Can Dietary Phytoestrogens Slow Down Prostate Tumor Proliferation?
Can Dietary Phytoestrogens Slow Down Prostate Tumor Proliferation?
The purpose of this study is to evaluate if a diet with a high content of phytoestrogens can
 slow down the prostate tumor proliferation. Phytoestrogens are found in food items such as
 soy, rye, and seeds.

 Two hundred thirty men with prostate cancer will be included in the study and followed until
 surgery (at least 6 weeks). Half of the study participants will receive general information
 about healthy food choices and a package of foods with high content of phytoestrogens to add
 to their food. The other half will get the same information but not receive the food-package.
Data from ecologic and experimental studies clearly show a protective effect of dietary
 phytoestrogens against prostate cancer. Genetic factors are also of etiologic importance in
 prostate cancer and there is growing evidence for the importance of a gene-diet interaction
 in prostate cancer progression. The investigators have recently found a putative genetic
 interaction for this protective role of phytoestrogens. The overall decreased risk of
 prostate cancer in men with a high intake of phytoestrogens was strongly modified by a
 nucleotide sequence variant in the estrogen receptor-beta (ERß) gene. An American study found
 a similar interactive effect between intake of phytoestrogens, as well as body mass index
 (BMI), and another nucleotide sequence variant in ERß.The phytoestrogens isoflavonoids and
 coumestans bind tightly to the estrogen receptor-beta (ERß) and are mainly found in soy and
 other beans. ERß expression has been found to be involved in progression of prostate cancer,
 suggesting that phytoestrogens may interact with ERß in the development of prostate cancer.
 With regard to potential carcinogenic mechanisms, phytoestrogens may be involved in the
 endocrine control of prostate cell growth by influencing the balance between AR and
 ERß.Testosterone and its metabolite 5α-dihydrotestosterone (DHT) cause proliferation of
 prostate epithelial cells through binding to the AR. In contrast, by binding to ERß, 5α
 androstane-3ß,17ß-diol (3ßAdiol), a metabolite of DHT, represses the expression of AR and
 thereby inhibits androgen-driven proliferation while promoting cell differentiation. In terms
 of proliferation, current data suggests a combined stimulatory role of ERα and AR in the
 prostate whereas ERß inhibits proliferation and stimulates differentiation. Both estrogen ERα
 and ß have affinity for estradiol whereas phytoestrogens and 3ßAdiol selectively activate
 ERß. Phytoestrogens should, as a result, be able to restrict cancer growth by acting as a
 substitute for 3ßAdiol. This is confirmed by experimental studies.

 Results from previous intervention studies have given promising results indicating that
 certain dietary or lifestyle modifications can influence the progression of prostate cancer,
 although most of these studies have been of fairly short of duration and based on relatively
 small numbers of patients. For example, a randomised study showed that men who were given a
 dietary addition of flaxseeds had a lower percentage of positive cells containing the
 proliferation marker Ki-67 than men who had not received this addition to their diet. Taken
 together, the present evidence points towards the possibility for prostate cancer patients to
 complement with lifestyle-related treatment alternatives to prevent or delay tumor
 progression.

 Specific aim: To perform a controlled randomized dietary intervention study in men with
 prostate cancer. The investigators will pursue the following specific aims/hypothesis:

 1. "Among men with prostate cancer of intermediate risk-level, a diet high in
 phytoestrogens will reduce tumor progression compared to men with a diet low in
 phytoestrogens".

 2. "The effects of a diet high in phytoestrogens on prostate cancer tumor progression
 differ between men with different genotypes of polymorphisms in the ERß gene". The men
 will be divided into two subgroups, those bearing TT or TC/CC alleles, of the
 single-nucleotide polymorphism (SNP) rs2987983-13950 T/C in the ERß-gene, in which the
 hypothesis will be tested.

 3. To determine whether RNA expression of the AR, ERα and ERβ in prostate tumor tissue, as
 well as levels of steroid hormones, differs between men with a diet high and low in
 phytoestrogens, respectively, or between different genotypes of the ERβ gene.

 4. To determine whether Cyclic citrullinated peptide (CCP) gene expression in prostate
 tumor tissue differs between men with a diet high and low in phytoestrogens,
 respectively, or between different genotypes of the ERβ gene.

 Study protocol: Trough treating physicians, the investigators will identify 240 men in the
 Västra Götaland healthcare region who have been diagnosed with prostate cancer of
 intermediate risk-level (T1-T2, Gleason score <8, PSA<20)scheduled for radical prostatectomy.
 The clinic will be informed of which patients have been enrolled in the study, and the
 surgery for these patients will be scheduled six weeks ahead of time from the date of
 inclusion. Once a patient has agreed to participate, he will be randomized to one of the two
 intervention arms. The patient will then receive additional information depending on which
 group he belongs to and will be asked to donate a blood sample. Under normal clinical
 routine, data on all prostate cancer cases is collected in the National Prostate Cancer
 Register (NPCR), for example information on Gleason score, tumor volume, Prostate-specific
 antigen (PSA) levels. The investigators will collect information from the registry in
 connection with the analysis of data.

 Intervention: Patients will be introduced to the intervention diet by a dietitian and will
 receive general information about healthy food choices (according to recommendations for the
 general public) and not to eat any dietary supplements, no other dietary restrictions will be
 given. The patients will be given a package containing food with a high amount of
 phytoestrogens (≥100 mg isoflavonoids and ≥100 mg lignans) that are to be consumed daily. The
 intervention continues until the day before the surgery (at least 6 weeks). The patients in
 the control group will be informed in the same way as the intervention group except that they
 will not receive any food-package.

