II Study of Azacitidine, Docetaxel, and Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer Previously Treated With Docetaxel-Based Therapy

Original Study

Phase I/II Study of Azacitidine, Docetaxel, and Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer Previously Treated With Docetaxel-Based Therapy Rakesh Singal, Kavitha Ramachandran, Edna Gordian, Carlos Quintero, Wei Zhao, Isildinha M. Reis Abstract DNA methylation of genes contributes to resistance to docetaxel in prostate cancer. We investigated the combination of azacitidine (demethylating agent), docetaxel, and prednisone with the aim of reversing docetaxel resistance. We treated 22 patients in a phase I/II study. This combination with growth factor support was active in metastatic prostate cancer patients previously treated with docetaxel. Introduction: Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. Patients and Methods: Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 þ 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. Results: In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 with growth factor support. Prostatespecific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. Conclusion: The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients. Clinical Genitourinary Cancer, Vol. 13, No. 1, 22-31 ª 2015 Elsevier Inc. All rights reserved. Keywords: Chemotherapy, GADD45A, Methylation, Taxotere, Vidaza

Introduction Docetaxel is the first Food and Drug Administration (FDA)-approved chemotherapeutic agent shown to improve survival in patients with metastatic castration-resistant prostate cancer (mCRPC).1,2 However, the median progression-free survival (PFS) University of Miami, Sylvester Comprehensive Cancer Center, Miami, FL Submitted: Jun 17, 2014; Revised: Jul 23, 2014; Accepted: Jul 29, 2014; Epub: Aug 1, 2014 Address for correspondence: Rakesh Singal, MD, Sylvester Comprehensive Cancer Center, 1475 NW 12th Ave, Miami, FL 33136 Fax: 305-243-5977; e-mail contact: [email protected]

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Clinical Genitourinary Cancer February 2015

with docetaxel is approximately 6 months. At the time of completion of the phase I study, there were no treatment options for patients with mCRPC whose disease had progressed during or after docetaxel chemotherapy. At present however, 4 new drugs, cabazitaxel, abiraterone, enzalutamide, and radium-223, have been approved by the FDA for use in the setting after docetaxel treatment. Even though cabazitaxel and prednisone have shown activity in docetaxel-refractory castration-resistant prostate cancer patients, the median overall and PFS improved only by 2.4 and 1.4 months over mitoxantrone and prednisone, respectively.3 The combination of abiraterone and prednisone showed a survival improvement of 4.6 months over placebo and prednisone.4 Enzalutamide also

1558-7673/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2014.07.008

improved survival by 4.8 months compared with placebo.5 Radium223 improved survival by 3.6 months compared with placebo.6 The mechanism of docetaxel resistance and the approaches to overcome docetaxel resistance in prostate cancer have been largely unexplored. We have found in vitro that growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic gene, that is induced on docetaxel treatment7 is downregulated by methylation in Du145 prostate cancer cells.8 Demethylation of the GADD45A promoter using treatment with azacitidine increased GADD45A expression, resulting in enhanced sensitivity of Du145 cells to docetaxel.8 These findings provided a strong scientific rationale to study a combination of azacitidine and docetaxel in the clinical setting.

The main objective of the single-center, single-arm, phase I study was to determine a safe and potentially efficacious dose of azacitidine that can be used in combination with docetaxel and prednisone for the treatment of docetaxel-resistant mCRPC. The secondary objective was to obtain an initial estimate of response to study therapy, which included objective response according to Response Evaluation Criteria In Solid Tumors (RECIST)9 in patients with measurable disease. The study was designed to alternately escalate or reduce azacitidine and docetaxel for each subsequent dose cohort. A total of 6 dose combinations were planned as shown in Table 1. The planned starting dose was at level 1, allowing 3 levels of dose escalation or 2 levels of dose reduction depending on the tolerability of the regimen. After completion of the phase I study, 7 patients were treated with this regimen in the phase II portion of the study. However, because of withdrawal of funding, the study was prematurely terminated.

months after cessation of at least 6 weeks of docetaxel-based therapy. There was no limitation on the number of previous chemotherapy regimens that the patients might have received. For patients with measurable disease, progression was defined according to RECIST criteria,9 demonstrating at least 1 visceral or soft tissue metastatic lesion (including new lesions). For patients with nonmeasurable disease, disease progression was defined as an increase in at least 2 consecutive serum prostate-specific antigen (PSA) values, each obtained at least 1 week apart and/or appearance of new lesions on bone scan. All patients were required to have Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2. The exclusion criteria included previous treatment with strontium89 (Metastron) (GE Healthcare, Pittsburg, PA), samarium153 (Quadramet) (Bristol Myers Squibb, N. Billerica, MA) radiation therapy within 8 weeks of enrollment, chemotherapy or radiotherapy within 4 weeks before entering the study, brain metastases, history of allergic reactions attributed to compounds with chemical or biological composition similar to azacitidine (Vidaza) (Celgene Corporation, Summit, NJ) or docetaxel or other drugs formulated with polysorbate 80 or mannitol. Required laboratory values included absolute neutrophil count (ANC) > 1500 cells per mm3, platelet count > 100,000 cells per mm3, hemoglobin > 10 g/dL, adequate liver function documented by total bilirubin  the upper limit of normal (ULN), aspartate transaminase (AST) and alanine transaminase (ALT)  2.5 times the ULN, and serum creatinine < 1.5 mg/dL. This clinical trial was sponsored by the University of Miami, approved by the institutional review board, and monitored by the institution’s data and safety monitoring board (DSMB). The trial was registered on the clinicaltrials.gov Web site (http://clinicaltrials. gov/ct2/show/NCT00503984).

