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Keywords:

  • HISTOMORPHOMETRY;
  • BISPHOSPHONATES;
  • ANDROGEN DEPRIVATION;
  • PROSTATE CANCER METASTASIS;
  • OSTEOCLASTS

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Qualitative and quantitative bone features were determined in nondecalcified and decalcified bone from 20 predetermined bone sites in each of 44 patients who died with castration-resistant prostate cancer (CRPC), some of which received bisphosphonate treatment (BP) in addition to androgen-deprivation therapy (ADT). Thirty-nine of the 44 patients (89%) had evidence of bone metastases. By histomorphometric analysis, these bone metastases were associated with a range of bone responses from osteoblastic to osteolytic with a wide spectrum of bone responses often seen within an individual patient. Overall, the average bone volume/tissue volume (BV/TV) was 25.7%, confirming the characteristic association of an osteoblastic response to prostate cancer bone metastasis when compared with the normal age-matched weighted mean BV/TV of 14.7%. The observed new bone formation was essentially woven bone, and this was a localized event. In comparing BV/TV at metastatic sites between patients who had received BP treatment and those who had not, there was a significant difference (28.6% versus 19.3%, respectively). At bone sites that were not invaded by tumor, the average BV/TV was 10.1%, indicating significant bone loss owing to ADT that was not improved (11%) in those patients who had received BPs. Surprisingly, there was no significant difference in the number of osteoclasts present at the metastatic sites between patients treated or not treated with BPs, but in bone sites where the patient had been treated with BPs, giant osteoclasts were observed. Overall, 873 paraffin-embedded specimens and 661 methylmethacrylate-embedded specimens were analyzed. Our results indicate that in CRPC patients, ADT induces serious bone loss even in patients treated with BP. Furthermore, in this cohort of patients, BP treatment increased BV and did not decrease the number of osteoclasts in prostate cancer bone metastases compared with bone metastases from patients who did not receive BP. © 2013 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Nearly all patients who die of advanced prostate cancer (PCa) have bone metastases.1, 2 Bone metastases are associated with bone pain, fracture, nerve and spinal cord compression, and myelophthisis.3–7 Androgen-deprivation therapy (ADT), mainstay treatment for metastatic disease in PCa patients, adversely induces osteoporosis and fragility fractures.8 Since 2004, bisphosphonates (BP) have been considered standard care for castration-resistant prostate cancer (CRPC) patients with bone metastases as they reduce the risk for skeletal related events (SREs). Zoledronic acid is the most potent of the chemical antiresorptives, and to date, no histomorphometric study evaluating the effects of an antiresorptive agent on human PCa bone metastases has been published.

To better characterize the bone changes induced by metastatic CRPC, ADT, and BP therapy on both tumor involved and uninvolved bone, we assessed the qualitative and quantitative bone parameters in 20 predetermined sites in 44 patients who died of metastatic CRPC. Each bone specimen from these 20 sites were divided into two halves; one was decalcified and paraffin embedded for qualitative assessment, and the other, nondecalcified, was directly embedded in methylmethacrylate (MMA) for quantitative bone histomorphometry. Our results confirm bone loss in uninvolved bone specimens from androgen-deprived patients and the existence of a spectrum of osteoblastic to mixed to osteolytic metastases, frequently in the same patient. Furthermore, BP increased bone volume (BV) in the osteoblastic metastases as expected but surprisingly did not decrease the number of osteoclasts in metastatic or uninvolved bone biopsy specimens. Giant osteoclasts were observed rarely in bone specimens of patients treated with alendronate and zoledronic acid. In addition, increased osteoid was observed in both high BV and low BV metastases, and osteoclast numbers were significantly higher in low BV BP-treated metastases versus high BV BP-treated metastases.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Tissue acquisition

Samples were obtained from patients who died of metastatic CRPC and who signed written informed consent for a rapid autopsy to be performed ideally within 2 hours of death, under the aegis of the Prostate Cancer Donor Program at the University of Washington.9 The study was approved by the institutional review board at the University of Washington. Bone biopsy specimens of all bone sites, except ribs (a dyke cutter was used for rib biopsies), were obtained using a drill with an attached trephine (11-mm-diameter coring drill bit). These specimens were obtained from 20 predetermined anatomical sites.2 Additional cores were obtained from either additional obvious sites of bone metastases or as indicated by the last bone scintigraphy survey.

Bisphosphonate therapy

Of the 44 patients in the study, 34 were treated with bisphosphonates. Based on notes from the medical charts of multiple patients, the usual BP dosing was as follows: For zoledronic acid (27 patients, 28 if you include one patient who received alendronate and zoledronic acid), the usual regimen was 4 mg iv monthly. This was varied based on patient factors, such as renal function. Some men were in a clinical trial and received zoledronic acid every 3 weeks. For pamidronate, (5 patients), the usual regimen was 80 mg iv monthly. One patient received alendronate 70 mg orally weekly.