 Endpoint: The effect of the diet on tumor proliferation rate will be measured by making
 comparisons of 1) percentage of positive cells containing the marker Ki-67, 2) PSA-levels, 3)
 CCP-score in the two groups. Tumor tissue from prostate biopsies taken during surgery (the
 end of the study) and the most recent biopsy taken before the beginning of the study will be
 collected. Blood samples will be taken at the beginning of the study, at the day before the
 surgery, and 3 months after surgery, and send to analysis of PSA (free and total). The
 absolute differences in PSA levels and the PSA doubling time will be calculated. In addition
 the investigators will measure the expression of AR, ERα and ERß in tumor tissue and the
 blood levels of hormones, testosterone, DHT, estradiol and 3ß-Adiol.

 Questionnaire: All patients will answer a password-protected questionnaire at baseline,
 surgery, and after 3 months, weight and height will be measured. Once during the 6-weeks
 period a 24-h recall interview will be performed, in which participants food intake during
 the preceding 24 h will be registered. The individual intake of energy and specific nutrients
 will be estimated by linking information from the questionnaire and diet record to the
 National Food Agency's nutritional database and our previously developed phytoestrogen
 database.

 Analysis of polymorphs:SNP in the ERß-gene will be analysed in blood samples, using the
 PCR-based method competitive allele specific PCR (KASP™). The allele-constitution will be
 identified and participants will be assigned to group TT, TC or CC. Analysis of Ki-67: The
 biopsy sample is fixed by immersion with 4 % paraformaldehyde in 0,1 M phosphate buffer
 during 24hours at +4°C. Then it is dehydrated with graded ethanol followed by xylene and
 paraffin embedding. Paraffin sections are heated for 30 minutes at 60ºC, pre-treated with
 citric acid buffer. Unspecific binding is blocked by donkey serum in phosphate buffered
 saline (PBS) containing Triton X-100 for 30 minutes. Incubating with Ki-67 antibody over
 night (4ºC) followed by incubation with the appropriate secondary biotinylated antibody. To
 enhance the signal, the sections are treated with avidin-biotin-complex solution, and to
 visualize the immunoreactivity Diaminobenzidine (DAB) is used. Stained tumor cells (Ki-67
 positivity) will be counted and the result will be reported as the ratio of positive nuclei
 divided by the total number evaluated × 100.

 Analysis of receptor-expression, CCP score, hormone levels and PSA: Tissue samples taken at
 surgery is placed in RNA-later before being frozen. The samples will be analysed using RNeasy
 plus Universal Mini Kits (QIAGEN), and Real-time polymerase chain reaction (PCR). ERα, ERβ
 and AR mRNA expression will be determined using TaqMan assay. For analysis of CCP score, the
 pathologist select regions of carcinoma and RNA will be extracted as above; thereafter gene
 expression will be assessed according to the manufactures (Prolaris®) instruction. PSA-blood
 levels will be analysed according to standard clinical protocol.

 Power calculation: In the Västra Götaland healthcare region in 2012, 321 men had intermediate
 risk-level prostate cancer for which they received curative treatment. Calculation using the
 primary outcome (Ki-67) gives for a study group consisting of 118 patients an 80 % power for
 a two-sided test with a level of significance at 0,05 and effect size of 0,5. The
 investigators found in earlier studies that approximately 42 % of the male population are
 heterozygous or homozygous for the variant allele (TC/CC) of the ERβ promoter region SNP
 (rs2987983-13950 T/C) and 58 % homozygous for the wild type allele (TT). Since the
 investigators expect the effect of the intervention to be greater among subjects with the
 variant allele, while smaller among subjects with the wild type allele, this latter group
 ought to be large enough to be able to find a difference if one exists. Thus a total sample
 size of 118/58%=203 patients is needed.

 Statistical Considerations: The investigators will utilize the intention-to-treat method. In
 the design and data collection testing of drugs as the template have been made
 (randomization, placebo, blinding, no attrition, no differential measuring errors, correct
 analysis); for the deviations from the perfect situation the investigators use
 epidemiological theory for guiding the analyses and interpreting the results. Preliminary
 analyses, based on means, medians, standard deviations and interquantile range will be
 provided to properly describe the outcomes of interest and stratification will be performed
 to assess the performance of the randomization of the assigned diet. Box-plots will therefore
 be produced to graphically evaluate the temporal trends in the response. The distribution of
 the outcomes will be studied and appropriate transformations will be applied, if needed, to
 improve symmetry and normality. Due to the longitudinal structure of the data, correlation
 among observations will be incorporated and linear mixed effects models will be carried out
 to study if (i) there is a temporal trend (ii) if the linear trend differs between treated
 and untreated individuals (iii) if the treatment effect between the intervention group and
 the control group is modified according to ERß genotypes, both for the common and the
 variant. A key step in analysis of longitudinal data is to identify the appropriate
 covariance structure that describes the correlations among the data points: independent
 covariance (no correlation), exchangeable covariance (equal correlation) or autoregressive
 covariance with correlation between responses decreasing with time between measurements. The
 investigators will identify the most appropriate structure, verified empirically with help of
 an information theoretic approach. Both statistical tests and confidence intervals will be
 produced to assess the significance of the effects, partial F test procedures will therefore
 be used to study the joined effect of main and interaction variables and appropriate
 statistical methods will be applied to study model fitting.
Inclusion Criteria:

 -  men in the Västra Götaland healthcare region

 -  diagnosed with prostate cancer of intermediate risk-level (T1-T2, Gleason score <8 and
 PSA<20)

 -  scheduled for radical prostatectomy

 Exclusion Criteria:

 -  ongoing hormonal treatment.

 -  other difficult physical or psychological conditions

 -  diminished cognitive function

 -  allergy to soy

 -  having taken dietary supplements regularly (daily) during the preceding three months

 -  does not understand written Swedish