Eligibility Criteria

Treatment Plan

All patients had histologically confirmed adenocarcinoma of the prostate and radiologically documented metastatic disease. Patients were required to have progressive disease despite castrate testosterone levels and have disease progression during or within 6

A treatment cycle consisted of 21 days, with an assigned dose of azacitidine administered on days 1 to 5, an assigned dose of docetaxel on day 6, and a continuous fixed dose of prednisone at 5 mg twice per day (b.i.d.) on days 1 to 21. An assigned dose of azacitidine was administered by intravenous (I.V.) infusion over 30 minutes on days 1 to 5 of each 3-week cycle. An assigned dose of docetaxel was given using I.V. infusion on day 6 of each 3-week cycle. Patients were premedicated with corticosteroids, and H1and H2-blockers before docetaxel administration to prevent hypersensitivity reactions. Because patients enrolled in an earlier part of the trial might receive a suboptimal dose, intrapatient dose escalation for the phase I patients was allowed, at the investigator’s discretion, in the absence of Grade > 2 toxicity. Patients had PSA levels (day 1), complete blood counts, liver function, and kidney function (days 1, 8, and 15) tested during each cycle and imaging using bone scan and computed tomography (CT) at baseline and after every 2 cycles. Patients were treated until disease progression or unacceptable toxicity. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria version 3.0. Up to 3 patients were enrolled at each dose level. If 1 of 3 patients experienced a dose-limiting toxicity (DLT) during the first cycle, up to 3 additional patients were enrolled at that dose level. If no additional DLTs were observed, then dose

Patients and Methods Study Design

Table 1 Dose Levels for Escalation/Deescalation in Phase I Part of Study

Dose Level Level L2 Level L1 Level 1 (Starting Dose) Level 2 Level 3 Level 4

Azacitidine (Vidaza) I.V. on Days 1 to 5 Every 21 Days

Docetaxel (Taxotere) I.V. on Day 6 Every 21 Days

50 mg/m2 50 mg/m2 75 mg/m2

50 mg/m2 60 mg/m2 60 mg/m2

75 mg/m2 100 mg/m2 150 mg/m2

75 mg/m2 75 mg/m2 75 mg/m2

The phase I study design included dose escalation/deescalation of azacitidine (Celgene Corporation, Summit, NJ) and docetaxel (Bridgewater, NJ) according to the standard design, to establish a phase II dose. The starting dose level was azacitidine 75 mg/m2 and docetaxel 60 mg/m2. Dose levels 2 and 1 were allowed for testing as a result of dose deescalation based on first cycle dose-limiting toxicity, or intrapatient dose reduction because of toxicity in any cycle. Intrapatient dose escalation was allowed at the discretion of the principal investigator.

Clinical Genitourinary Cancer February 2015

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Azacitidine, Docetaxel, and Prednisone in CRPC escalation proceeded. Patients were enrolled sequentially so that no more than 2 patients per dose cohort could experience a DLT. If 2 patients in a cohort experienced a DLT, then the maximum tolerated dose was considered as exceeded. DLT was defined as treatment-related toxicity occurring within the first 21 days of therapy (cycle 1) that included  Grade 3 nonhematologic toxicity or  Grade 4 hematologic toxicity. Grade 4 neutropenia was not considered a DLT unless lasting longer than 1 week. No prophylactic granulocyte growth factors were allowed during the first cycle of treatment. In subsequent cycles, growth factors were permitted as per the investigator’s discretion. Dose modifications were defined according to protocol. Grade > 3 thrombocytopenia required a 1-level dose reduction for azacitidine/docetaxel. If the patient had < 1000 per mm3 neutrophils before day 1, then chemotherapy was delayed for 1 week. If patients recovered the neutrophil count to  1000 per mm3 in time for the next scheduled treatment, a dose reduction was not required and the patient received the starting or previous treatment dose level of azacitidine and docetaxel and resumed the every 3 weeks dosing schedule. Only patients with afebrile Grade 4 neutropenia that lasted for  7 days, or Grade 4 febrile neutropenia (1 oral reading of > 38.5 C, or 3 oral readings of > 38.0 C in a 24-hour period) were treated at the next lower dose level for the next treatment cycle. A maximum treatment delay of 3 weeks was allowed. If the febrile neutropenia recurred despite 2 levels of dose reductions, chemotherapy was stopped. For Grade 2 neuropathy, the azacitidine/ docetaxel dose was reduced by 1 level. Treatment was discontinued for Grade 3 or 4 neuropathies. For Grade 3 and 4 nonhematologic toxicities, treatment was withheld until the toxicity resolved to  Grade 1, then reinstituted (if medically appropriate) with a level 1 dose reduction.

