DETAILED DISCUSSION OF CANDIDATE GENES IN SEVEN NEW LOCI
(see also Figure 1)
rs1465618 on chromosome 2p21 is in intron 30 of THADA, a gene disrupted by rearrangement in thyroid adenomas and part of the death receptor pathway1, 2. Another SNP, rs7578597 is in exon 24 of THADA and has been associated with type 2 diabetes3. Alleles in TCF2 and JAZF1 have also been shown to be susceptibility loci for both diabetes and PrCa. This locus may provide another example of this phenomenon. rs12621278 on 2q31 is in intron 1 of ITGA6, the gene encoding integrin alpha 6. Integrins control cell attachment to the extracellular matrix and mediate cell proliferation, migration and survival. ITGA6 may also be considered as a potential therapeutic target, as its up-regulation is associated with a metastatic phenotype and an increase in cancer cell motility, including for PrCa4. rs12500426 and rs17021918 on 4q22 are in introns 7 and 9, respectively, of PDLIM55. LIM domain-containing proteins are scaffolds involved in cytoskeleton organization, cell lineage specification, organ development, and oncogenesis. rs7679673 on 4q24 lies 90kb upstream of TET2, coding for a DNA binding zinc finger protein. TET2 is widely expressed, with highest expression levels found in prostate and bone marrow, and mutations of TET2 have been reported in myelodysplasia cases6. rs2928679 and rs1512268 are 90kb apart on 8p21. Thus, they may be markers for the same causal variant, but given that they are in separate LD blocks, they could also indicate the presence of two distinct causal alleles. An interesting candidate gene in this general region is NKX3.1 (rs1512268 is 10kb downstream of NKX3.1), which codes for the androgen-regulated homeobox protein NKX3.1. This is important for maintenance of normal prostate tissue and is expressed at all stages of prostate development and in adult prostate7. Loss of heterozygosity at 8p21 is frequently observed in early PrCa. NKX3.1 is up-regulated by androgens and simultaneous loss of NKX3.1and PTEN is common in PrCa initiation8. NKX3.1stabilises TP53 via Akt independent mechanisms8; it associates with HDAC1, preventing deacetylation and destabilisation of TP53 by HDAC1/MDM2, thereby promoting apoptosis. Of note, HDAC inhibitors have been developed as targeted therapies and are currently in trial in metastatic PrCa9. NKX3.1also downregulates expression of PSA by PDEF10 (an epithelium-specific Ets transcription factor, which plays a role in PrCa), therefore loss of NKX3.1results in increased PSA expression. rs7127900 is in a region on 11p15 that includes several plausible candidate genes: IGF2, IGF2AS, INS and TH. The first three are members of the insulin family of polypeptide growth factors11. A SNP in H19, a regulator of IGF2 that lies telomeric to this region, has been associated with breast cancer risk12. rs5759167 lies on chromosome 22q13, for which evidence of linkage to PrCa has been found13,14,15. This SNP is ~7Mb downstream from the previously reported linkage peak. There are several genes of interest in the LD block (see figure 1).
SUPPLEMENTARY TEXT: DESCRIPTION OF THE PRACTICAL CONSORTIUM GROUPS
Subjects were included from 21 studies comprising PrCa cases and controls: eight from Europe, nine from North America, one from China, and three from Australia. These comprised 16, 332 PrCa cases and 16, 344 male controls. Details are shown in supplementary Table 4. Some have previously been described as part of work in the PRACTICAL Consortium16. The Mayo Clinic and Utah studies oversampled cases from multiple case families and only one case per family was genotyped. Three of the studies contained men of Asian ancestry (China and Japan); six of the studies contained men of African American ancestry and two studies contained men of Latino (Hispanic) origin; one study was from Hawaii (see Figure 2). The remaining studies were predominantly of men of European ancestry. All studies have the relevant IRB approval in each country in accordance with the principles embodied in the Declaration of Helsinki. Details of each study set are given below, and a summary of the studies is given in Supplementary Table 4.
BiPAS: The Birmingham Prostatic Neoplasms Association Study (BiPAS) – A Genetic and Environmental Case Control Study
The Birmingham Prostatic Neoplasms Association Study base consists of men living in the south Birmingham area, United Kingdom aged 50 years. The study recruited men with lower urinary tract symptoms (LUTS) and/or high serum prostate specific antigen (PSA) levels referred for prostate biopsies between March 2007 until October 2008. PrCa cases were recruited from the Queen Elizabeth Medical Centre, Birmingham. Cases are defined as men with histologically confirmed adenocarcinoma of the prostate. Controls were also recruited from the Queen Elizabeth Medical Centre and SellyOakHospital, Birmingham. Men with a normal repeat PSA and a negative biopsy were categorized as benign controls.
