|Prostate cancer; Bone metastasis; Denosumab;
|PSA: Prostate Specific Antigen; CRPC: Castration-Resistant Prostate
Cancer; NTx: N-Terminal Telopeptide; BCE: Bone Collagen
Equivalents; Cr: Creatinine; ALP: Alkaline Phosphatase; P: Phosphate;
EOD: Extent of Disease; CKD: Chronic Kidney Failure; OR: Odds
Ratio; CI: Confidence Interval; SREs: Skeletal-Related Events; CCR:
|Prostate cancer is diagnosed in more than 670,000 men yearly
worldwide [1,2], and compared to other malignancies, urological
malignancies are the most common cause of bone metastases (70–
80%) [3,4]. Patients with bone metastases may experience local
irreversible skeletal complications, including pathologic fractures and
spinal cord compression, and may require bone irradiation or surgery.
Such complications, referred to as skeletal-related events (SREs), are indicators of poor prognosis and cause substantial pain and morbidity
that frequently lead to hospitalization, poor quality of life, and
increased utilization of medical resources [5-9]. In recent decades,
patients with metastatic castration-resistant prostate cancer have been
living longer, owing to the advent of newer, targeted therapies [10,11].
Therefore, they are more likely to experience SREs and the role of
bone-targeted therapies to prevent and treat SREs is becoming more
|The functional mechanism of Denosumab differs from that of
bisphosphonates, which have been the first-line choice for prevention
of SREs for more than a decade. Denosumab, a fully human
monoclonal antibody with high affinity and specificity for the human
receptor activator of nuclear factor-κB ligand, was shown to inhibit
bone re-absorption in early studies in patients with advanced cancer,
including those who did not respond to prior bisphosphonate
treatment [12-15], and these results suggest a potential new approach
for the treatment of bone metastasis. However, according to drug
safety information released by the European Medicines Agency ,
hypocalcemia can occur at any time during Denosumab therapy but
occurs most commonly within the first 6 months of therapy. Patients with severe renal impairment (creatinine clearance of <30 ml/min) or
who are on dialysis are at greater risk of developing hypocalcemia. The
United States Food and Drug Administration released prescribing
information indicating that Denosumab can cause severe symptomatic
hypocalcemia, and fatal cases have been reported . In addition, the
Japanese Ministry of Health, Labour and Welfare called attention to
serious hypocalcemia resulting from Denosumab administration .
However, information about the incidence of and risk factors for
hypocalcemia associated with Denosumab treatment is limited. In this
study, we evaluated the risk factors for the development of
hypocalcemia after treatment with Denosumab for the prevention of
skeletal complications in prostate cancer patients with bone
|Patients and Methods
|We analyzed data for 69 consecutive patients who had radiologically
documented bone metastases from prostate cancer and who were
treated with initial subcutaneously injected Denosumab (120 mg) on
day 1 at Kitasato University Hospital or Sagamidai Hospital from May
2012 to May 2013.
|Patients were excluded if they were receiving calcitonin
hydrochloride, thalidomide, a calcium salt, vitamin D, or vitamin K or
if they had a history of parathyroid disorder or hypercalcemia
(adjusted serum calcium concentration of >10.2 mg/dl) before
administration of Denosumab. A total of 48 patients were deemed
eligible for the study (Figure 1).
|The study protocol and informed consent documents were reviewed
and approved by an Institutional Review Board of Kitasato University
Hospital and Sagamidai Hospital. The study was conducted in
accordance with the ethical principles outlined in the Declaration of
Helsinki, in accordance with ICH Good Clinical Practice guidelines
and with applicable regulatory requirements, and in compliance with
|This study was a single-arm, open-label, prospective multicenter
study of patients with prostate cancer with bone metastases. Fortyeight
eligible patients received Denosumab injected subcutaneously at
a dose of 120 mg on day 1. Androgen-deprivation therapy was used in
all cases. During the observation, vitamin D and calcium supplements
were introduced as soon as any grade of hypocalcemia was found and
the drug had been withdrawn until serum calcium level returned to
normal range (Figure 1).