Tissue processing

Bone samples for qualitative histology were fixed in formalin, decalcified in 10% formic acid, and embedded in paraffin. Bone samples for quantitative histomorphometric analysis were fixed in 70% ethanol and embedded in a methylmethacrylate (MMA) medium without decalcification.10

Histology and histomorphometry

Five-micron sections of decalcified paraffin-embedded bone samples were stained with hematoxylin and eosin and assessed by two pathologists for bone and tumor content (MR and LT). Five-micron sections of nondecalcified MMA-embedded bone samples were stained with Goldner's method for histomorphometric analysis.11 The reagent NBT/BCIP was used to evaluate alkaline phosphatase (ALP) activity. An aqueous coupling azo reactive dye mixture was used to evaluate osteoclast number (acid phosphatase [TRAcP] positivity). All histomorphometric measurements are expressed in accordance with the American Society for Bone and Mineral Research.12 Histomorphometry was performed on a 13.55-mm2 area at 20x magnification. Image analysis was conducted with an Olympus BX41 microscope using Bioquant Nova Prime software (Bioquant Image Analysis Corp., Nashville, TN, USA). In each biopsy specimen where the number of osteoclasts was >1, osteoclasts were counted and separated into <9 nuclei, 9 to 12 nuclei, 13 to 20 nuclei, or >20 nuclei groups. Woven bone area was measured using two polarizing filters on Goldner's and TRAcP-stained bone sections where BV/TV was equal or more than 45%. Mineralization was not studied in this cohort of patients.

Statistical analysis

Significance of differences was evaluated using a two-sample equal variance Student's t test with paired observations when appropriate and a two-tailed distribution. p values ≤ 0.05 indicated statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We processed bone samples from 44 rapid autopsies of patients who died with metastatic CRPC. Patients' demographics and treatments are presented in Supplemental Table S1. The mean age at autopsy was 72.8 years. All patients had received ADT with a median treatment duration of 65.9 months. Thirty-four patients (77%) received BP therapy. Zoledronic acid was the most common BP treatment; among those treated with BP, the median treatment duration was 17.2 months (range 1 month to 70 months). One patient received alendronate and zoledronic acid, and one patient received only alendronate for 42 months. Five patients received pamidronate for 6 to 22 months. In addition, 29 patients (66%) received secondary hormonal manipulation, and 31 patients (71%) received at least one form of chemotherapy.

From a potential of 894 decalcified paraffin-embedded samples, 873 were suitable for histological analysis. Five hundred eighty-seven had tumor involvement (67%), accounting for 38 of 44 patients (82%) having at least one bone metastasis. Qualitatively, of the decalcified paraffin-embedded metastatic samples, 30% were predominantly osteoblastic, 38% were osteoblastic with a small osteolytic component, 9% were osteolytic with a small osteoblastic component, 14% were predominantly osteolytic, and 9% had no bone response to tumor (Fig. 1A). Two patients were predominantly osteolytic in all bone metastatic sites.

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Figure 1. (A) Summary of qualitative analysis of bone response in ADT- and bisphosphonate-treated PCa decalcified paraffin-embedded bone metastatic specimens from 44 patients. Histologically, we determined the predominant phenotype of each bone metastasis sample. Five categories were used: predominantly osteoblastic (red), osteoblastic with a small component of osteolysis (pink), osteolytic with a small component of osteoblastic (light green), osteolytic (dark green), and no bone change (blue). The number in each colored square represents the number of bone samples having each of the bone phenotypes. White squares indicate no bone metastases were observed. Asterisks indicate those patients who were treated with BP. In the majority of patients, there exists a spectrum of bone phenotypes from osteoblastic to osteolytic or no bone change in response to tumor. Six patients had no bone metastases. Two patients (nos. 3 and 19) were predominantly osteolytic. There appears to be no major qualitative difference in bone response between BP- and NBP-treated patients. (B) Summary of the quantitative analysis in methylmethacrylate (MMA)-embedded bone specimens of bone response in 44 ADT- and BP-treated patients. Bone specimens were obtained at rapid autopsy from the right femoral neck (RF), left femoral neck (LF), right iliac (RI), left iliac (LI), lumbar vertebrae 1 to 5 (L1 to L5), thoracic vertebrae 8 to 12 (T8 to T12), sternum (St), rib, right humeral head (RH), and left humeral head (LH) in each patient. White squares identify samples that could not be measured because of technical reasons. In gray, the bone sample did not contain tumor. Blue squares represent metastatic specimens with BV less than the age-matched control. The majority of sites are red, representing metastatic specimens with BV greater than the age-matched control. Asterisks indicate patients treated with BP. Crosses indicate samples that were positive for tumor in the paraffin-embedded cores but negative or had no sample available for analysis in MMA. Eight patients had no bone metastases. An additional six bone sites with no tumor and eight with tumor were also analyzed.