Correlative Studies Growth arrest and DNA damage-inducible alpha methylation levels were analyzed in the 15 phase I patients. Blood was collected on days 1 and 6 before azacitidine and docetaxel treatment, respectively. DNA was extracted from 2 mL serum using QIAamp UltraSens virus Kit (Qiagen) (Valencia, CA). DNA from buffy coat was extracted using a MasterPure DNA Purification Kit Blood Version II (Epicentre) (Madison, WI). Bisulfite conversion of the DNA was carried out as previously described.10 DNA was amplified using bisulfite polymerase chain reaction. Methylation levels of GADD45A were quantitated using a methylation-sensitive single nucleotide primer extension assay as previously described.8

Statistical Analysis

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Patient demographic characteristics (age, race/ethnicity), performance status, laboratory parameters, and disease characteristics were summarized using descriptive statistics: counts and percentages, range, median, mean, and standard deviation. Toxicities were tabulated according to type and Grade, and attribution to treatment. PSA response was evaluated according to Prostate Cancer Working Group 1 (PCWG1) criteria (PSA decline > 50% from baseline that was maintained for at least 3 weeks).11 In patients with measurable disease, objective response was also evaluated using the RECIST criteria.9 Response rates were estimated according to the percentage of patients achieving the response criterion and the

Clinical Genitourinary Cancer February 2015

corresponding 95% confidence interval (CI) using the binomial method. PFS was defined as the elapsed time from start of treatment until the first documented or confirmed disease progression, according to PSA or RECIST criteria, or death related to prostate cancer, whichever was earlier. In the absence of any event defining progression, follow-up time was censored at the date of the last documented progression-free status. Overall survival (OS) was defined as the elapsed time from start of treatment until date of death from any cause. In the absence of death, follow-up was censored at the date of last contact. The KaplaneMeier method was used to estimate PFS and OS, and corresponding medians. Analyses were performed using SAS version 9.2 (SAS institute, Cary, NC).

Results Patient Characteristics Phase I patients were enrolled between June 2007 and April 2009, and phase II patients between October 2009 and February 2013. The study was closed to enrollment in June 2013 because of lack of funding. After a robust accrual in the phase I and earlier part of phase II, the accrual rate dropped because of FDA approval of second-line treatments for prostate cancer. Fifteen patients with mCRPC previously treated with docetaxelbased therapy were treated in the phase I part of the study. The median patient age was 66 years. 6 patients (40%) had a Gleason score of 7 and 8 patients (53%) had a Gleason score of 8 to 10. 4 patients (27%) had an ECOG performance status of 1 to 2, and 11 patients (73%) had an ECOG performance status of 0. Seven patients were previously treated with docetaxel chemotherapy only and 8 patients had treatment with 2 or more regimens. Most of patients had disease progression either during docetaxel chemotherapy (73.3%) or within 6 weeks of last docetaxel dose (20%). With regard to type of disease progression, 3 had shown PSA progression only, 11 had PSA and bone or soft tissue, and 1 had PSA and symptomatic progression during docetaxel chemotherapy (Table 2). Seven patients were treated in the phase II of the study. The median patient age was 69 years. There were 2 patients with a Gleason score of 7, and 5 with a Gleason score of 8 to 10. ECOG performance status was 2 for 1 patient, 1 for 2 patients, and 0 for 4 patients. Five patients were previously treated with docetaxel chemotherapy only and 2 patients had treatment with 2 regimens. Six patients had disease progression during docetaxel chemotherapy and 1 patient between 6 and 12 weeks of last docetaxel dose. Disease progression during or after docetaxel treatment was seen in CT and/or bone scan in addition to PSA progression in 6 patients and 1 patient had PSA progression alone (Table 2).

