and bone turnover markers. Nineteen of 20 patientsdemonstrated a decline in the SUV max and mean pre- andposttherapy. Seventeen patients had the FMAU PET scan pre- andposttherapy and 14 showed a SUV decline of�20%. Two patientsshowed a decline of <20% at 14.2% and 18.7%. Only 1 patientshowed a 23% increase in SUV max. Maximum SUV decline was63.7%. Examples are shown in Fig. 3. The NaF-PET scan wasevaluated in 19 patients and 18 (1 patient showed a 5% decline)showed a decline in SUV max of >20%. Maximum change inuptake was a 85.4%decrease. The timeline of changes and declinein tracer activity on both imaging techniques was very rapid andseen within 1 to 2 weeks of therapy. SerumNTX showedminimalchange from pretherapy to week 4 of therapy, median decline of2.8% from pretherapy median levels of 12.5 to 11.4 at week 4 oftherapy.UrineNTX revealed anappreciable decrease frommedianof 29 pretherapy to 15 posttherapy, median decrease of 41.2%.Unfortunately, no correlation was noted between the magnitudeof decline in SUV max by imaging with clinical benefit. Elevenpatients had CTC > 5 pretherapy, of which 5 patients converted toCTC< 5 after therapy. Table 1 summarizes the changes dichoto-mized by patients receiving fewer than 8 cycles, or 8 ormore cyclesof therapy.

As an exploratory analysis only, Spearman rank correlationcoefficients (rho values and their CI) for all pairs of 5 variables(percent change in eachof FMAUPETSUV,NaFSUV, BSAP, serumNTx, and urine NTx) are given in Supplementary Appendix TableS2. The 2 pairs of imaging change and bone marker change withthe largest (and positive) correlation (rho > 0.20) were NaF SUVwith BSAP (rho ¼ 0.22), and NaF SUV with urine NTx (rho ¼0.26). The nonparametric LOESS curve fit for percent changein NaF SUV as a function of percent change in BSAP is shownin Fig. 4A. There is a positive relationship overall, especially in therange of negative percent changes in BSAP. There, large decreasesin BSAP tended to relate to large decreases in NaF SUV. A similarbut weaker positive relationship was found in which largedecreases in urine NTx tended to relate to only modest decreasesin NaF SUV (Fig. 4B).

Tissue testingTumor biopsies were conducted pretherapy and 2 weeks after

therapy. C-MET testing was conducted by IHC on tumor biopsies(Fig. 5A and B). No consistent changes in c-MET expression were

noted pretherapy and posttherapy, and the extent of CMETexpression did not correlate with clinical benefit (Fig. 5C and D).

DiscussionRadiologic response and progression represent key decision-

making endpoints in oncology therapeutics. Frequently, deci-sions to continue or change therapy hinge on changes noted inimaging. In the case of bone metastases, the predominant site ofspread in prostate cancer, this endpoint is flawed and leads toerroneous decisions. Aprime example of thiswasnotedwithin theimaging changes after cabozantinib administration. Convention-al (Technetium) 99mTc-MDP bone scans showed remarkableresponse in a majority of the patients and led to excitement inphase II trials, which was subsequently not matched by clinicalefficacy seen in the randomized setting (25). The current studywasdesigned to incorporate other imaging techniques such as NaF-PET and FMAU PET scans to evaluate correlation with clinicalendpoints. The results reveal that these scanning methods werenot predictive of efficacy. Novel imaging techniques, such ascholine/acetate scans and fluciclovine scans based on amino acidradiotracer, have demonstrated exquisite sensitivity to detectrecurrent disease at low PSA levels, and high positive predictivevalues (26). These scans are able to detectmetastases in both boneand soft tissue; however, they have not yet been assessed formonitoring effects of systemic therapy (27). Bone turnover mar-kers have been explored as potential predictors of response orclinical benefit from therapy. Unfortunately, the results of moststudies have been disappointing, and these biomarkers are notutilized in routine clinical practice. Alkaline phosphatasechanges have been reported to be predictive of response inradium-223 therapy (28). In fact, changes in alkaline phospha-tase are likely to be better predictors of benefit than changes inPSA for radium-223 treatment. Cabozantinib had a majorimpact on the bone turnover in prostate cancer bone metastasesin initial studies. It also demonstrated promising clinicalresponse rates in measurable disease (5). Unfortunately, laterstudies showed that the remarkable efficacy noted did not leadto an OS impact. Randomized double-blind controlled trialsrevealed lack of OS benefit when compared with prednisonetherapy alone. However, investigator-assessed PFS, which wasan exploratory endpoint of the study, was statistically signifi-cantly improved by cabozantinib treatment (HR ¼ 0.48; P <0.001). The result was that the clinical efficacy of cabozantinibin randomized trials could not be proven.

