Concurrent, but not sequential, PD-1 blockade with a DNA vaccine elicits anti-tumor responses in patients with metastatic, castration-resistant prostate cancer.

T-cell checkpoint inhibitors have demonstrated dramatic clinical activity against multiple cancer types, however little activity in patients with prostate cancer. Conversely, an anti-tumor vaccine was approved for the treatment of prostate cancer, having demonstrated an improvement in overall survival, despite few objective disease responses. In murine studies, we found that PD-1 expression on CD8+ T cells increased following anti-tumor vaccination, and that PD-1/PD-L1 blockade at the time of immunization elicited greater anti-tumor responses. Based on these data we initiated a pilot trial evaluating the immunological and clinical efficacy of a DNA encoding prostatic acid phosphatase (PAP) when delivered in combination with pembrolizumab. 26 patients were treated for 12 weeks with vaccine and received pembrolizumab either during this time or during the subsequent 12 weeks. Adverse events included grade 2 and 3 fatigue, diarrhea, thyroid dysfunction, and hepatitis. Median time to radiographic progression was not different between study arms. 8/13 (62%) of patients treated concurrently, and 1/12 (8%, p=0.01) of patients treated sequentially, experienced PSA declines from baseline. Of these, two were over 50% and one was a complete PSA response. No confirmed CR or PR were observed, however 4/5 patients treated concurrently had measurable decreases in tumor volume at 12 weeks. PSA declines were associated with the development of PAP-specific Th1-biased T cell immunity and CD8+ T cell infiltration in metastatic tumor biopsy specimens. These data are the first report of a clinical trial demonstrating that the efficacy of an anti-tumor vaccine can be augmented by concurrent PD-1 blockade.

Prime-boost vaccination targeting prostatic acid phosphatase (PAP) in patients with metastatic castration-resistant prostate cancer (mCRPC) using Sipuleucel-T and a DNA vaccine.

BACKGROUND: Prostatic acid phosphatase (PAP) is a prostate tumor antigen, and the target of the only FDA-approved anti-tumor vaccine, sipuleucel-T. We have previously reported in two clinical trials that a DNA vaccine encoding PAP (pTVG-HP) could elicit PAP-specific, Th1-biased T cells in patients with PSA-recurrent prostate cancer. In the current pilot trial we sought to evaluate whether this vaccine could augment PAP-specific immunity when used as a booster to immunization with sipuleucel-T in patients with metastatic, castration-resistant prostate cancer (mCRPC). METHODS: Eigthteen patients with mCRPC were randomized to receive sipuleucel-T alone or followed by intradermal immunization with pTVG-HP DNA vaccine. Patients were followed for time to progression, and immune monitoring was conducted at defined intervals. RESULTS: Overall, patients were followed for a median of 24 months. 11/18 patients completed treatments as per protocol. No treatment-associated events > grade 2 were observed. Th1-biased PAP-specific T-cell responses were detected in 11/18 individuals, and were not statistically different between study arms. Higher titer antibody responses to PAP were detectable in patients who received pTVG-HP booster immunizations. Median time to progression was less than 6 months and not statistically different between study arms. The median overall survival for all patients was 28 months. CONCLUSIONS: These findings suggest that prime-boost vaccination can augment and diversify the type of immunity elicited with anti-tumor vaccination in terms of T-cell and humoral immunity. Future studies will explore DNA as priming immunization rather than a booster immunization. TRIAL REGISTRATION: NCT01706458 .

Comparison of NaF and FDG PET/CT for assessment of treatment response in castration-resistant prostate cancers with osseous metastases.