Dose Escalation, DLT, and Other Toxicities In the phase I portion of study, no DLT was observed. The highest level reached was level IV (azacitidine 150 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 along with growth factor support and fixed prednisone 5 mg b.i.d. on days 1-21), which was considered as the recommended phase II dose (RPTD). A total of 76 cycles were administered at this dose level (Table 3). As anticipated, treatment-related hematologic toxicity was frequently observed (Table 4). There were 6 episodes of treatment-related serious adverse events (SAEs) including febrile neutropenia,

Rakesh Singal et al Table 2 Patient Characteristics Phase I (n [ 15) Characteristic

Phase II (n [ 7)

n

%

n

%

<60

3

20.0

1

14.3

60-69

8

53.3

3

42.9

70 Mean (SD)

4

26.7

3

Age, Years

Median (range)

42.9

63.9 (8.9)

70.6 (8.0)

66 (41-73)

69 (58-80)

Ethnicity Hispanic or Latino Not Hispanic or Latino

5

33.3

1

14.3

10

66.7

6

85.7

4

26.7

1

14.3

11

73.3

6

85.7

Race Black or African American White Gleason Score 7

6

40.0

2

28.6

8-10

8

53.3

5

71.4

Missing

1

6.7

e

e

0

11

73.3

4

57.1

1

3

20.0

2

28.6

ECOG Performance Status

2

1

6.7

1

14.3

15

100.0

7

100.0

7

46.7

5

71.4

2 Regimens

3

20.0

2

28.6

3 Regimens

5

33.3

e

e

Stage IV prostate cancer Previous Chemotherapy 1 Regimen

Previous Progression During docetaxel treatment

11

73.3

6

86.7

6 Weeks after last docetaxel dose

3

20.0

0

0

>6 Weeks to 12 weeks after last docetaxel dose

1

6.7

1

14.3

PSA progression only

3

20.0

1

14.3

PSA and bone or soft tissue progression

11

73.3

6

86.7

PSA and symptomatic progression

1

6.7

e

e

Type of Previous Disease Progression

Laboratory tests Median LDH (range), U/L

504 (27-1559)

740 (448-775) (n ¼ 3)

Median alkaline phosphatase (range), U/L

97 (42-174)

145 (49-213) (n ¼ 5)

12.1 (9.5-14.8)

12.6 (11.5-13.8) (n ¼ 6)

97.6 (12.6-758.6)

123.6 (4.9-433.6)

Median hemoglobin (range), g/dL Median PSA (range), ng/mL

Abbreviations: ECOG ¼ Eastern Cooperative Oncology Group; LDH ¼ lactate dehydrogenase; PSA ¼ prostate-specific antigen.

anemia, and catheter-related infection with normal ANC. There were 4 episodes of Grade 3 febrile neutropenia in 4 (26.7%) patients, 1 episode of Grade 3 anemia in 1 (6.7%) patient, and 1 episode of catheter-related infection with normal ANC in 1 (6.7%) patient. There was 1 death due to stroke which was not treatment-related.

The most common treatment-related adverse event (AE) was neutropenia. This was less frequent with administration of growth factor support. There were 4 episodes of neutropenia lasting more than 7 days (1 Grade 3 and 3 Grade 4, all with growth factor support); this corresponds to a rate of 3.9% of 103 cycles given with granulocyte-colony

Clinical Genitourinary Cancer February 2015

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Azacitidine, Docetaxel, and Prednisone in CRPC Table 3 Treatment and Response Treatment Response According to:

Cycles per Dose Level No. of Cycles

I: 75 Aza and 60 Doc

II: 75 Aza and 75 Doc

1

6

2

1

2

2

1

1

3

2

1

1

e

4

11

e

1

5

1

e

1

6

2

e

1

1

e

7

12

e

e

1

11

8

6

e

e

2

4

2-6

9

4

e

e

1

3

2, 4

10

17

e

e

e

17

2-17

11

14

e

e

5 (Cycles 10-14)

9 (Cycles 1-9)

2-4, 6-9, 11-14

12

7

e

e

e

7

2-7

13

3

e

e

e

3

2-3

14

21

e

e

14 (Cycles 8-21)

7 (Cycles 1-7)

2-21

15

19

e

12 (Cycles 8-19)

3 (Cycles 5-7)

4 (Cycles 1-4)

2-7, 9, 11-19

Total (G-CSF)

127 (103)

4 (1)

18 (13)

29 (25)

76 (64)

(103)

16

5

e

e

e

5

1-5

PCWG1 (3)

NA

SD

17

8

e

e

e

8

1-8

None

PR

PD

18

20

e

e

e

20

1-20

PCWG1 (11)

NA

SD

19

7

e

e

e

7

1-7

PCWG1 (3)

PD

SD

20

5

e

e

e

5

1-5

None

SD

SD

21

1

e

e

e

1

1

e

NE

NE

22

3

e

3

e

e

1-3

None

NA

SD

Total (G-CSF)

49 (49)

e

3 (3)

e

46 (46)

(49)

Phase Cohort Patient I

1

2

3

4

II

III: 100 Aza and 75 Doc

IV: 150 Aza and 75 Doc

G-CSF, Cycle

PSA (Week)

1

2

2-6

None

PD

e

e

2

None

NA

PD

e

2

None

SD

PD

1

9

2-11

PCWG1 (13)

