Phase II trial of isoflavone in prostate-specific antigen recurrent prostate cancer after previous local therapy

Sample size determination to detect a reduction of 10% in PSA from initial levels [actual calculation as log₁₀ (PSA) with a standard deviation of 37% of PSA level] required 27 patients to have a significance of p = 0.05 and a power of 60%. However, due to poor accrual, the study was closed after the enrollment of 20 patients. With the sample size of 20, a reduction of 12% in PSA can be detected with the same significance level and power.

Twenty patients were enrolled in this study from May 2004 to January 2007. Eligibility criteria included: able to give informed consent, adenocarcinoma of the prostate (no small cell component), and prior treatment with radiation (at least 6200 cGy, n = 9) or radical prostatectomy (n = 11) for clinically localized disease (clinical T1 or T2). Table 1 shows the demographic, clinical, and pathologic characteristics of the study cohort. All patients had demonstrated evidence of biochemical failure within a median of 6 ± 3 years after treatment. Biochemical failure was defined as a) detectable and rising PSA after surgery or b) three consecutive PSA rises after radiation therapy [11].

Patients could not have demonstrable and/or histologically confirmed metastatic or locally recurrent disease demonstrated on bone scan, computed tomography or transrectal ultrasound, or be clinically symptomatic at the time of enrollment. Patients could have received androgen deprivation therapy (ADT), but not within 12 months of entry into the study. In addition, patients had to have a life expectancy of at least one year and performance status of <2 of Zubrod scale. Patients with a known allergic reaction to milk or soy products were excluded.

Twenty men who have had evidence of biochemical relapse after radiation therapy and/or prostatectomy comprised the study cohort. These patients ingested Soy Dream Enriched, Original or Vanilla, soy milk which provided 47 mg of isoflavonoid per 8 oz serving. The patients received three 8 oz servings per day in an open label, nonblinded fashion. No dose escalation or reduction was planned. Compliance was assessed by counting empty soy milk containers and verified by presence of soy components in serum samples.

Pretreatment evaluation included a complete medical history, physical examination (including digital rectal examination), serum PSA, free/total testosterone, lipids, serum isoflavone levels (genistein, daidzein, and equol), and assessment of quality of life (Functional Assessment of Cancer Treatment-Prostate, FACT-P questionnaire). Furthermore, whole blood was obtained prior to initiation of the study to assess for DNA polymorphism. Follow-up serum PSA levels to assess efficacy were obtained at 3, 6, 9, and 12 months after initiation of treatment. Medical history, physical examination, serum testosterone, lipids, isoflavone, and quality of life were assessed at 6 and 12 months after initiation of treatment.

Quality of life prior to and during therapy was measured by the FACT-P questionnaire, which consists of 38 items calculated to evaluate functional impairment and the perceived effect of that impairment on quality of life. The instrument assesses overall and specific aspects of quality of life in five domains (i.e. physical, functional, social, emotional, and prostate related). These include self-reported ability to perform normal physical and social activities, attitude towards self and future, level of physical well-being, and quality of support from friends, family and health care providers. The measure was used to assess the impact of the dietary intervention on quality of life.

At least three pretherapy serum PSAs were available for each subject to compute pretherapy PSA slope. Next, PSA had been measured within days of the time the patient was enrolled in the study. This level was used as study entry level. PSA serum level was measured again 3 months after enrollment in the study, after 6 months, and after 12 months. PSA outcome was evaluated in two ways: a) how response changed over time [i.e. slope of line log (PSA) study entry versus 3 months, versus 6 months, versus 12 months of treatment], and b) as a change on the calculated PSA doubling time before treatment versus PSA doubling time after 3 months, 6 months, and 12 months of treatment. PSA doubling time was calculated using the following formula: PSA doubling time = log 2 × t/[log(final PSA) - log (initial PSA)]. Because pretherapy PSA levels were compared to posttherapy PSA levels, each subject could serve as his own control.

Patients were allowed to continue on therapy until a) they exhibited evidence of PSA progression (as defined by two successive increases in their PSA with an absolute increase of at least 30% above baseline), b) they had clinical evidence or radiographic evidence of distant metastases, or c) patient/physician opted to stop therapy.

A complete LC-MS analysis of patient serum samples for genistein, daidzein and equol (total and aglycone) was performed. The serum samples were both sulfate and glucuronide conjugates which requires hydrolysis with a mixed β-glucuronidase and sulfatase enzyme preparation (Helix pomatia; Sigma Chemicals, St Louis, MO) to determine total versus free isoflavone. For each sample, an aliquot was analyzed directly for free isoflavones, while another was enzymatically hydrolysed to determine total isoflavones. Quantitative isoflavone analysis was accomplished using reversed-phase HPLC with UV and mass spectral detection in series [12]. Briefly, samples were prepared for analysis by mixing with ammonium acetate buffer and formic acid prior to multiple extractions in ethyl acetate. Pooled extracts were dried under nitrogen and resuspended in 0.5 ml of 50:50 acetonitrile: 0.2% aqueous formic acid. Samples were then injected onto an Apollo C18 column (Alltech, Deerfield, IL) with Alltech Adsorbosphere HS C18 guard column in a Hewlett-Packard 1100 series liquid chromatograph system (Wilmington, DE). Separation occurred under a linear gradient with mobile phase B (0.1% formic acid in acetonitrile) increasing from 40% to 60% over 60 min. Mobile phase A was 0.1% formic acid in water. The flow rate was 0.40 ml/min with column temperature of 40°C. Free isoflavones and deconjugated glucosides were detected for quantitation using an HP variable wavelength UV detector at 260 nm and quantified against an external standard series. Identification was confirmed using the Finnigan LCQ Ion Trap Mass Spectrometer (Finnigan MAT, San Jose, CA) in positive ion mode with electrospray ionization. Extraction efficiency was determined using the isoflavone biochanin A, with phenolphthalein-glucuronide as deconjugation surrogate.