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For nondecalcified MMA-embedded samples, of 894 potential samples, 233 (26%) of the bone samples could not be evaluated because of technical reasons. The remaining 661 MMA-embedded samples from 44 patients were analyzed. The main histomorphometric parameters that we measured were: BV, new unmineralized bone formation (ie, osteoid volume), osteoclast numbers, and ALP. Four hundred forty-three of the 661 MMA-embedded samples had tumor involvement (67%), accounting for 37 of 44 patients (84%) with at least one bone metastasis (Fig. 1B, showing bone volume in all specimens, metastatic or not). This is similar to but not identical to the 64% tumor involvement noted in the paraffin-embedded samples because of different portions of each bone core being used for these two processing methods.

We calculated a weighted mean for BV based on the most comprehensive published analysis of BV in the aging male.13 This weighted mean was based on the age profile of the patients. The weighted mean average BV for our cohort based on age was 14.7%. Of the 661 available MMA-embedded samples, 3 were excluded as statistical outliers, 215 (33%) had no tumor with a mean BV of 10.5% (range 2.33% to 25.42%). Of those, 176 (82%) had <14.7% BV and 39 (18%) had >14.7% BV, indicating that ADT induces bone loss. In addition, of the 661 available samples, 443 (67%) had tumor as described above, with 147 (33%) containing tumor with <14.7% BV and 296 (67%) containing tumor with >14.7% BV, indicating a predominantly osteoblastic response in the PCa bone metastases (Supplemental Table S2).

The 443 tumor-containing samples were compared with the 215 samples without tumor for various histomorphometric characteristics (Table 1). The mean tumor volume as a percent of tissue volume in the metastatic sites was 24.7%. Overall, there was a considerable amount of bone present in the metastatic sites (range 2.8% to 76.6%; mean 25.7%; %BV/TV), compared with uninvolved bone (range 2.3% to 25.4%; mean 10.5%; %BV/TV) (Table 1). The significant increases in bone volume (p < 0.001), bone surface (p < 0.001), trabecular thickness (p < 0.001), and trabecular number (p < 0.001) in metastatic bone sites were the result of the addition of woven bone and osteoid onto preexisting trabeculae and in between a nest of cancer cells within the bone marrow microenvironment. Osteoid but no woven bone was observed in purely osteolytic lesions. As expected, BV (p < 0.001), osteoid area (p < 0.001), and ALP (p < 0.001) activity were also higher in the metastatic sites versus uninvolved sites, with 10-fold more osteoid area and more than double the percentage of metastatic sites being positive for ALP compared with the uninvolved bone sites. The osteoid volume compared with total BV was fivefold higher in the metastatic sites (Table 1). No difference was found in osteoid volume between BP-treated and untreated bone specimens. As expected, osteoclast numbers were significantly higher (p = 0.005) in metastases than in uninvolved bone (Table 1). However, the osteoclast number per bone surface unit was no different (p = 0.660) in metastases and uninvolved bone, revealing that both involved and uninvolved bone had similar osteoclast coverage on the bone surface (Table 1).

Table 1. Histomorphometric Analysis of Metastatic and Nonmetastatic Bone Specimens From 44 Patients From the CRPC Rapid Autopsy Series
Total sitesTumorNo tumorp Value
  1. Bone sites with tumor (n = 443) and no tumor (n = 215) were assessed for quantitative tumor volume and bone response. The percentage of bone volume in tissue volume (%BV/TV), bone surface (BS; mm), percentage of bone surface in tissue volume (%BS/TV), trabecular thickness (Tb.Th; µm), trabecular number (Tb.N; 1/nm), trabecular separation (Tb.Sp; µm), percentage of tumor volume in tissue volume (%TuV/TV), osteoid volume (OV; mm2), total bone volume (Tt.BV; mm2), ratio of osteoid volume to total bone volume (OV/Tt.BV; %), alkaline phosphatase activity (ALP), osteoclast number (N.Oc), and ratio of osteoclast number to bone surface (N.Oc/BS) were measured in each sample. BV/TV demonstrates that metastatic bone biopsy specimens have an overall osteoblastic phenotype (25.7% versus 14.7%, which is considered as normal BV for age-matched patients). Nonmetastatic bone biopsy specimens have an osteoporotic phenotype with a BV/TV of 10.5%. Greater osteoid but similar osteoclast numbers on the bone surface in involved bone as in uninvolved bone demonstrates higher bone accumulation is associated with metastasis when compared with uninvolved bone.