BACKGROUND: Assessment of skeletal metastases' response to therapy is a highly relevant but unresolved clinical problem. The main goal of this work was to compare pharmacodynamic responses to therapy assessed with positron emission tomography-computed tomography (PET/CT) using fluorine-18 sodium fluoride (NaF) and fluorine-18 fluorodeoxyglucose (FDG) as the tracers. MATERIALS AND METHODS: Patients with prostate cancer with known osseous metastases were treated with zibotentan (ZD4054) and imaged with combined dynamic NaF/FDG PET/CT before therapy (baseline), after 4 weeks of therapy (week 4), and after 2 weeks of treatment break (week 6). Kinetic analysis allowed comparison of the voxel-based tracer uptake rate parameter Ki, the vasculature parameters K1 (measuring perfusion/permeability) and Vb (measuring vasculature fraction in the tissue), and the standardized uptake values (SUVs). RESULTS: Correlations were high for the NaF and FDG peak uptake parameters (Ki and SUV correlations ranged from 0.57 to 0.88) and for vasculature parameters (K1 and Vb correlations ranged from 0.61 to 0.81). Correlation was low between the NaF and FDG week 4 Ki responses (ρ = 0.35; P = .084) but was higher for NaF and FDG week 6 Ki responses (ρ = 0.72; P < .0001). Correlations for vasculature responses were always low (ρ < 0.35). NaF and FDG uptakes in the osseous metastases were spatially dislocated, with overlap in the range from 0% to 80%. CONCLUSION: This study found that late NaF and FDG uptake responses are consistently correlated but that earlier uptake responses and all vasculature responses can be unrelated. This study also confirmed that FDG and NaF uptakes are spatially dislocated. Although treatment responses assessed with NaF and FDG may be correlated, using both tracers provides additional information.

Real-time immune monitoring to guide plasmid DNA vaccination schedule targeting prostatic acid phosphatase in patients with castration-resistant prostate cancer.

PURPOSE: We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial, we sought to evaluate whether prolonged immunization with regular booster immunizations, or "personalized" schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT). EXPERIMENTAL DESIGN: Sixteen patients with castration-resistant, nonmetastatic prostate cancer received six immunizations at 2-week intervals and then either quarterly (arm 1) or as determined by multiparameter immune monitoring (arm 2). RESULTS: Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment >grade 2 was observed. Six of 16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12 of 16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least 1 year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5 of 13 individuals at 1 year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pretreatment to posttreatment was 1.6 (range, 0.6-7.0; P = 0.036). CONCLUSIONS: Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine.

High resolution ultrasound features of prostatic rib metastasis: a prospective feasibility study with implication in the high-risk prostate cancer patient.

OBJECTIVE: In a prior study, high resolution ultrasound (US) was shown to be accurate for evaluating rib metastasis detected on bone scan. However, that study did not address the specific US appearance typical of osteoblastic rib metastasis. Our objective was to determine the specific US imaging appearance of osteoblastic prostate carcinoma rib metastasis using osteolytic renal cell carcinoma rib metastasis as a comparison group. MATERIALS AND METHODS: The Institutional Review Board approval and informed consent were obtained for this prospective feasibility study. We performed high resolution US of 16 rib metastases in 4 patients with prostate carcinoma metastases and compared them to 8 rib metastases in 3 male patients with renal cell carcinoma. All patients had rib metastases proven by radiographs and computed tomography (CT). High resolution US scanning was performed by a musculoskeletal radiologist using a 12-5 MHz linear-array transducer. Transverse and longitudinal scans were obtained of each rib metastasis. RESULTS: All 16 prostate carcinoma metastases demonstrated mild cortical irregularity of the superficial surface of the rib without associated soft tissue mass, cortical disruption, or bone destruction. 7 of 8 (88%) renal cell carcinoma rib metastases demonstrated cortical disruption or extensive bone destruction without soft tissue mass. One of 8 (12%) renal cell carcinoma rib metastases demonstrated only minimal superficial cortical irregularity at the site of a healed metastasis. CONCLUSION: Osteoblastic prostate carcinoma rib metastases have a distinctive appearance on US. Our success in visualizing these lesions suggests that US may be a useful tool to characterize isolated rib abnormalities seen on a bone scan in high-risk prostate cancer patients who are being evaluated for curative surgery or radiation treatment.

A randomized phase II trial evaluating different schedules of zoledronic acid on bone mineral density in patients with prostate cancer beginning androgen deprivation therapy.