NA

SD

e

e

NE

NA

NE

NE

NE

NE

CR

NA

PCWG1 (9)

SD

SD

None

NA

SD

None

SD

SD

PCWG1 (8)

NA

SD

PCWG1 (10)

NA

SD

None

SD

PD

PCWG1 (6)

PR

SD

PCWG1 (10)

NA

SD

2

3-7, 9, 11 PCWG1 (9)

CT Bone Scan Scan PD

Treatment cycle of 3 weeks, with azacitidine given on days 1 through 5, docetaxel on day 6, and prednisone 5 mg twice per day on days 1 through 21. All 15 patients in phase I received growth factor support (G-CSF) in cycles other than cycle 1. All 7 phase II patients received G-CSF in all cycles. In phase I, intrapatient dose escalation was allowed in the absence of Grade 2 toxicity at the previous cycle. DLT was defined as first cycle treatment-related Grade 4 hematologic or  Grade 3 nonhematologic toxicity. Grade 4 neutropenia was not a DLT unless lasting longer than 1 week. No DLTs were observed. PSA response (PCWG1) was defined as PSA > 50% decline from baseline measured twice 3 to 4 weeks apart (in parenthesis), is reported the week corresponding to the first measurement with > 50% decline. Note that all, except 1, PSA responses occurred after at least 1 cycle at dose level IV. Abbreviations: Aza ¼ azacitidine; CT ¼ computed tomography; DLT ¼ dose-limiting toxicity; Doc ¼ docetaxel; G-CSF ¼ granulocyte-colony stimulating factor; NA ¼ no measurable disease; NE ¼ not evaluable (lesion present, but the patient did not complete the required 2 cycles of chemotherapy; PCWG1 ¼ Prostate Cancer Working Group 1; PSA ¼ prostate-specific antigen.

26

-

stimulating factor (G-CSF) as growth factor support. In addition, there were 21 episodes of neutropenia lasting < 7 days: 6 of Grade 3 and 2 of Grade 4 with growth factor support (7.8% of 103 cycles with G-CSF), and 5 of Grade 3 and 8 of Grade 4 without growth factor support (54.2% of 24 cycles given without G-CSF). In the phase II portion of the study, 6 patients received the initial RPTD of azacitidine 150 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 along with growth factor support. A total of 46 cycles were administered at this dose level (Table 3). At this dose level, there were 2 episodes of  Grade 3 hematologic toxicities and 3  Grade 3 nonhematologic toxicities that were treatment-related. Among the patients treated at the highest dose, 1 patient had Grade 3 leukopenia. One patient died from neutropenic sepsis (SAE), which was considered treatment-related (Table 4). This patient also had

Clinical Genitourinary Cancer February 2015

other SAEs leading to his death such as renal failure, diarrhea, hyperkalemia, and hyponatremia that were not treatment-related. None of the other patients had SAEs. After the death of this patient, the study was reviewed by the DSMB and the RPTD was reduced to azacitidine 75 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 with growth factor support for subsequent patients. One patient was treated at this reduced dose level and had no treatment-related  Grade 3 AEs or SAEs. When we analyzed the treatment-related  Grade 3 AEs at different dose levels, in the phase I portion of the study, there were 40 events at dose level IV in 11 patients who received 76 treatment cycles (53%) and 13 events at dose level III in 9 patients who received 29 treatment cycles (45%). Comparatively, at dose level II, there were fewer events: 5 events in 18 cycles of treatment (28%).

Rakesh Singal et al Table 4 Grade ‡ 3 Adverse Events According to Received Dose Level Phase I: Received Dose level All Patients, n

Phase II: Received Dose level

I: 75 Aza II: 75 Aza III: 100 Aza IV: 150 Aza and 60 Doc and 75 Doc and 75 Doc and 75 Doc

All

II: 75 Aza IV: 150 Aza and 75 Doc and 75 Doc

15

3

7

9

11

7

127

4

18

29

76

24

3

5

4

12

103

1

13

25

64

Patients With Grade ‡3 AEs

15

3

3

5

10

3

1

2

Episodes of Grade ‡3 AEs

68

3

5

16

44 (9 SAEs)

9

1

8 (1 SAE)

Treatment Cycles Received, n Without G-CSF With G-CSF

1

6

49

3

46

e

e

e

49

3

46

Hematologic

56

3

5

11

37 (5 SAEs)

2

e

2 (1 SAE)

Nonhematologic

12

e

e

5

7 (4 SAEs)

7

1

6

61

3

5

13

40 (6 SAEs)

5

e

5 (1 SAE)

7

e

e

3

4 (3 SAEs)

4

1

3

61

3

5

13

40

5

e

5

Treatment Attribution Definite/probable/possible Unrelated/unlikely Adverse Event With Treatment Attribution Definitive, Probable, or Possible Episodes of Grade 3 Hematologic