A blood sample from every hospital based subject was obtained using standard venepuncture methods, and collected in a 5ml tube containing EDTA. Samples were transported to the laboratory immediately in a cool bag with cool packs and stored at 4°C. DNA was extracted using the QIAGEN maxi blood kit.
CHSH: China
All samples were from men of Chinese Han origin from Shanghai and its surrounding city. Patients with PrCa enrolled in the study were diagnosed by transrectal ultrasonographic prostate biopsy, and confirmation of the pathologic diagnoses for those who underwent radical prostatectomy. They were from two hospitals; Changhai hospital and ChangzhengHospital in Shanghai, China.
Controls are hospital based and are from other clinical divisions which are treating non cancer patients; these have a PSA level of < 3.0ng/ml, and are age matched (+/-3 years).
FHCRC: FredHutchinsonCancerResearchCenter, SeattleUSA
The study population consists of participants from two population-based case-control studies in Caucasian and African American residents of King County, Washington (Study I and Study II), which have been previously described. Incident cases with histologically confirmed PrCa were ascertained from the Seattle-Puget Sound Surveillance, Epidemiology and End Results cancer registry. In Study I, cases were diagnosed between January 1, 1993, and December 31, 1996 and were 40-64 years of age at diagnosis. In Study II, cases were diagnosed between January 1, 2002, and December 31, 2005 and were 35-74 years of age at diagnosis. Overall, 2,244 eligible PrCa patients were identified and 1,754 (78%) were interviewed. Blood samples yielding sufficient DNA for genotyping were drawn from 1,457 (83%) cases who completed the study interview.
A comparison group of controls without a history of PrCa, residing in King County, Washington, was identified for each study using random digit telephone dialing. Controls were frequency-matched to cases by five-year age groups and recruited evenly throughout each ascertainment period for cases. A total of 2,448 men were identified who met the eligibility criteria and 1,645 (67%) completed a study interview. Blood samples were drawn and DNA prepared from 1,352 (82%) interviewed controls.
FMHS: Michigan, US
The Flint Men’s Health Study (FMHS) is a community-based, case-control study of PrCa in African American men living in Genesee County, MI, US conducted from 1996 to 2002. Controls were recruited from a probability sample of African American men aged 40-79 years with intentional over-sampling from older age groups. Cases were identified from the Genesee County Community-Wide Hospital Oncology Program registry. Participants provided blood samples from which DNA and serum were isolated and completed detailed interviews which addressed potential risk factors for PrCa, urinary symptoms, PrCa screening history and general medical history, socio-economic factors, and access to and use of health care. Additionally, controls underwent urological examinations and PSA screening and cases provided access to medical records pertaining to their PrCa diagnoses. A total of 383 controls participated in all portions of the study. Nineteen of those controls were diagnosed with PrCa during the time of the study and subsequently recruited as cases. DNA is currently available for 356 of the remaining controls. A total of 136 cases participated in all portions of the study and DNA is currently available for 133 cases.
HaPCS:Hannover, Germany
A hospital-based series of 499 unselected Caucasian patients with PrCa who were treated with brachytherapy between October 2000 and September 2007 at Hannover Medical School, were enrolled for this study17. All patients had biopsy-proven adenocarcinoma of the prostate. Indication for permanent brachytherapy was clinically localized low risk early PrCa (cT2a or less with a PSA serum level < 10 ng/ml and a Gleason score < 7) following the European Society for Therapeutic Radiology and Oncology/European Assocation of Urology/European Organization for Research and Treatment of Cancer recommendations. The median age at diagnosis was 67 years in this patient series (range 42-82 years). For comparison, a series of 504 genomic DNA samples was established from ethnically matched adult male blood donors at HannoverMedicalSchool in the period from 2006-2007.
MAYO: The MAYO clinic: Rochester, Minnesota, USA
The Mayo Clinic study consisted of hospital-based cases, including 476 affected men from 185 families with PrCa, 445 men with sporadic PrCa, 199 with aggressive (Gleason score > 7) PrCa, and 500 population-based controls. The controls (all males) were randomly selected from a sampling frame of Olmsted County, Minnesota, provided by the Rochester Epidemiology Project. The methods used to ascertain familial and sporadic PrCa patients, as well as controls, have been described previously18. All individuals from the Mayo Clinic study included in this report were of self-reported European descent.