OBJECTIVE: To assess the effects of timing and schedule of zoledronic acid (ZA) administration on bone mineral density (BMD) in patients beginning androgen deprivation therapy (ADT) for the treatment of recurrent prostate cancer. PATIENTS AND METHODS: In this randomized, 3-arm trial, we evaluated changes in BMD after 3 different ZA administration schedules in men with recurrent prostate cancer who were beginning ADT. Forty-four patients were enrolled and randomized to receive a single dose of ZA given 1 week before beginning ADT (arm 1), a single dose of ZA given 6 months after beginning ADT (arm 2), or monthly administration of ZA starting 6 months after beginning ADT, for a total of 6 doses (arm 3). RESULTS: Patients who received ZA before ADT had a significant improvement in BMD at the total proximal femur and trochanter after 6 months compared with the other groups. In addition, only patients in the arm that received multiple doses improved lumbar spine BMD while on ADT, with these findings persisting to 24 months. However, this group also experienced more grade 1 adverse events. CONCLUSIONS: Analysis of these data suggests that ZA administration before initiation of ADT was superior to treatment 6 months after starting ADT in maintaining BMD. In addition, monthly ZA administration can increase BMD above baseline but is associated with more adverse events. Further study is needed to examine whether the timing and frequency of ZA therapy in patients on ADT can reduce fracture risk.

Phase I trial of tremelimumab in combination with short-term androgen deprivation in patients with PSA-recurrent prostate cancer.

CTLA-4 blockade has demonstrated antitumor efficacy in human clinical trials. The antitumor mechanism is presumably mediated in part by the expansion of tumor-specific T cells. Androgen deprivation, the cornerstone of treatment for patients with metastatic prostate cancer, has been shown to elicit prostate tissue apoptosis and lymphocytic inflammation. We hypothesized that treatment with androgen deprivation, followed by an anti-CTLA-4 antibody, could augment a tumor-specific immune response elicited by androgen deprivation. We report here the results of a phase I trial evaluating a humanized monoclonal antibody targeting CTLA-4, CP-675,206 (tremelimumab), in combination with androgen deprivation using an antiandrogen. Eligible patients were those with PSA-recurrent prostate cancer after primary surgery and/or radiation therapy, not previously treated with androgen deprivation, and without radiographic evidence of metastatic disease. Subjects were treated in two cycles, 3 months apart, in which they received bicalutamide 150 mg daily days 1-28 and tremelimumab on day 29. The primary endpoint of the trial was safety. Secondary endpoints included measures of PSA kinetics and identification of a maximum tolerated dose. Eleven patients were enrolled and completed at least 1 year of follow-up. Dose-limiting toxicities included grade 3 diarrhea and skin rash. No favorable changes in PSA doubling time were observed in a period shortly after completing treatment; however, three patients experienced a prolongation in PSA doubling time detectable several months after completing treatment. The identification of delayed, prolonged favorable changes in serum PSA suggests that future studies could explore this combination in studies evaluating time to disease progression.

Pilot trial of interleukin-2 and zoledronic acid to augment γδ T cells as treatment for patients with refractory renal cell carcinoma.

Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human Vγ9 Vδ2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these Vγ9 Vδ2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of Vγ9 Vδ2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in Vγ9 Vδ2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of Vγ9 Vδ2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of Vγ9 Vδ2 T cell in patients with metastatic RCC.

Safety and immunological efficacy of a DNA vaccine encoding prostatic acid phosphatase in patients with stage D0 prostate cancer.

PURPOSE: Prostatic acid phosphatase (PAP) is a prostate tumor antigen. We have previously demonstrated that a DNA vaccine encoding PAP can elicit antigen-specific CD8+ T cells in rodents. We report here the results of a phase I/IIa trial conducted with a DNA vaccine encoding human PAP in patients with stage D0 prostate cancer. PATIENTS AND METHODS: Twenty-two patients were treated in a dose-escalation trial with 100 microg, 500 microg, or 1,500 microg plasmid DNA, coadministered intradermally with 200 microg granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant, six times at 14-day intervals. All patients were observed for 1 year after treatment. RESULTS: No significant adverse events were observed. Three (14%) of 22 patients developed PAP-specific IFN gamma-secreting CD8+ T-cells immediately after the treatment course, as determined by enzyme-linked immunospot. Nine (41%) of 22 patients developed PAP-specific CD4+ and/or CD8+ T-cell proliferation. Antibody responses to PAP were not detected. Overall, the prostate-specific antigen (PSA) doubling time was observed to increase from a median 6.5 months pretreatment to 8.5 months on-treatment (P = .033), and 9.3 months in the 1-year post-treatment period (P = .054). CONCLUSION: The demonstration that a DNA vaccine encoding PAP is safe, elicits an antigen-specific T-cell response, and may be associated with an increased PSA doubling time suggests that a multi-institutional phase II trial designed to evaluate clinical efficacy is warranted.