40

3

3

5

29

1

e

1

36

3

3

3

27

1

e

1

Neutropenia lasting 7 days without G-CSF

5

3

1

e

1

e

e

e

Neutropenia lasting 7 days with G-CSF

6

e

e

e

6

e

e

e

Neutropenia lasting >7 days with G-CSF

1

e

e

e

1

e

e

e

Febrile neutropenia (with G-CSF)

4

e

e

e

4

e

e

e

12

e

2

e

10

1

e

1

Lymphopenia

4

e

e

1

3

e

e

e

Hemoglobin

4

e

e

2

2

e

e

e

Nonhematologic

4

e

e

2

2

e

e

e

Allergic reaction

2

e

e

1

1

e

e

e

Chest pain

1

e

e

1

e

e

e

e

Fatigue

1

e

e

e

1

e

e

e 4

Leukopenia

Episodes of Grade 4 Hematologic Neutropenia lasting 7 days without G-CSF

21

e

2

8

11

4

e

20

e

2

8

10

1

e

1

8

e

1

2

5

e

e

e

Neutropenia lasting 7 days with G-CSF

2

e

e

2

e

e

e

e

Neutropenia lasting >7 days with G-CSF

3

e

1

e

2

e

e

e

7

e

e

4

3

e

e

e

e

e

e

e

1

e

1 SAE

1

e

e

e

1

3

e

3

1

e

e

e

1

e

e

e

Leukopenia Febrile neutropenia Nonhematologic Infection, normal ANC, catheter-related

e

Acidosis

e

e

e

e

e

1

e

1

Cardiac ischemia/infarction

e

e

e

e

e

1

e

1

Hypotension

e

e

e

e

e

1

e

1

7

e

e

3

4

4

1

3

6

e

e

3

3

4

4

3

Chest pain

1

e

e

e

1

e

e

e

Dyspnea

1

e

e

e

1

e

e

e

Hyponatremia

4

e

e

3

1

1

e

1

Diarrhea

1

e

e

e

e

1

e

1

Renal

1

e

e

e

e

1

e

1

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Adverse Event With Treatment Attribution Unlikely or Not Related Episodes of Grade 3

Grade 5 CNS-cerebrovascular ischemia

Abbreviations: AE ¼ adverse event; ANC ¼ absolute neutrophil count; Aza ¼ azacitidine; Doc ¼ docetaxel; G-CSF ¼ granulocyte-colony stimulating factor; SAE ¼ serious adverse event.

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Azacitidine, Docetaxel, and Prednisone in CRPC Six treatment-related SAEs occurred at dose level IV and there were no SAEs at other dose levels. The phase II patients did relatively better at dose level IV. Among the phase II patients, there were 5  Grade 3 treatment-related events in 6 patients who received 46 cycles (11%) of dose level IV (Table 4).

Of all the patients treated in this study, there were 10 evaluable patients with measurable disease on CT scan. One patient showed complete response, 2 patients showed partial response, 5 had stable disease, and 2 had progressive disease defined according to RECIST criteria.

Response Evaluation

Correlative Studies

Anticancer activity was assessed as a secondary end point of this study. Change in PSA from baseline (%) was measured over 18 weeks and over all follow-up. PSA response according to PCWG1 (defined as > 50% decline in PSA from baseline measured twice 3-4 weeks apart) was observed in 10 of 19 evaluable patients treated in this study, yielding an estimated PSA response rate of 52.6% (95% CI, 28.8%-75.6%; Figure 1). For these 10 patients, time to initial response ranged between 3 and 13 weeks. The median duration of response for these 10 patients was 20.5 weeks (range, 3-63.4 weeks; Table 3). As per PCWG2 recommendations,12 we report the percentage of change in PSA from baseline to 12 weeks (or earlier for those who discontinued therapy), and the maximum decline in PSA that occurred at any point after treatment (Figure 1). Thirteen patients showed PSA decline from baseline to 12 weeks and until the end of treatment.

There was a significant reduction in the level of GADD45A methylation in buffy coat DNA from day 1 (baseline) to day 6 of cycle 1 (P < .05) and the effect was not related to dose. Six PSA responses were observed among 10 patients (60%) with a postazacitidine GADD45A methylation decrease from baseline. GADD45A methylation data were not available for 1 additional PSA responder (patient 12) because of lack of sample. None of the 4 patients (patients 5, 6, 9, and 13) with an increase in post-azacitidine GADD45A methylation achieved PSA response. The difference in PSA response according to change in methylation (6 of 10 vs. 0 of 4) was marginally significant (P ¼ .085 using Fisher exact test; Figure 2). Free circulating DNA in serum is thought to be derived from tumor cells.13 In 3 patients in whom buffy coat and serum DNA were available, we observed a higher methylation level in serum DNA compared with buffy coat DNA. Demethylation of serum