No. of samples443215 
BV/TV (%)25.710.5<0.001
BS (mm)64.231.0<0.001
BS/BV (mm2/mm3)23.424.90.700
Tb.Th (µm)112.688.3<0.001
Tb.N (nm−1)2.41.2<0.001
Tb.Sp (µm)440.6869.7<0.001
TuV/TV (%)24.70.0 
OV (mm2)0.100.01<0.001
Tt. BV (mm2)3.51.4<0.001
OV/Tt. BV (%)3.60.7<0.001
ALP (% positive)65.530.7<0.001
N.Oc (cm−2)7.94.00.005
N.Oc/BS (mm−1)0.20.20.660

Our patients had an extremely wide range of BP treatment times (0.8 to 70 months). An analysis was completed for all BP versus nonbisphosphonate-treated (NBP) samples. Metastatic sites in patients receiving BP had a similar tumor volume as equivalent metastatic sites in patients not receiving BP treatment (Table 2). BV as a percentage of tissue volume (%BV/TV) was significantly greater in BP-treated patient metastatic sites (p < 0.001) when compared with metastatic sites of NBP patients (Supplemental Fig. S1A—D). There was a significant increase in mean osteoid volume in BP-treated metastatic lesions (p = 0.005), with fewer ALP-positive sites present in patients receiving BP. There was no significant difference in the number of TRAcP-positive osteoclasts in NBP and BP metastatic specimens, but there was a significant difference in osteoclast coverage of bone surface (p = 0.048) in metastatic sites from patients receiving BP treatment versus metastatic sites from NBP patients (Table 2). In uninvolved samples, BP treatment had no effect on BV (p = 0.209), with no significant effect on osteoid volume, ALP-positive sites, and osteoclast numbers (Table 2). In addition, we also elected to confine this analysis to those patients who had been treated for a minimum of 12 months. The results were similar (Supplemental Table S3).

Table 2. Histomorphometric Analysis of Bone Specimens From Bisphosphonate- and Nonbisphosphonate-Treated Patients
 TumorNo tumor
BPNBPp-valueBPNBPp-value
  1. Metastatic and nonmetastatic specimens from bisphosphonate (BP)- and nonbisphosphonate (NBP)-treated patients were assessed by histomorphometry. The histomorphometric parameters are described in Table 1. Bone volume (BV) is greater in metastatic specimens treated with BP than in metastatic NBP specimens with no change in the number of osteoclasts. In uninvolved specimens, there is a trend to greater BV in BP-treated specimens than in NBP specimens with no change in the number of osteoclasts.

No. of samples345100 15957 
BV/TV (%)27.419.3<0.00111.310.10.209
BS (mm)65.060.70.27131.531.60.945
BS/BV (mm2/mm3)22.825.70.02023.927.10.005
Tb.Th (µm)118.591.90.00491.982.90.063
Tb.N (nm−1)2.52.30.2121.21.20.986
Tb.Sp (µm)423.8516.20.025869.1823.60.431
TuV/TV (%)24.824.20.7950.00.0 
OV (mm2)0.110.050.0050.010.010.507
Tt. BV (mm2)3.72.6<0.0011.51.30.205
OV/Tt. BV (%)3.92.30.0170.70.80.636
ALP (% positive)61.479.00.00128.935.10.404
N.Oc (cm−2)8.46.30.3423.35.80.088
N.Oc/BS (mm−1)0.180.090.0480.10.20.117

To determine if the site of skeletal metastasis influenced the relationship between the tumor and the corresponding bone response, bone histomorphometric parameters in the long bones, vertebrae, sacrum, and other sites, ie, sternum, ribs, and clavicle, were compared (Supplemental Table S4). Fifty-four percent of the long bones had tumor compared with 73%, 74%, and 69% in vertebrae, sacrum, and other sites, respectively. Larger numbers of osteoclasts were present in the sternum, ribs, and clavicle specimens versus the other bone specimens, reaching 67 per specimen in a 7-month zoledronic acid–treated patient (Supplemental Table S4). The high osteoclast numbers may be associated with increased bone remodeling in these more flexible bone sites (Supplemental Table S4).