The anti-VEGF therapies have demonstrated a consistent pat-tern of promising efficacy in single-arm trials, which subsequentlydid not translate into OS benefit in the randomized setting.Sunitinib demonstrated lack of OS benefit in a double-blindplacebo-controlled trial with median OS of 13.1 months withsunitinib and 11.8months with placebo (29). CALGB 90401 was

Table 1. Percent change in imaging, bone markers, and CTC pre- and post cabozantinib therapy

Variable Median change Minimum Maximum<8 cycles(median change)

�8 cycles(median change)

FMAU PET scan �45% �63.4% þ23.2% �45% �40.1%NaF scan �48.1% �85.4% �24.5% �48.1% �58.8%BSAP 21.3% �55.8% 250.7% 21.3% �7.5%Serum Ntx �13% �68.2% 522.6% �13.0% 18.5%Urine Ntx �41.7% �77.4% 66.7% �41.7% 50.0%CTC (week2) �33.2% �97.8% 100% �66.7% �16.7%

CTC (prog) 423% �61.5% 2633% 423% 300%

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a trial that compared the combination of bevacizumab anddocetaxel to docetaxel alone, and although the combinationshowed improved response rates (43.4% vs. 35.4 %) andmedianPFS of 9.9 vs. 7.5 months (stratified log-rank P < 0.001), the OS(medianof 22.6months for the combination vs. 21.5months,P¼

�0.18) was no different (30). A meta-analysis of 9 randomizedcontrol trials of docetaxel and antiangiogenic therapy as com-pared with docetaxel and prednisone confirmed lack of clinicalbenefit and possibly increased toxicity with combination therapyin mCRPC (31).

Figure 4.

A, Nonparametric regression LOESS curve (solidline) of percentage change (pre/post XL 184)therapy in NaF SUV as a function of percentagechange (from day 1 to week 4) in serum BSAP. Theshaded area indicates the 90%CI for the predictedmean percentage change in NaF SUV over therange of observed levels of percentage change inserum BSAP. B, Nonparametric regression LOESScurve (solid line) of percentage change (pre/postXL 184) therapy in NaF SUV as a function ofpercentage change (from day 1 to week 4) in urineNTx. The shaded area indicates the 90% CI for thepredicted mean percentage change in NaF SUVover the range of observed levels of percentagechange in urine NTx.






The current study was designed to detect the mechanism of thebone changes and to explore if bone biomarkers and imagingchanges could select a patient population that was likely tobenefit. The results of our study show that initial response andsustained clinical benefit for over 8 months was noted in 30% ofthe patients. The observed reduction in serum marker levels waslikely due to the reduced activity of cathepsin K, a protease highlypresent in metastatic prostate cancer (32) and the only enzymecapable of completely degrading helical andnonhelical collagen I,the main component of bone matrix (33). We also suspect theinvolvement of DDR1 and DDR2, 2 novel RTKs that are ligandsfor bone matrix collagen (34, 35). Whether cabozantinib med-iates its action on bone turnover via this axis needs furtherinvestigations.

Table 1 reveals that the serumandurine biomarkers tested, suchas CTC, BSAP, and serum and urine Ntx, were not distinctlyindicative of early prediction of clinical benefit with cabozantinibtherapy. The MET overexpression and phosphorylated MET alsodid not reveal any clear trend of being predictive markers ofefficacy. C-terminal MET protein expression was absent in hor-mone-na€�ve prostate cancer and, in contrast, was present in CRPCin 23%of palliative transurethral resection specimens and in 72%of bone metastases (36). This was also not related to METpolysomy or amplification. C-MET is phosphorylated after nucle-ar translocation, and the staining noted indicates the activity;

however, the level of expression was not associated with clinicalbenefit (36). On imaging, the decline in SUV was very rapid andoccurred within a short period of time, in less than 1 week. Thiswas in concordance with that observed in preclinical studies, butunfortunately no differential emerged to predict clinical out-comes. MET overexpression on IHC staining was also not predic-tive of clinical benefit. Thirty percent of patients had a durablebenefit and continued on therapy for longer than 32 weeks. Theimaging changes were seen in a majority of the treated patients,regardless of clinical benefit with cabozantinib.