|Study endpoints and evaluations
|The primary endpoint was the occurrence of hypocalcemia 1 week
and 1 month after the administration of Denosumab. Then risk factors
for Denosumab-induced hypocalcemia in patients with bone
metastases from prostate cancer were evaluated.
|The following patient characteristics and laboratory data were
collected at baseline: age at the start of Denosumab treatment; Gleason
score; androgen sensitivity status (castration-resistant prostate cancer
or not); history of treatment or prevention of SREs (zoledronic acid
hydrate or none); serum concentrations of calcium, albumin alkaline
phosphatase (ALP), phosphate and prostatic specific antigen (PSA);
urine N-terminal telopeptide (uNTX) level; extent of disease (EOD;
i.e., extent of bone metastasis on initial bone scan); and chronic kidney
disease (CKD) stage. The EOD was classified by the method of Soloway
et al. : EOD 0, normal; EOD 1, bone metastases, <6 lesions; EOD 2,
bone metastases, 6–20 lesions; EOD 3, bone metastases, >20 but less
than superscan; and EOD 4, superscan (75% of ribs, vertebrae, and
pelvic bones had lesions). CKD was classified according to the
guidelines introduced by the National Kidney Foundation Kidney
Disease Outcomes Quality Initiative in 200220). Then serum concentrations of calcium, albumin, ALP and phosphate were
monitored 1 week and 1 month after the admistration of Denosumab.
Urine NTx and serum PSA were examined 1 month after that.
|If the serum albumin level was less than 4.0 mg/dl, an adjusted
serum calcium level was calculated according to the following
equation: adjusted serum calcium level=serum calcium level (mg/dl)
+4.0-serum albumin level (mg/dl). Serum calcium level was assessed
according to the National Cancer Institute Common Toxicity Criteria
(ver. 4.0); the two institutes set the lower limit of normal for serum
calcium as 8.8 mg/dl and the upper limit as 10.2 mg/dl.
|Data are reported as number of patients and percentage or as
median or mean. At 1 week after Denosumab administration and
again at 1 month after Denosumab administration, patients were
divided into 2 groups on the basis of whether or not hypocalcemia had
developed. The t test was used for 2-group comparisons with respect to
age; serum PSA, calcium, ALP, and phosphate levels; and u-NTx level.
Pearson’s chi-square test was adopted for comparisons with respect to
the number of patients with castration-resistant prostate cancer, with
an EOD of ≥ 3, or with a history of receiving zoledronic acid hydrate.
The Mann-Whitney U test and the Fisher test were used for comparisons with respect to Gleason score and number of patients
with CKD ≥ 3, respectively. In addition, the associations between
hypocalcemia caused by Denosumab and various clinical factors were
evaluated by means of multivariate logistic regression analysis. Odds
ratios and 95% confidence intervals were determined for each factor. A
P value of <0.05 was considered statistically significant in all the
analyses. Statistical analyses were performed using SPSS statistical
software (version 13.0; SPSS Japan Inc., Tokyo, Japan).
|Characteristics at baseline of 48 consecutive patients treated with
Denosumab were evaluated (Table 1). The mean age was 72.3 years
(range 49-89 years). 25% of the patients had CKD ≥ 3, and 37.5% had
an EOD of ≥ 3.
|The numbers of patients with hypocalcemia at 1 week and 1 month
after Denosumab administration are shown in Table 2. Nineteen
patients (39.6%) demonstrated hypocalcemia at 1 week and 16 patients (33.3%) at 1 month. Symptomatic hypocalcemia was not found in any
of the patients.
|The patients with hypocalcemia at 1 week after Denosumab
administration had higher baseline serum ALP than the patients who
did not have hypocalcemia at 1 week (658 ± 1694.1 [mean ± SD] vs.