Histomorphometric features of metastatic sites from patients treated with BP with >14.7% or <14.7% BV were compared to determine if features other than BV could explain differences in bone formation (Supplemental Table S5). Besides a decrease in ALP activity in the <14.7% BV samples, no obvious factors were observed. A similar analysis mentioned above was done for patients who received BP or NBP with <14.7% or >14.7% BV, and again, no significant differences were observed (Supplemental Table S2). Based on these data, the challenge to identify histomorphometric parameters that are significantly different between the >14.7% and <14.7% BV samples reaches an unanticipated level of complexity.

Therefore, to better define the role of the osteoblasts and osteoclasts in the bone remodeling process, extreme ends of the spectrum were examined, ie, highly osteoblastic (>35.3% BV) and highly osteolytic (<9.1% BV) metastases in BP-treated patients (Table 3). We selected these cutoffs of 35.3% and 9.1% because they were the mean BV measurements in metastatic sites from patients who received BP with >14.7% and <14.7% BV (Supplemental Table S5). The mean %BV/TV was 49.9% in extremely osteoblastic samples and 6.5% in extremely osteolytic sites (Table 3). Remarkably, even with this significant difference in %BV/TV, osteoid volume and ALP positivity were similar between these two groups. However, the osteoid bone coverage was nearly fourfold higher in the osteolytic metastatic sites, indicating an important bone-forming process in BP-treated bone.

Table 3. Histomorphometric Analysis of Highly Osteoblastic and Highly Osteolytic Metastatic Bone Specimens From Bisphosphonate-Treated Patients
 Bone volume
>35.3%<9.1%p Value
  1. This subgroup of 16 patients received BP treatment for a minimum of 1 year. Bone volume >35.3% versus <9.1% assesses the significant differences between highly osteoblastic and highly osteolytic castrate-resistant BP-treated metastatic bone sites. Histomorphometric parameters are described in Table 1. Two extremes of the bone phenotype spectrum in prostate cancer metastasis are shown with no difference in osteoid volume between the two extremes; however, significantly more osteoclasts relative to bone surface were present in osteolytic lesions.

Tumor samples7132 
BV/TV (%)49.96.5<0.001
BS (mm)78.930.8<0.001
BS/BV (mm2/mm3)13.235.3<0.001
Tb.Th (µm)188.662.5<0.001
Tb.N (nm−1)3.01.1<0.001
Tb.Sp (µm)186.4963.3<0.001
TuV/TV (%)17.935.2<0.001
OV (mm2)0.070.050.430
Tt. BV (mm2)6.60.9<0.001
OV/Tt. BV (%)1.14.3<0.001
ALP (% positive)50.750.00.948
N.Oc (cm−2)3.59.30.057
N.Oc/BS (mm−1)0.060.330.003

A marked increase in osteoclast number (p = 0.057) and osteoclast coverage of the bone surface (p = 0.003) was observed in the extremely osteolytic sites together with a high osteoid bone coverage, indicating a higher bone turnover in osteolytic lesions compared with osteoblastic lesions (Table 3; Supplemental Fig. S1E, F; Supplemental Fig. S2). Similar results were observed when we assessed patient-paired osteoblastic and osteolytic bone biopsy specimens from a much smaller subset of 5 BP-treated patients with metastatic bone sites that had both extremely osteoblastic (>35.3% BV) and extremely osteolytic (<9.1% BV) metastases (Supplemental Table S6).

To determine BP effects on osteoclasts, we compared the number of osteoclasts found in metastatic biopsy samples from patients untreated and treated with pamidronate, alendronate, and zoledronic acid for <1 year, <2 years, <3 years, and >3 years. There was no significant difference between the number of osteoclasts in biopsy samples from each of the groups (Fig. 2A). Pamidronate-treated metastases had the lowest number of osteoclasts. Alendronate had the highest number of osteoclasts, but could not be statistically compared with the other groups because only one patient was treated with alendronate. It is noteworthy that all metastases of one patient in each BP and NBP group had no osteoclasts, suggesting that individual PCa's may induce no osteoclasts at all.

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Figure 2. Osteoclasts were still observed in bone metastases of patients treated with zoledronic acid. (A) Metastatic osteoclast numbers were not significantly different in the NBP no treatment (No Tt) biopsy group compared with any of the zoledronic acid (Zol) treatment duration groups. In the pamidronate (Pam) group, a low number of osteoclasts were observed. In an alendronate patient (Aln), osteoclast number was high. (B) When the osteoclast data were sorted by the number of their nuclei, giant osteoclasts were observed in the bone metastases of the alendronate (Aln)-treated patient and in bone metastases of patients with <1 year through >3 years of zoledronic acid treatment. Only 9 to 12 nuclei (or less) containing osteoclasts were found in bone metastases from NBP patients and in pamidronate-treated patients.