The cabozantinib experience in mCRPC highlights the majorchallenge of using imaging response as a surrogate for clinicaloutcomes in this disease. Bone as a site of metastases alonecomprises about 90% of the patients with CRPC. The conven-tional bone scan is severely limited in response determination, asthe extent of tracer uptake and changes thereof does not correlatewith clinical response. TheMDPbone scan also cannot be utilizedformeasurement of lesions. In addition, due to the bone targeted,osteoclast inhibitory activity of cabozantinib, bone marker, andbone scan changes were misleading and not useful to predictclinical outcomes. Our study depicts that even novel imagingmethods such asNaF-PET and FMAUPET scanswere unsuccessfulin detecting responses that would predict clinical benefit with thisagent. NaF-PET is thought to detect bone metabolism and for-mation and may also bind to calcium phosphate, and hence not

Figure 5.

Changes in membrane and cytoplasmic C-MET expression on tumor tissue by IHC, pre- and post-cabozantinib therapy. A, Membrane cMET scores pre- andposttherapy. B, Cytoplasm cMET scores pre- and posttherapy. C, Pretherapy cMET expression by immunohistochemistry. D, Posttherapy cMET expression byimmunohistochemistry.


Vaishampayan et al.




likely to provide a clear measure of treatment response (37).FMAU PET was developed with the intent to image tumor pro-liferation, butmay be trapped in the cell bymitochondria by TK2,limiting the ability to assess changes in tumor growth (18). Withthe advent of genomic testing, next-generation sequencing–basedresults should be investigated as guides to therapeutic decisions.

Cabozantinib-based combinations are being explored inmCRPC, such as a clinical trial conducted by the NCI withnivolumab and an ongoing trial in combination with atezolizu-mab (NCT03170960). Synergy has been reported with multipleagents, such as immune-checkpoint inhibitors, and with radio-therapy. ThephenotypeofmCRPC is likely to changedramaticallyover the next decade. The advent of early indication and utiliza-tion of chemotherapy and abiraterone in themetastatic hormone-na€�ve setting and use of agents such as enzalutamide and apalu-tamide in nonmetastatic disease will alter the configuration of themCRPC state. Incidence of neuroendocrine features withinmCRPC is likely to increase. Cabozantinib has demonstratedefficacy in neuroendocrine tumors and is worthy of evaluationin prostate cancers that manifest neuroendocrine features (38).Application of next-generation sequencing will provide futureclues in predicting clinical benefit with cabozantinib. With reviewof specific activity of cabozantinib in medullary thyroid cancer, itcan be hypothesized that the RET gene mutations in prostatecancers could possibly predict for response (39). Future investiga-tions of cabozantinib in mCRPC should focus on genomic mar-kers that are representative of MET upregulation and RETmutations.

In conclusion, cabozantinib represents a uniquemechanism ofaction that is distinct from currently approved therapies inmCRPC and continues to be worthy of deeper investigation. Itholds potential in the treatment of patients with refractorymCRPC. The imaging changes occurred indiscriminately andwereunable to indicate clinical benefit with the agent.

Disclosure of Potential Conflicts of InterestU. Vaishampayan reports receiving commercial research grants from and is a

consultant/advisory board member for Exelixis. E.I. Heath reports receivingspeakers bureau honoraria from Sanofi. A.F. Shields reports receiving commer-cial research grants from Exelixis. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: U.N. Vaishampayan, I. Podgorski, L.K. Heilbrun,E.I. Heath, A.F. ShieldsDevelopment of methodology: U.N. Vaishampayan, I. Podgorski, J. Boerner,K. Stark, E.I. Heath, A.F. ShieldsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): U.N. Vaishampayan, J.M. Lawhorn-Crews, J. Boerner,E.I. Heath, A.F. ShieldsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): U.N. Vaishampayan, L.K. Heilbrun, J.M. Lawhorn-Crews, D.W. Smith, E.I. Heath, A.F. ShieldsWriting, review, and/or revision of the manuscript: U.N. Vaishampayan,I. Podgorski, L.K. Heilbrun, J.M. Lawhorn-Crews, K.C. Dobson, E.I. Heath,J.A. Fontana, A.F. ShieldsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): U.N. Vaishampayan, J.M. Lawhorn-Crews,K.C. Dobson, J. BoernerStudy supervision: U.N. Vaishampayan, A.F. Shields

AcknowledgmentsThis study was partially supported by Department of Defense National

Oncogenomics and Molecular Imaging Center grant W81XWH-11-1-0050,Exelixis Inc., and the NIH Cancer Center Support grant CA-22453.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 18, 2018; revised July 31, 2018; accepted October 10, 2018;published first October 16, 2018.

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2019;25:652-662. Published OnlineFirst October 16, 2018.Clin Cancer Res Ulka N. Vaishampayan, Izabela Podgorski, Lance K. Heilbrun, et al. Castrate-Resistant Prostate CancerOutcomes of Oral Cabozantinib Therapy in Metastatic Biomarkers and Bone Imaging Dynamics Associated with Clinical

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