Randomized, double-blinded phase II evaluation of docetaxel with or without doxercalciferol in patients with metastatic, androgen-independent prostate cancer.

PURPOSE: Docetaxel is standard of care for androgen-independent prostate cancer (AIPC). Doxercalciferol (1 alpha-hydroxyvitamin D2) had modest activity in phase I/II trials. Preclinical data support combining vitamin D analogues with docetaxel to treat AIPC. EXPERIMENTAL DESIGN: Chemotherapy-naive men with metastatic AIPC were randomized 1:1 to receive, on a 4-week cycle, docetaxel (35 mg/m2 i.v., days 1, 8, and 15) with or without doxercalciferol (10 microg orally, days 1-28). The primary end point was prostate-specific antigen (PSA) response. Secondary end points were progression-free survival, overall survival, objective response, and toxicity. Survival was analyzed as intent to treat. RESULTS: Seventy patients were randomized. Median follow-up was 17.6 months (range, 3.3-45.2). PSA response rate was 46.7% [95% confidence interval (95% CI), 30-64] in the doxercalciferol arm and 39.4% (95% CI, 25-56) with placebo (P = 0.560). Median progression-free survival in the doxercalciferol arm was 6.17 months (95% CI, 4.20-10.7) versus 6.20 months (95% CI, 4.83-9.07) with placebo (P = 0.764). Median overall survival in the doxercalciferol arm was 17.8 months (95% CI, 14.9-23.6) versus 16.4 months (95% CI, 11.9-23.8) with placebo (P = 0.383). Twenty-four patients in the doxercalciferol arm and 23 in the placebo arm were evaluable for objective response. No complete responses were observed. Partial objective response rate was 12.5% with doxercalciferol versus 8.7% with placebo (P = 0.672). Rate of grade > or =3 toxicity was 46% with doxercalciferol versus 42% with placebo (P = 0.785). CONCLUSIONS: Daily doxercalciferol with weekly docetaxel did not enhance PSA response rate or survival. Toxicity was similar between arms. Despite the disappointing results of this study, other vitamin D analogues remain under active investigation.

Effect of medical castration on CYP3A4 enzyme activity using the erythromycin breath test.

PURPOSE: Testosterone administration can lead to increased antipyrine clearance in humans. Medical or surgical castration is a standard treatment of progressive prostate carcinoma, but the effect of the subsequent fall of testosterone concentrations upon drug metabolism has not been reported. METHODS: Eleven men with a biopsy-proven diagnosis of progressive prostate cancer were enrolled after providing informed consent. CYP3A4 activity was determined using the erythromycin breath test (EBT) in each patient prior to their beginning with an LHRH-agonist (leuprolide or goserelin). No patients had elected to undergo orchiectomy during the period of subject accrual. Each subject underwent a second EBT 2 months after beginning LHRH suppression. Blood samples were collected at these time points to determine changes in testosterone and leutinizing hormone. RESULTS: All subjects had a predictable drop in serum testosterone concentrations over the 8-week course of the study, but concentrations in three did not fall below castrate levels (<50 ng/dl). There was no statistically significant change in CYP3A4 activity using the EBT method (p = 0.88). The extent and direction of changes in CYP3A4 activity was highly variable, with three subjects experiencing an increase in activity, and five demonstrating a decrease in activity. CONCLUSION: There is no clinically significant change in CYP3A4 activity after medical castration. No changes in the clearance of docetaxel or other CYP3A4 substrates are likely during and after medical castration. Although similar findings are expected after orchiectomy, we were not able to test this presumption because of patient preference for medical castration.