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To define giant osteoclasts, we sorted the osteoclasts by the number of nuclei visible on the metastatic sections. When we compared the BP groups defined above, we found that 8-or-less-nuclei and 9-to-12-nuclei osteoclasts were observed in all groups representing the bulk of the osteoclasts present in the metastases (Fig. 2B). Giant osteoclasts, defined as either containing 13 to 20 nuclei and more than 20 nuclei, were observed in the patient treated with alendronate, as well as in all zoledronic acid–treated patient groups (Fig. 3C, E–G), they were absent in the pamidronate-treated and in NBP-treated groups. Concerning osteoclast apoptosis, we found only 3 osteoclasts (out of 1971 reviewed) with pyknotic nuclei in a 4-year zoledronic acid–treated metastasis (Fig. 3H). However, we noticed that many osteoclasts from the zoledronic acid group had atypical nuclei with angulated nuclear membrane, clear open chromatin, and one large irregular nucleolus in each nucleus (Fig. 3E).

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Figure 3. Morphologic characteristics of bone and osteoclasts of ADT- and BP-treated bone metastases. (A) The osteolytic aspect of a bone metastasis in an 86-year-old patient androgen ablated for 20 years and receiving zoledronic acid for more than 3 years before death. (B) The osteoblastic aspect of a bone metastasis in the same patient. In both biopsies, tumor fills the spaces between bone trabeculae. Goldner's stain x2.5 magnification. (C) A 20-nuclei osteoclast untreated with BP. (D) Goldner's staining of a giant osteoclast with more than 20 nonapoptotic nuclei in the right iliac bone of a patient treated with alendronate for 42 months. (E) Goldner's staining of a nest of osteoclasts with a variable number of nonapoptotic nuclei in a biopsy specimen of the right iliac bone in a patient treated with zoledronic acid for 10 months. Note the osteoclast with a single large nucleus with a large nucleolus (arrow). (F) Twelve nuclei (or fewer) osteoclasts in a sacrum biopsy specimen of a patient treated with pamidronate for 6 months. (G) Sixteen-nuclei giant osteoclast in a right iliac bone of a patient treated with zoledronic acid for 4 years. (H) Six pyknotic nuclei in an osteoclast in a left iliac bone of the same patient as in (G). Goldner's stain, x40 magnification. (I) Osteoblastic bone metastasis of a patient treated with zoledronic acid for 6 months. TRAcP staining x5 magnification. (J) Same as in (I), observed under polarization showing small areas of lamellar bone and mainly woven bone, representing 89% of the total bone. TRAcP staining x5 magnification with polarization.

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To further analyze bone formation in the metastatic specimens from CRPC patients, we measured the woven bone component in 78 biopsies that had more than 45% BV/TV. This was chosen as a cut-off because 45% BV/TV and above is without a discussion an increase of bone mass. Woven bone was found occupying from 15.6% to 98.5% of total bone in osteoblastic biopsies with an average of 88.1%. No difference was observed in woven bone between metastatic specimens from BP- and NBP-treated patients. Woven bone was formed de novo from the tumor stroma in the bone marrow or deposited directly on lamellar trabeculae (Fig. 3I, J).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

At least four processes are known to influence bone structure in PCa bone metastasis: (1) normal bone physiology, (2) systemically and locally secreted tumor factors, (3) androgen deprivation therapy, and (4) BP treatment.

The physiological and perpetual systemic bone remodeling involves balanced and coupled actions of osteoblasts and osteoclasts. All 44 patients in our study were on ADT, and the results show that ADT induces serious bone loss similar to postmenopausal osteoporosis in women by systemic activation of osteoclasts. As expected, we observed a huge increase in the number of osteoclasts per uninvolved biopsy specimen.13 BPs, because of their high affinity for skeletal mineralized bone matrix, induce a detrimental effect on osteoclast biology and a systemic and local loss of osteoclast activity. Thus BP treatment has been shown to prevent bone loss in men receiving ADT.14 However, in our study, in uninvolved bone biopsy specimens, BPs were not able to significantly decrease osteoclast number. Consequently, %BV/TV averaged 11.0% in those patients with BP treatment, which was not significantly different from %BV/TV (10.1%) measured in bone specimens from NBP-treated patients. These low numbers contribute to the fragility fractures observed in ADT patients (Table 2).