MCCS: Melbourne Collaborative Cohort Study, Melbourne, Australia
MCCS/RFPCS/EOPCFS: Cancer Council Victoria, Melbourne, Australia
The Melbourne Collaborative Cohort Study (MCCS) is a prospective cohort of 17,154 men aged 40 to 69 years at recruitment in 1990-4. MCCS participants were diagnosed with PrCa during follow-up to mid 2006 and were ascertained through linkage with the Victorian Cancer Registry and the National Cancer Statistics Clearing House that includes diagnoses from other States in Australia. The Risk Factors for Prostate Cancer Study (RFPCS) is a population based case-control study that in the period 1994-1997 recruited through State Cancer Registries men resident in Perth and Melbourne diagnosed with prostate cancer at age less than 70 years. The Early-Onset Prostate Cancer Family Study (EOPCFS) is a population-based study of prostate cancer in men diagnosed at age less than 56 years ascertained through the Victorian Cancer Registry. Controls for stage 3 were a random sample of the MCCS participants that were not diagnosed with PrCa during follow-up. All study subjects were of Caucasian origin. Participants in the three studies whose samples were used for stage 2 were excluded from stage 3.
MEC: The Multiethnic Cohort Study
The Multiethnic Cohort Study19is apopulation-based prospective cohort study that was initiated between1993 and 1996 and includes subjects from various ethnic groups -African-Americans and Latinos primarily from California (mainly LosAngeles) and Native Hawaiians, Japanese-Americans, and EuropeanAmericans primarily from Hawaii. State drivers’ license fileswere the primary sources used to identify study subjects in Hawaii andCalifornia. Additionally, in Hawaii, state voter's registration fileswere used, and, in California, Health Care Financing Administration
(HCFA) files were used to identify additional African American men.All participants (n=215,251) returned a 26-pageself-administered baseline questionnaire that obtained generaldemographic, medical and risk factor information. In the cohort,
incident cancer cases are identified annually through cohort linkage topopulation-based cancer Surveillance, Epidemiology, and End Results(SEER) registries in Hawaii and Los AngelesCounty as well as to theCaliforniaState cancer registry. Information on stage and grade ofdisease are also obtained through the SEER registries.Blood sample collection in the MEC began in 1994 and targetedincident PrCa cases and a random sample of study participantsto serve as controls for genetic analyses. This nested PrCacase-control study in the MEC consists of 2,792 invasive PrCacases and 2,377 controls. This study was approved by the Institutional
Review Boards at the University of Southern California and at theUniversity of Hawaii and informed consent was obtained from all studyparticipants.
MOFFITT: Moffitt Study, Tampa, Florida, US
This is a hospital-based incident study of 646 patients with primary adenocarcinoma of the prostate (560 whites, 55 African Americans, 28 white Hispanics and 3 others). They were recruited from 2002 to 2007 at the H. Lee Moffitt Cancer Center (Tampa, FL, US) and JamesA.HaleyVeteransAffairsHospital (Tampa, FL, US). Ninety-five percent of the case subjects who were asked to participate in the study agreed. All cancer cases were histologically confirmed by the Department of Pathology at each institution.
The controls consisted of 320 subjects (302 whites, 10 African Americans, 6 white Hispanics and 2 others) who were visiting the LifetimeCancerScreeningCenter, which is affiliated with the H. Lee Moffitt Cancer Center. At this center, routine screenings are offered to men for cancers of the prostate, colorectum, and skin. Men could have been self-referred or directed for screening by their primary healthcare provider. All control subjects were male and had had no previous diagnosis of cancer. The control subjects were frequency matched to the patients by age at diagnosis (± 5 years). Eighty-three percent of the control subjects who were asked to participate in the study consented.
Non-genetic risk factor data for the present study were obtained through in-person interviews with the patients and controls at enrollment. The questionnaire covered demographic information, family history of cancer (ie, whether they have one or more first-degree family member with PrCa), medical history, and detailed tobacco consumption. For the patients, data on cancer stage, Gleason score, and prostate specific antigen level were abstracted from the medical records. The subjects were asked to provide a blood or buccal sample after the interview as a source of genomic DNA20.