Phase II study of interferon-alpha and doxycycline for advanced renal cell carcinoma.

OBJECTIVE: To assess the efficacy and toxicity of the combination of interferon-alpha and doxycycline in patients with metastatic renal cell carcinoma and to assess the effect of this treatment on serum vascular endothelial growth factor (VEGF) levels. PATIENTS AND METHODS: Seventeen patients with Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and life expectancy greater than 4 months with radiologically evident advanced renal cell carcinoma were enrolled. Eight patients had prior nephrectomy and 10 patients were treated within 4 months of their diagnosis. Treatment consisted of interferon-alpha up to 9 million units subcutaneously three times per week and doxycycline 300 mg orally twice per day for weeks one and three of each four-week cycle. Toxicity was evaluated on a biweekly basis and response on a bimonthly basis. VEGF plasma levels were assessed monthly as a measure of potential antiangiogenic effect. RESULTS: No objective responses were seen. The mean duration of study was 2.6 cycles (range: 0.8-6.0 cycles). Three patients (17%) tolerated therapy and displayed stable disease for greater than four months. Five patients withdrew from study before the first response evaluation. Ten patients experienced grade 2 gastrointestinal toxicity requiring dose reduction of doxycycline. Eight patients experienced grade 2 fatigue requiring dose reduction of interferon. VEGF plasma levels were initially suppressed in patients who demonstrated progressive disease but not in patients with stable disease. CONCLUSION: This regimen of doxycycline and interferon-alpha was not efficacious as treatment for renal cell carcinoma. Plasma VEGF levels were significantly decreased during the first two cycles of treatment, but this does not correlate with clinical outcome.

A phase II multicenter, randomized, double-blind, safety trial assessing the pharmacokinetics, pharmacodynamics, and efficacy of oral 2-methoxyestradiol capsules in hormone-refractory prostate cancer.

PURPOSE: To determine whether the preclinical antitumor and antiangiogenic activity of 2-methoxyestradiol can be translated to the clinic. EXPERIMENTAL DESIGN: Men with hormone-refractory prostate cancer were enrolled into this phase II randomized, double-blind trial of two doses of oral 2-methoxyestradiol capsules (400 and 1,200 mg/d) given in 4-week cycles. Pharmacokinetic sampling was done on day 1 of cycles 1 and 2 and trough samples were obtained weekly. RESULTS: Thirty-three men were accrued between February and September 2001. The notable toxicity related to therapy was one grade 2 and two grade 3 episodes of liver transaminase elevation, which resolved with continued treatment in two patients. There were two cases of deep venous thromboses. The drug had nonlinear pharmacokinetic, rapid conversion to 2-methoxyestrone and approximately 85% conjugation. Trough plasma levels of unconjugated 2-methoxyestradiol and 2-methoxyestrone were approximately 4 and 40 ng/mL, respectively. Prostate-specific antigen declines between 21% and 40% were seen in seven patients in the 1,200 mg group and in one patient in the 400 mg group. The higher-dose group showed significantly decreased prostate-specific antigen velocity (P = 0.037) and compared with the 400 mg dose had a longer median time to prostate-specific antigen progression (109 versus 67 days; P = 0.094) and time on study (126 versus 61 days; P = 0.024). There was a 2.5- and 4-fold increase in sex hormone-binding globulin for the 400 and 1,200 mg dose levels, respectively, at days 28 and 56. CONCLUSION: 2-Methoxyestradiol is well tolerated and, despite suboptimal plasma levels and limited oral bioavailability with this capsule formulation, still showed some anticancer activity at 1,200 mg/d.

Phase II trial of perillyl alcohol (NSC 641066) administered daily in patients with metastatic androgen independent prostate cancer.