Regarding the elevated osteoclast numbers in BP-treated patients (Table 2 and Fig. 2), we found that a small percentage of those osteoclasts were giant osteoclasts, defined here as hypernucleated osteoclasts with greater than 13 nuclei. Previously, it was reported that long-term alendronate treatment is associated with an increase in the number of osteoclasts, which include distinctive giant hypernucleated detached osteoclasts that are undergoing protracted apoptosis.15 Similarly, we found in a single alendronate-treated patient that the number of osteoclasts per biopsy specimen was the highest of all BP-treated bone metastases, with 157/mm2 osteoclasts in a metastatic sample. We did not morphologically observe pyknotic nuclei in those alendronate-treated osteoclasts but did not use immunohistochemistry to test DNA fragmentation in our plastic embedded sections. In addition, we did not often observe giant osteoclasts with pyknotic nuclei in bone biopsies from patients treated long term with zoledronic acid. One explanation for the presence of the giant osteoclasts in BP-treated specimens is that they may have lost their basic functions by perturbation of the mevalonate pathway but have not as yet disappeared from the bone surface.15, 16 The study of apoptosis markers in osteoclasts in paraffin sections of metastasis specimens of this series is warranted.

Local tumor-secreted factors, still to be identified, induce the characteristic osteoblastic phenotype of PCa metastasis. Our study, confirming a pilot study previously reported from 12 patients,2 show that the osteoblastic phenotype can manifest as a spectrum of osteoblastic to osteoblastic/osteolytic (mixed) and osteolytic bone responses observed in the same patient at different biopsy sites. Not surprisingly, this study clearly demonstrates that PCa in the bone is predominantly osteoblastic, with an osteolytic component (Fig. 1). However, this spectrum of lesions has not been previously reported in such detail. Bone scans preferentially expose osteoblastic lesions, thereby underestimating osteolytic lesions. The existence of predominantly osteolytic lesions in a bone site together with osteoblastic lesions in another bone site can be explained either by (1) a maturation of lesions, ie, osteolytic first and then osteoblastic, or osteoblastic and then osteolytic, or (2) the presence of multiple tumor clones secreting different bone acting factors. This study lacks the knowledge from sampling different bone metastases in each patient at different times as the sampling was made for all patients at death.

In addition to the osteoblastic response of the bone to PCa cells, a high bone turnover is observed in tumor-involved bone biopsy specimens as demonstrated by the presence of a high volume of osteoid and a high number of osteoclasts, both in BP-treated and NBP-treated patients. Osteoid, defined as collagen 1 before mineralization, is fourfold higher in metastases than in uninvolved bone, indicating the ongoing osteoblastic process in response to the local presence of prostate tumor cells. This suggests that bone response is a localized event. If physiologic bone coupling was maintained, then we would expect that an increase in new bone formation would be associated with an increase in osteoclast number. However, in low BV metastases, osteoid bone coverage and osteoclast number are much higher than in high BV metastases, suggesting that normal physiologic coupling is perturbed by the tumor (Table 3; Supplemental Fig. S2).

Results of this study clearly demonstrate the complex biology taking place in CRPC bone metastases, including the dramatic loss of bone induced by ADT, the dramatic addition of woven bone, the limited efficacy of BPs to change the bone responses, and the need to adjust BP treatment to individual tumor-related resorption. Finally, this study highlights the persistent need to identify the factor(s) that promote osteoblast activity so that better targeted therapies can be delivered clinically to improve the quality of life of patients suffering from CRPC skeletal metastases.17, 18–20

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

MPR was employed by Amgen Inc. within the past 36 months before submission of this manuscript. All other authors state that they have no conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank the patients and their families who were willing to participate in the Prostate Cancer Donor Program, for without them, research of this nature would not be possible. We also acknowledge Bruce Montgomery, MD; Peter Nelson, MD; Susan Ott, MD; the rapid autopsy teams; and students in the Urology Department at the University of Washington. This material is the result of work supported by resources from the VA Puget Sound Health Care System, Seattle, WA (RLV is a Research Career Scientist; PHL is a staff physician), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the NIH grant PO1CA085859, and the Richard M LUCAS Foundation. CM is a recipient of the Career Development Award from the Pacific Northwest Prostate Cancer SPORE (P50CA097186).