339 ± 0.4). Univariate logistic regression analysis of data obtained at 1
week after the administration of Denosumab demonstrated that a
baseline u-NTx level of ≥ 100 nmol bone collagen equivalents /mmol
creatinine (nmol BCE/mmol Cr) and a baseline serum ALP level of ≥
500 IU/L were significant risk factors for hypocalcemia (odds ratio
[OR]=7.875, 95% confidence interval [CI]=1.421-43.640 and
OR=10.710, 95% CI=2.620-43.810, respectively). Multivariate logistic
regression analysis showed that a baseline serum ALP level of ≥ 500
IU/L was an independent risk factor for hypocalcemia at 1 week after
the administration of Denosumab (OR=7.596, 95% CI=1.542-37.410).
|At 1 month after Denosumab administration, compared to patients
who did not have hypocalcemia, patients who did have hypocalcemia
had higher baseline serum ALP and baseline u-NTx (1455.5 ± 1694.1 [mean ± SD] vs.190.9 ± 63.9, respectively). In addition, the percentage
of patients with EOD of ≥ 3 at baseline was higher in the hypocalcemic
group than in the non-hypocalcemic group (10 patients [62.5% ] vs 8
patients [25.0%]). Univariate logistic regression analysis of the data at 1
month after Denosumab administration demonstrated that a u-NTx
level of ≥ 100 nmol BCE/mmol Cr, a serum ALP level of ≥ 500 IU/L,
and an EOD of ≥ 3 were significant risk factors for hypocalcemia
(OR=31.000, 95% CI=3.368–285.300; OR=7.222, 95%
CI=1.879-27.750; and OR=5.000, 95% CI=1.376-18.170, respectively).
Upon multivariate analysis, only a u-NTx level of ≥ 100 nmol BCE/
mmol Cr was an independent risk factor for hypocalcemia
(OR=12.410, 95% CI=1.059-145.600).
|In patients with grade 2 or 3 hypocalcemia at either 1 week or 1
month after Denosumab administration (8 patients, 16.7%), serum
calcium levels returned to the normal range within 1 to 4 months after
Denosumab administration (Figure 2). The median time from first
observation of hypocalcemia to return to the normal range was 42 days
(range 28-105 days).
|Hypocalcemia is a common adverse effect of bone-modifying agents
such as bisphosphonates and Denosumab [20-22]. In a randomized
phase 2 study of Denosumab and a bisphosphonate (pamidronate or
zoledronic acid) in patients with multiple malignancies with bone
involvement, 6 of 74 patients (8%) treated with Denosumab developed
grade 3 hypocalcemia, and 1 patient (1%) developed grade 4
hypocalcemia; in contrast, there was only 1 patient with grade 3
hypocalcemia among 37 patients (3%) treated with a bisphosphonate
. In a phase 3 trial comparing Denosumab with zoledronic acid,
hypocalcemia developed in 13% of patients on Denosumab and in 6%
of patients on zoledronic acid; grade 3 hypocalcemia occurred in 5%
and 1% of the Denosumab and zoledronic acid groups, respectively
. The results of these randomized control trials indicate that
Denosumab use may increase the risk of hypocalcemia more than
|Recently, Lechner et al.,  retrospectively examined the incidence
and management of hypocalcemia among patients with bone metastases treated with Denosumab. As indicated by Common
Terminology Criteria for Adverse Events grading, 17 (32.7%) patients
experienced up to grade 1 hypocalcemia, 4 (7.7%) patients experienced
up to grade 2 hypocalcemia, 4 (7.7%) patients experienced up to grade
3 hypocalcemia, and 1 (2.9%) patient experienced up to grade 4
hypocalcemia. In our study of Japanese patients, 39.6% developed
hypocalcemia of grade 1 or greater after Denosumab administration.