Figure 1 Prostate-Specific Antigen (PSA) Response in 21 Study Patients. (Top) PSA Waterfall Plots of Changes Over 18 Weeks (Left), and Overall Follow-Up (Right); Solid Lines for Patients Who Reached PSA Reduction of ‡ 50%. (Bottom) Waterfall Plots of PSA Change at 12 Weeks (Left) and of Maximum PSA Change Over All Follow-Up (Right); * Patients Who Discontinued Therapy Before 12 Weeks

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Rakesh Singal et al Figure 2 Effect of Azacitidine Treatment (First Cycle) on Methylation of GADD45A. Difference in Methylation Level is Indicated as Percentage Grouped According to Azacitidine Dose. R Denotes PSA Response According to PCWG1 Criteria. GADD45A Methylation Data at First Treatment Cycle Were Not Available for the Seventh Responder (Patient ID 12) Because of Nonavailability of a Sample

Overall Survival 18 of 22 patients (81.8%) had died. The 4 patients alive at last follow-up experienced progression at 2.6, 3.1, 7.4, and 15.5 months, respectively, from diagnosis and had been followed for 8.3, 14.5, 14, and 54.5 months, respectively. KaplaneMeier estimate of median survival was 19.5 months (95% CI, 13.6-26.4 months), and the estimated survival rates at 6, 12, 18, and 30 months were 90.9% (68.3%-97.6%), 81.8% (58.5%-92.8%), 61.4% (37.2%-78.7%), and 27.9% (10.4%-48.8%), respectively (Figure 3).

Discussion

Abbreviations: GADD45A ¼ Growth Arrest and DNA Damage Inducible Alpha; PCWG1 ¼ Prostate Cancer Working Group 1; PSA ¼ Prostate-Specific Antigen.

DNA corresponded with that observed in buffy coat in these patients (data not shown).

Progression-Free Survival All 22 patients (100%) had disease progression within 24 months. KaplaneMeier estimate of median PFS was 4.9 months (95% CI, 1.4-9.5 months), and the estimated PFS rates at 6, 12, and 18 months were 40.9% (20.9%-60.1%), 27.3% (11.1%46.4%), and 4.5% (0.3%-18.9%), respectively (Figure 3).

Although docetaxel chemotherapy is associated with an OS benefit for patients with mCRPC, the median time to progression remains 6 to 8 months, and OS remains less than 2 years.1,2 Because of limited therapeutic options in patients whose disease progresses during or after docetaxel chemotherapy, new therapeutic approaches that can counteract docetaxel resistance for these patients are urgently needed. The current study evaluated a combination of azacitidine, docetaxel, and prednisone in patients with mCRPC whose disease had progressed during or after a docetaxel-based chemotherapy regimen. Azacitidine 75 mg/m2 administered subcutaneously for 7 consecutive days every 4 weeks improves outcomes in myelodysplastic syndromes.14 Quagliana et al evaluated a phase II study of azacitidine in solid tumors.15 Patients were started at an initial dose of 225 mg/m2 I.V. on day 1 through 5 every 3 weeks. Because of toxicity, the dose was subsequently reduced to 175 mg/m2 and later to 150 mg/m2. Docetaxel is presently approved at a dose of 75 mg/m2 every 3 weeks for the treatment of mCRPC patients. In view of these factors, dose escalation above level IV was not pursued in our study. On completion of the phase I part of our study, the RPTD was azacitidine 150 mg/m2 daily for 5 days and docetaxel 75 mg/m2 on day 6 every 21 days, along with prednisone 5 mg orally b.i.d. continuously. Treatment was well tolerated in most patients. The main toxicity seen in the phase I part of the study was hematological

Figure 3 Progression-Free Survival (A) and Overall Survival (B) in All 22 Phase I/II Patients