Authors' roles: CM accepts responsibility for the integrity of the data. MPR, LDT, PHL, CSH, and RLV were responsible for the conception of the experiments. CM, AD, and MPR were responsible for conducting the experiments. CM, AD, MPR, MK, CW, EC, and RLV were responsible for analyzing data. CM, MPR, AD, LDT, MK, CW, EC, PHL, CSH, and RLV all participated in the drafting of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
  • 1
    Bubendorf L, Schopfer A, Wagner U, Sauter G, Moch H, Willi N, Gasser TC, Mihatsch MJ. Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol. 2000;31:57883.
  • 2
    Roudier MP, Morrissey C, True LD, Higano CS, Vessella RL, Ott SM. Histopathological assessment of prostate cancer bone osteoblastic metastases. J Urol. 2008;180:115460.
  • 3
    Lange PH, Vessella RL. Mechanisms, hypotheses and questions regarding prostate cancer micrometastases to bone. Cancer Metastasis Rev. 1998;17:3316.
  • 4
    Olson KB, Pienta KJ. Pain management in patients with advanced prostate cancer. Oncology (Williston Park). 1999;3:153749.
  • 5
    Shahinian VB, Kuo YF, Freeman JL, Goodwin JS. Risk of fracture after androgen deprivation for prostate cancer. N Engl J Med. 2005;352:15464.
  • 6
    Alibhai SM, Duong-Hua M, Cheung AM, Sutradhar R, Warde P, Fleshner NE, Paszat L. Fracture types and risk factors in men with prostate cancer on androgen deprivation therapy: a matched cohort study of 19,079 men. J Urol. 2010;184:91823.
  • 7
    Coleman RE. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin Cancer Res. 2006;12:6243s9s.
  • 8
    Higano CS. Androgen-deprivation-therapy-induced fractures in men with nonmetastatic prostate cancer: what do we really know?. Nat Clin Pract Urol. 2008;5:24434.
  • 9
    Roudier MP, True LD, Higano CS, Vesselle H, Ellis W, Lange P, Vessella RL. Phenotypic heterogeneity of end-stage prostate carcinoma metastatic to bone. Hum Pathol. 2003;34:64653.
  • 10
    Lebeau A, Muthmann H, Sendelhofert A, Diebold J, Lohrs U. Histochemistry and immunohistochemistry on bone marrow biopsies. A rapid procedure for methyl methacrylate embedding. Pathol Res Pract. 1995;191:1219.
  • 11
    Recker RR. Bone histomorphometry: techniques and interpretation. Boca Raton, FL: CRC Press; p. 239. 1983.
  • 12
    Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987;2:595610.
  • 13
    Bartl R, Frisch B. Biopsy of bone in internal medicine: an atlas and sourcebook (current histopathology) Vol. 21. Boston, MA: Kluwer Academic Publishers; 1993.
  • 14
    Serpa NA, Tobias-Machado M, Esteves MA, Senra MD, Wroclawski ML, Fonseca FL, Dos Reis RB, Pompeo AC, Giglio AD. Bisphosphonate therapy in patients under androgen deprivation therapy for prostate cancer: a systematic review and meta-analysis. Prostate Cancer Prostatic Dis. 2012;15:3644.
  • 15
    Weinstein RS, Roberson PK, Manolagas SC. Giant osteoclast formation and long-term oral bisphosphonate therapy. N Engl J Med. 2009;360:5362.
  • 16
    Rogers MJ, Gordon S, Benford HL, Coxon FP, Luckman SP, Monkkonen J, Frith JC. Cellular and molecular mechanisms of action of bisphosphonates. Cancer. 2000;88:296178.
  • 17
    Hatoum HT, Lin SJ, Guo A, Lipton A, Smith MR. Zoledronic acid therapy impacts risk and frequency of skeletal complications and follow-up duration in prostate cancer patients with bone metastasis. Curr Med Res Opin. 2011;27:5562.
  • 18
    Glantschnig H, Hampton RA, Lu P, Zhao JZ, Vitelli S, Huang L, Haytko P, Cusick T, Ireland C, Jarantow SW, Ernst R, Wei N, Nantermet P, Scott KR, Fisher JE, Talamo F, Orsatti L, Reszka AA, Sandhu P, Kimmel D, Flores O, Strohl W, An Z, Wang F. Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo. J Biol Chem. 2010;285:4013547.
  • 19
    Miller RE, Roudier M, Jones J, Armstrong A, Canon J, Dougall WC. RANK ligand inhibition plus docetaxel improves survival and reduces tumor burden in a murine model of prostate cancer bone metastasis. Mol Cancer Ther. 2008;7:21609.
  • 20
    Tian X, Jee WS, Li X, Paszty C. Ke HZ. Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model. Bone. 2011;48:197201.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr_1749_sm_SupplFig1.tif11980KSupplementary Figure 1
jbmr_1749_sm_SupplFig2.tif5363KSupplementary Figure 2
jbmr_1749_sm_SupplTab1.doc26KSupplementary Table 1
jbmr_1749_sm_SupplTab2.doc26KSupplementary Table 2
jbmr_1749_sm_SupplTab3.doc34KSupplementary Table 3
jbmr_1749_sm_SupplTab4.doc56KSupplementary Table 4
jbmr_1749_sm_SupplTab5.doc25KSupplementary Table 5
jbmr_1749_sm_SupplTab6.doc27KSupplementary Table 6
jbmr_1749_sm_SupplLegends.doc24KSupplementary Legends

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