OBJECTIVE: We conducted a phase II multicenter trial of perillyl alcohol in patients with advanced hormone refractory prostate cancer (HRPC). The primary endpoint was to evaluate the 6-month progression-free survival given the potential cytostatic nature of the drug. Secondary objectives included assessing acute and chronic toxicities, as well as measuring objective response rates. METHODS: Patients with metastatic androgen-independent prostate cancer that failed at least one prior chemotherapeutic or experimental regimen were eligible. Perillyl alcohol was administered orally at 1200 mg/m2/dose four times daily and continued until disease progression or development of unacceptable toxicity. RESULTS: Fifteen patients were eligible. Six patients received less than one cycle (4 weeks) of drug, four of which stopped because of drug intolerance. Only six patients received more than two cycles of therapy and were considered evaluable for response. Main toxicity included grade 1-2 gastrointestinal intolerance (nausea/vomiting in 60% of the patients) and fatigue (47%). One patient developed a grade 4 hypokalemia that was felt likely attributable to the drug. No objective responses were seen. All patients either progressed or withdrew from the study secondary to drug intolerance before the 6-month time period. CONCLUSION: Perillyl alcohol administered at this dose and formulation did not have any objective clinical activity in this patient population.

Phase II study of 1alpha-hydroxyvitamin D(2) in the treatment of advanced androgen-independent prostate cancer.

PURPOSE: In this single institution Phase II trial, we evaluated the efficacy of the vitamin D analogue, 1alpha-OH-D(2), in patients with advanced hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: The patients initially received 1alpha-OH-D(2) at 12.5 micro g p.o. every day, which was dose adjusted for hypercalcemia. Given the cytostatic nature of the drug, the primary study end point was progression-free survival for a minimum of 6 months. The secondary end point was further characterization of drug toxicity. RESULTS: A total of 26 patients was enrolled. Using the intent-to-treat population, stable disease was seen for an average of 19.2 weeks (median 12 weeks, range 3-108 weeks). Twenty patients were evaluable for response. The one patient that achieved disease stabilization for >2 years elected to come off-study because of patient preference. His last disease evaluation showed no evidence of progression. No objective responses were seen. Previous and ongoing clinical observations strongly imply that PSA could be a misleading surrogate marker for clinical effect with this type of drug. Therefore, prostate-specific antigen was not used as a marker for disease response. Toxicity was as expected with mild hypercalcemia and associated symptoms like constipation and prerenal azotemia seen in some patients. Six (30%) evaluable patients experienced stable disease for >6 months, suggesting possible cytostatic activity. CONCLUSION: The results of this and other trials suggest further clinical investigation in this disease with vitamin D analogues alone or in combination with other agents, such as chemotherapy, should be pursued.

Phase I trial of 1alpha-hydroxyvitamin d(2) in patients with hormone refractory prostate cancer.

This Phase I study of 1alpha-hydroxyvitamin D(2), an p.o. administered vitamin D analogue, in patients with advanced hormone-refractory prostate cancer was designed to assess the toxicity, pharmacokinetic and biological markers of drug activity, and lastly tumor response data to recommend a dose for Phase II studies. 1alpha-Hydroxyvitamin D(2) was administered daily at doses ranging from 5 to 15 microg/day. Patients were monitored for toxicity and tumor response, and blood and urine samples were collected for pharmacokinetics (1alpha,25-dihydroxyvitamin D(2) levels) and other parameters of biological activity (bone markers, parathyroid hormone, urine calcium, and serum phosphorus levels). Twenty-five patients were enrolled. Main toxicities were hypercalcemia with associated renal insufficiency. No other significant toxicity was seen. Pharmacokinetics showed an increase in the active metabolite 1alpha,25-dihydroxyvitamin D(2) that reached a plateau by week 4 despite continuous drug dosing. Elevation in daily urinary calcium excretion and serum phosphorus levels was seen, whereas a decrease in serum parathyroid hormone was evident. Two patients showed evidence of a partial response, whereas 5 others achieved disease stabilization for > or =6 months. 1alpha-Hydroxyvitamin D(2) was well tolerated with main toxicities being hypercalcemia and renal insufficiency. All of the toxicity was reversible with drug discontinuation. Evidence for drug activity was seen in surrogate markers, and pharmacokinetic analysis showed substantial increases in vitamin D metabolite levels among the various cohorts. Whereas the defined maximum tolerated dose was not reached, the recommended Phase II dose was 12.5 microg/day given continuously.