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Azacitidine, Docetaxel, and Prednisone in CRPC with a high incidence of Grade 3 or 4 neutropenia. The incidence of Grade 4 neutropenia decreased significantly with the use of growth factor support. In contrast, the incidence of Grade 3/4 neutropenia with docetaxel and prednisone was rather limited when used as a first-line chemotherapy regimen in patients with mCRPC.2 The higher incidence of neutropenia in the present study is likely related to the addition of azacitidine to the therapeutic regimen and possibly diminished bone marrow reserve related to previous chemotherapy and/or radiation therapy. In the phase II portion of the study, the first 6 patients were treated at level IV. The first 5 patients tolerated treatment well. However, the SAE (neutropenic sepsis) leading to death of the sixth patient warranted dose reduction. After the DSMB recommendation to continue the study at dose level II, 1 patient received treatment with no significant side effects. The DSMB recommendation of the use of dose level II was based on the lesser incidence of  Grade 3 AEs at this level. The efficacy of demethylating agents in treatment of prostate cancer as single agent is fairly limited. A small phase II trial had previously evaluated a demethylating agent, decitabine, in 14 men with progressive metastatic prostate cancer after combined androgen blockade and flutamide withdrawal.16 Decitabine was administered intravenously at 75 mg/m2 per dose every 8 hours for 3 doses and repeated every 5 to 8 weeks. 2 of 12 patients evaluable for response had stable disease with a time to progression of > 10 weeks. In another study, chemotherapy naive patients with castration-resistant prostate cancer taking combined androgen blockade and PSA-doubling time (PSA-DT) < 3 months were treated with azacitidine 75 mg/m2 administered subcutaneously on days 1 to 5 of each 28-day cycle up to 12 cycles or until clinical progression or intolerable toxicities.17 A PSA-DT  3 months was attained in 19 patients (55.8%). Fourteen patients had some PSA decline during therapy and 1 patient had a  30% decline compared with baseline. The median clinical PFS was 12.4 weeks. In our study, we observed a significant PSA response, and a favorable PFS, and OS in patients who received a combination of azacitidine, docetaxel, and prednisone in the present study. Because 91% of the patients had disease progression during docetaxel treatment or within 6 weeks of docetaxel treatment, it is likely that pretreatment with azacitidine reversed docetaxel resistance. Biologic hypomethylating activity was suggested by demethylation of the GADD45A promoter in buffy coat DNA. The serial sampling of prostate tumor tissue can facilitate the determination of intratumoral hypomethylating activity, but is problematic because of its invasiveness. Free circulating DNA in serum is thought to be derived from tumor cells.13 In 3 patients in whom both buffy coat and serum DNA were available, we observed a higher methylation level in serum DNA compared with buffy coat DNA. Similar to demethylation seen in buffy coat DNA, we observed demethylation in serum DNA after azacitidine treatment (data not shown), suggesting that treatment with azacitidine might result in demethylation in tumor-derived DNA. No relationship of GADD45A demethylation with azacitidine dose was seen in this study. However, 6 PSA responses were observed among 10 patients who showed demethylation of the GADD45A promoter region after azacitidine treatment and there were no PSA responders among the 4 patients with no demethylation. Therefore,

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it is possible that lack of response to azacitidine and docetaxel might relate to resistance to azacitidine. The mechanism of resistance to azacitidine remains to be defined. The effectiveness of azanucleoside might be influenced by the relative transport capacity of target tissue and nucleoside transporters such as equilibrative nucleotide transporter-1 (ENT-1) might play a role in azacitidine sensitivity.18 Although efficacy was not a primary end point of this study, the frequency of PSA declines and objective response observed is encouraging. One limitation of our study, however, is that most of the responses were observed at dose level IV, which was discontinued toward the end of the study according to DSMB recommendations. Hence, we do not have sufficient data to determine response at dose level II, which was the final recommended dose. However, we did observe demethylation of GADD45A at this dose in buffy coat DNA. Further studies will be required to determine the effectiveness of this treatment in the management of prostate cancer. There is recent evidence suggesting that azacitidine treatment can reverse drug resistance in ovarian cancer.19 Our data suggest reversal of resistance in prostate cancer. Preclinical studies from our laboratory has indicated that pretreatment of cells with azacitidine can reverse chemoresistance in pancreatic and bladder cancer.20,21 Future clinical trials could be designed to test whether azacitidine combined with chemotherapy can reverse drug resistance in other cancers.

Conclusion In summary, the combination of azacitidine 75 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 along with growth factor support and fixed prednisone 5 mg b.i.d. on days 1 to 21 is active in mCRPC patients whose disease progressed during or after docetaxel therapy.

Clinical Practice Points  Methylation-mediated silencing of genes has been shown to

contribute to resistance to docetaxel in prostate cancer.  Our hypothesis was that azacitidine might reverse resistance to











docetaxel by demethylation of these genes. However, the combination of azacitidine and docetaxel had not been evaluated in prostate cancer patients. Our study showed that the combination of azacitidine 75 mg/m2 daily for 5 days followed by docetaxel 75 mg/m2 on day 6 with growth factor support is active in mCRPC patients whose disease progressed during or after docetaxel therapy. Our study provides a novel and effective treatment option for patients with mCRPC previously treated with docetaxel chemotherapy. The results from this study might also help in designing a new clinical trial comparing this regimen to the existing standards of care. Further trials might be designed based on the absence or presence of methylation markers of docetaxel resistance such as the GADD45A gene. Patients who exhibit methylation patterns indicative of docetaxel resistance could be treated with alternate approaches such as a combination of azacitidine and docetaxel chemotherapy to reverse docetaxel resistance.

Rakesh Singal et al Acknowledgments Research support was provided by Celgene Corp, Sanofi-aventis, and Sylvester Cancer Center, University of Miami.

Disclosure The authors have stated that they have no conflicts of interest.

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