NC_CCPC: San Francisco, California USA
This population-based case-control study of advanced PrCa in non-Hispanic white and African-American men was conducted in the San Francisco Bay Area. Newly diagnosed cases aged 40-79 years were identified through the regional cancer registry, which is part of the Surveillance, Epidemiology and End Results (SEER) Cancer Registry Program. Non-Hispanic white cases were diagnosed between July, 1 1997 and February 28, 2000, and African-American cases were diagnosed between July 1, 1997 and December 31, 2000. Overall, 1,015 patients with a first primary advanced PrCa were identified. Of these, 106 were deceased at the time of contact, 33 were enrolled in another study and thus not available, 12 were declined contact by their physician, and 76 no longer lived in the San FranciscoBay area or did not meet other eligibility criteria. Of 788 eligible cases contacted, 568 (72%) completed the interview and 533 (68%) provided a biospecimen sample. DNA from blood samples was available for 389 cases.
Non-Hispanic white and African-American population controls aged 40-79 years were identified through random-digit dialing. In addition, controls aged 65-79 years were randomly selected from the rosters of beneficiaries of the Health Care Financing Administration (HCFA). Controls were frequency-matched to cases by five-year age group and race. Of 1,081 controls selected into the study 16 were deceased at the time of contact, 41 had a history of PrCa, and 156 did not meet other eligibility criteria. Of 868 eligible controls contacted, 545 (63%) completed the interview and 525 (60%) provided a biospecimen sample. DNA from blood samples was available for 256 controls [John et al., 2005].
PCMUS: Bulgaria
The Bulgarian sample of PrCa patients consist mainly of newly diagnosed cases, which are histopathologically confirmed. The patients (N=114, age range 51-91) are of Bulgarian origin. Transrectal biopsy was performedat the Urology Clinic, AlexandrovskaUniversityHospital, mainly because of an elevated PSA. Some of the patients were referred from other centers to the tertiary university hospital after being previously diagnosed with PrCa. A small subset of patients had previously had definitive treatment (mainlyradical prostatectomy), and they werecalled retrospectively for invitation to join the study.The control group is matched to the patients by sex, age, and ethnicity. It consists of two groups: (i) 96 healthy males, age range 51-86, presenting to our institution with lower urinary tract symptoms caused by benign prostatic hypertrophy (BPH) who had a PSA <3.5. The majority of themsubsequently underwent surgical treatment with histological verification of the BPH; (ii) an additional healthy control group of 110 anonymous males matched to the PrCa patients by age and ethnicity, but with no PSA data.
All participants gave written informed consent and anonymous controls have been selected from the available DNA bank at MolecularMedicineCenter, Medical University Sofia.
PENN, (SCORE), University of Pennsylvania, Philadelphia, US
Incident PrCa cases were identified through Urologic Clinics between 1995 and 2008. Case status was confirmed by medical records review using a standardized abstraction form. Cases were excluded from this study if they reported having exposure to finasteride (Proscar) at the time of their PrCa diagnosis. Patients who were non-incident cases (i.e., those diagnosed more than twelve months prior to the date of study ascertainment), or had a prior diagnosis of cancer at any site except non-melanoma skin cancer, were also excluded.
Risk factor, medical history, PrCa screening history, and PrCa diagnostic information was obtained by using a standardized questionnaire and review of medical records. Information collected included personal history of benign prostatic hyperplasia (BPH) and vasectomy, previous cancer diagnoses, and demographic information, and PrCa screening history. Existence of BPH was confirmed by medical records review.
Genomic DNA for the present study was self-collected by each study participant using sterile cheek swabs(Cyto-Pak Cytosoft Brush, Medical Packaging Corporation, Camarillo, CA), and processed using either a protocol modified from Richards et al. (1993)21 as described previously22 or using a modified protocol on the Qiagen 9604B robot with the QIAamp 96 DNA Buccal Swab Biorobot Kit (Valencia, CA).
ProtecT, UK
ProtecT [ Donovan et al, 2003] is a national study of community-based PSA testing and a randomised trial of subsequent PrCa treatment. Approximately 200,000 men between the ages of 50 and 69 years, ascertained through general practices in nine regions in the UK, were approached and 100, 000 were recruited. Men known to be non-white were excluded. For this study, 1800 cases identified by PSA screening within the ProtecT study were analysed. Controls (1800) with normal PSA levels (<3ng/ml) were selected from the same GP register and 5 year age band as the cases.