Whole blood was obtained from patients and DNA extracted utilizing QiAamp DNA blood kit (Qiagen, Valencia, CA). Nested PCR reaction was utilized to amplify the CAG polymorphism on the AR gene. Primers for AR gene were designed using Primer Express software (PE-Applied Biosystems). Outside primers for CAG was constructed: 5'-GTGCGCGAAGTGATCCAGAA-3' and 5'-TCTGGGACGCAACCTCTCTC-3' and inside primers 5'-AGAGGCCGCGAGCGCAGCACCTC-3'-fam and 5'-GCTGTGAAGGTTGCTGTTCCTCAT-3'. The inside forward primer was labeled with fluorescent 6-carboxy-fluorescein (FAM). The first PCR reaction consisted of 17 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 30 sec). The nested PCR used 1 μl of the first PCR product and amplified further for 28 cycles (94°C for 1 min, 66°C for 1 min, and 72°C for 1.5 min). Screening for AR mutations was performed using single-strand conformation polymorphism (SSCP) of PCR amplified AR genomic DNA. The radio-labeled PCR fragment identified above was analyzed by nondenaturing polyacrylamide gel electrophoresis [13]. Fragments showing an aberrant PCR-SSCP pattern on gel were subjected to direct DNA sequencing to document the exact nucleotide base deletion, addition, or change.

A mixed regression model was used to compare the slope of PSA after study entry to that before study entry. The PSA levels for a given patient were treated as repeated measurements and the uniform correlation between PSA measurements was assumed. The slopes before and after study entry were globally compared using all data points in the mixed model. In addition, the slope of PSA after study entry was compared to that before study entry for each individual patient by regular regression analysis. The PSA doubling time before study entry was computed by tlog2/[log(PSA at entry) - log(initial PSA)] and the PSA doubling time after study entry was computed by tlog2/[log(last PSA) - log(PSA at entry)]. Note that the PSA doubling time can be negative if the denominator is negative. The PSA doubling times before and after study entry were compared by the sign test based on paired data. The Wilcoxon signed rank test was performed for other chemistries (Table 2) and QOL. The correlation between AR gene CAG polymorphisms and PSA levels at 12 months after study entry was evaluated by the Spearman's correlation coefficient. All reported p-values were 2-sided. All data were analyzed using SAS version 9.1.3 software.

Twenty men with biochemical recurrent prostate cancer were enrolled. Table 1 shows the demographic, clinical, and pathologic characteristics of the cohort. Compliance was estimated to be high with only two subjects noted not to ingest the required amount of soy milk. Six men did not complete the study: 1 due to side effects, 2 were lost to follow-up, and 3 began ADT. One of the men who had initiation of ADT did so despite no significant increase in serum PSA. The other two patients had a significant increase in serum PSA (PSA doubling time < 3 months). The two non-compliant patients were among the six men who dropped out of the study.

Six patients achieved 8%, 9%, 20%, 23%, 30% and 70% reduction in PSA after the treatment, respectively. In addition, one patient had no change in PSA. The remaining 13 patients had increased PSA.

The slope of PSA after study entry was also compared to that before study entry for each individual patient. It was found that the slope of PSA after study entry was significantly lower than that before study entry in 6 patients (p < 0.05) and the slope of PSA after study entry was significantly higher than that before study entry in 2 patients (p < 0.05). For the remaining 12 patients, the change in slope was insignificant (Figure 1). In two drop-outs, the PSA slopes after study entry were significantly higher than that before study entry; in other two drop-outs, the PSA slopes after study entry were significantly lower than that before study entry; in the remaining two drop-outs, the differences in PSA slope were not statistically significant. Drop-outs are noted by "D" on Figure 1.

Free testosterone decreased while on therapy (median 10.3 vs. 9.7 ng/ml, p = 0.031). However, neither total testosterone nor cholesterol level was significantly manipulated during isoflavone therapy (Table 2).

Median total genistein, daidzein, and equol prior to the start of therapy were <0.002, 0.012, and 0.023 μg/ml, respectively. Serum isoflavone levels measured at 6 months are depicted in Table 3. Among 16 men with available data, 12 men (75%) demonstrated significant (> 2 × CTL, CTL = 0.023 μg/ml) levels of total equol and 10 of 16 (63%) had significant (> 2 × CTL, CTL = 0.009 μg/ml) levels of free equol present in serum. In the study cohort, median genistein level was 0.524 μg/ml (range < 0.002 to 2.071), median daidzein levels was 0.468 μg/ml (range 0.005 to 0.817), and median equol level was 0.115 μg/ml (range <0.001 to 0.523). Median free genistein level was 0.012 μg/ml (range <0.002 to 0.019), median free daidzein level 0.016 μg/ml (range 0.003 to0.434), and median free equol level was 0.040 μg/ml (range <0.001 to 1.328). The correlations of free equol and total equol with PSA level at 12 months were 0.11 and 0.39, respectively (p-values = 0.71 and 0.19). Correlations of genistein (free and total) and daidzen (free and total) with PSA level at 12 months were not significant either.

There were 9 documented AR gene CAG polymorphisms in our cohort (Table 3). Eight men had ≤ 20 CAG repeat. A longer CAG polymorphism was associated with a lower PSA level at 12 months after study entry (Spearman's correlation coefficient = -0.63, p = 0.05) and a slower rise in PSA over time (Figure 2).

There was no significant difference in the five domains of the FACT-P between pretherapy and on therapy (Figure 3). Side effects were minimal. One patient of 20 (5%) reported diarrhea and withdrew from the study. No other side effects were reported.