The incidence of hypocalcemia in the Japanese patients in our study
was higher than that in previously reported randomized control trials
[14,23] and close to that reported by Lechner et al., indicating that in
standard practice, patients with bone metastases may have a higher
likelihood of developing hypocalcemia.
|As reported by the US Food and Drug Administration , an
increased risk of hypocalcemia has been observed in clinical trials of
patients with renal dysfunction, and the risk is particularly high among
patients with severe dysfunction (that is, patients with a creatinine
clearance of <30 ml/min or who were on dialysis). The Japanese
Ministry of Health, Labour and Welfare  has reported that patients
with severe renal dysfunction have an increased risk of hypocalcemia
after Denosumab injection. However, the risk factors for the
development of hypocalcemia following Denosumab in patients with
bone metastases have not been studied in detail. In a retrospective
review of the records of patients who had received Denosumab,
univariate logistic regression analysis illuminated that the patients
without a history of receiving zoledronic acid before Denosumab or
with low creatinine clearance (CCr) were found to have a high risk of
hypocalcemia (P=0.040 and 0.030, respectively) . The cut off value
of CCr was 50.4 mL/min calculated by receiver-operator
characteristics curves. Furthermore, multivariate logistic regression
analysis showed that non-administration of zoledronic acid
(OR=10.430, P =0.040) and CCr less than 50.0 ml/min (OR= 5.900,
P=0.040) were independent risk factors for hypocalcemia caused by
|Chronic renal failure with a reduction in glomerular filtration rate
may present with decreased production of 1,25-dihydroxyvitamin D,
and absorption of calcium in the enteron can be reduced owing to the
inactivation of vitamin D. However, in our study, low CCr did not
correlate with an increased risk of hypocalcemia. Therefore, although
renal dysfunction may be a risk factor for hypocalcemia, it may not be
a strong risk factor compared to massive bone metastatic burden that
was often observed in patients with elevated bone turnover as our
multivariate analysis results revealed that u-NTx level was a significant
independent risk factor for the development of hypocalcemia.
|The body calcium pool is made up of the plasma calcium in rapid
exchange largely with the calcium in bone mineral , and this
dynamic equilibration verges to bone mineral under accelerated bone
metabolism. In patients with elevated bone turnover, we believe that
Denosumab administration rapidly suppresses bone turnover, and the
result is a decrease in the supply of calcium to the plasma from bone.
In fact, the results of the present study demonstrates that pretreatment
factors of a serum ALP level of ≥ 500 IU/ml, an EOD of ≥ 3, and a u-
NTx level of ≥ 100 nmol BCE/mmol Cr were positively correlated with
hypocalcemia induced by Denosumab (Tables 3 and 4). Therefore,
massive bone metastatic burden may affect serum calcium homeostasis
after Denosumab administration. However, serum calcium levels
recovered to normal in all cases within 16 weeks (Figure 2). This
finding indicates that early monitoring of serum calcium is crucial for
determining whether the continued use of Denosumab is safe.
|The timing of the occurrence of hypocalcemia after Denosumab
administration is becoming clearer. Lechner et al.,  reported the
median time from the first Denosumab injection to the occurrence of
grade 2 hypocalcemia was 16 days. We found that 8 patients developed
grade 2 or 3 hypocalcemia during the study period, and among these, 6
patients (75%) had confirmed hypocalcemia at 1 week after
Denosumab administration. This is a key point for clinicians; that is,
our findings suggest that calcium levels should be monitored not only
before the first administration but also in the early phase of treatment,
especially at around 1 week after the first dose of Denosumab.
|The current study had some limitations. First, prophylactic
administration of calcium and vitamin D supplements was not covered
by health insurance in Japan during the study period, and this fact
might have resulted in a high incidence of hypocalcemia. Second, the
sample size was relatively small, and there was only a single arm.
Large-scale prospective trials are needed.
|This is the first report on the risk factors contributing to the
development of hypocalcemia after Denosumab administration in
patients with bone metastases from prostate cancer. Multivariate
analysis revealed that beseline u-NTx level was an independent risk
factor for hypocalcemia and that patients with high baseline u-NTx
levels should be monitored for hypocalcemia after the first dose of
|Conflict of Interest
|The authors declare the following financial potential conflicts of
interest with respect to the research, authorship, and/or publication of
this article: Dr. Satoh received speaking fees from Daiichi Sankyo Co
Ltd., Astra Zeneca, and Janssen Pharmaceutical K.K.
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