Introduction to P-Mark
In Europe, prostate cancer (Pca) is the second most frequent lethal malignancy in men. Yearly about 40,000 men die of Pca in the EU countries. There is a slow increase of mortality and in addition, due to an ageing population, a 50% increase in incidence is expected by the year 2020. So far, the only chance for cure is early detection and treatment by either surgery or radiotherapy. Diagnosis of Pca is made by ultrasound guided transrectal biopsy of the prostate for histology. An increased level of the serum marker prostate specific antigen (PSA) predominantly indicates such a biopsy. A major disadvantage of this diagnostic marker is its low specificity, resulting in a significant amount of false biopsy indications. PSA is a normal excretion product of the prostate cells and is therefore not only found in the circulation of men with prostate cancer but also of men with a normal prostate and men with benign prostatic hyperplasia, a phenomenon that is associated with ageing. Nevertheless, PSA is the standard marker for Pca diagnosis and has demonstrated to be effective in advancing the diagnosis by detecting Pca at earlier stages. A growing number of men choose to be screened for Pca by PSA analysis, even up to 60-70% of men in the USA. However, the value of screening for Pca has not been established yet and is currently subject of investigation in the European Randomized Study of Screening for Prostate Cancer (ERSPC). A major drawback of the standard diagnostic tools for Pca is the detection of small non-aggressive or non-life threatening cancers, leading to overdiagnosis and overtreatment, as well as the detection of tumours that are too advanced to cure. Currently, there are no serum or urine markers available for the prognosis of Pca at the time of early disease stages apart from PSA.
It is apparent that improved diagnostic and prognostic serum or urine markers are required that can discriminate men with clinically irrelevant Pca, curable Pca, or life threatening Pca. The P-Mark project will address this growing need.

P-Mark project outline
For a period of 3 years, P-Mark will search for improved diagnostic and prognostic Pca markers by the identification and evaluation of novel markers as well as the evaluation and validation of recently developed promising markers. Novel serum and urine markers will be identified in clinically well-defined biomaterials using innovative mass spectrometry tools, and antibody-based immunoassays will be developed for these markers. The novel markers will be evaluated for their clinical importance using these assays. Recently developed promising markers that prove their clinical value during the evaluation will be validated on a sample set derived from two European screening studies. Eventually, the markers arising from this project will be offered to SMEs for commercialisation and to ongoing large European clinical studies for clinical implementation.
The project is broken down into 5 subprojects, called workpackages (WP), which are described in more detail below.

WP1 Management of biosamples
In WP1, a serum and a urine biorepository including matching tissue and clinical data from well-characterised Pca patients will be constructed and managed. These biomaterials are a prerequisite for the discovery and evaluation of novel Pca markers (WP2) and for the evaluation and validation of recently developed promising Pca markers (WP3 and WP4), as well as for the introduction of the new markers into routine clinical use. In addition, a Pca tissue array will be developed in WP1.

WP2 Discovery and evaluation of novel Pca markers
For the discovery of novel Pca markers in serum and urine, peptide or protein profiles will be generated using SELDI-TOF, MALDI FT-MS and nanoLC FT-MS. The relevant proteins or peptides will be identified followed by analysis of the presence of Pca-related peaks in these profiles by bioinformatics. Subsequently, immunoassays against the selected Pca-related proteins or peptides or combinations thereof will be developed. These assays will then be used for the evaluation of the markers. To facilitate continuation of developing novel Pca markers into the clinic beyond P-Mark, a strategy for clinical implementation will be designed as an endpoint of WP2.

WP3 Evaluation of recently developed promising Pca markers
In this WP, six recently developed Pca markers will be evaluated for their clinical value, including PCA3DD3, bone morphogenetic protein-6 (BMP-6), osteoprotegerin (OPG), nicked PSA, human kallikrein 2 (hK2) and cytochrome P450 3A5*3 polymorphism (CYP3A5*3). Based on the outcome of the evaluation, markers will be selected for clinical implementation in WP4.

WP4 Clinical implementation of recently developed promising Pca markers
Recently developed promising markers selected from WP3 will be validated in a mono-centre or multi-centre setting. This will lead to guidelines for cost-efficient strategies for detection and treatment as well as recommendations for marker application, that have to be discussed in the public domain of related European professional societies. Validated markers will be offered to the principal investigators of ongoing screening studies in Europe for implementation in the study. Taken the duration of P-Mark into consideration (three years), clinical marker implementation will continue beyond this project.

WP5 Identification of risk groups in the general population
This WP contains the access to essential biomaterials and data within the European Randomized Study of Screening for Prostate Cancer (ERSPC) for marker evaluation and validation. The ERSPC analyses the effect of early detection of Pca on mortality and quality of life in over 200,000 men aged 55-74 (see www.erspc.org for more information). Similarly, data and materials obtained from the Prostate testing for cancer and Treatment (ProtecT) study (see www.epi.bris.ac.uk/protect/ for more information) in the United Kingdom are available for validation of new markers. The ERSPC study is in an advanced stage and has in 2007 60 to 90% power to show a difference in Pca mortality of 20 versus 30%. The ERSPC data are essential for P-Mark to complete its search, evaluation and validation of Pca markers in high risk groups. ERSPC contributes to the value of P-Mark by defining the value of markers when identifying risk groups for early diagnosis, stratification between aggressive and non-aggressive cancers and prognosis, and subsequently developing related screening guidelines. The ERSPC will give access to data and biorepositories to P-Mark, and will share its co-ordination and epidemiologic expertise in order to perform epidemiological analysis and validation of markers.

Summarised, the five main objectives of the P-Mark project are:
I. Establishment of a serum biorepository and a urine biorepository
II. Discovery of novel Pca markers in human body fluids by innovative mass spectrometry tools
III. Establishment of the clinical utility of recently developed promising Pca markers
IV. Validation of Pca markers and identification of risk groups in the general population in Europe
V. Development of guidelines for cost-efficient strategies for Pca detection and therapy

A schematic overview of the P-Mark project design is presented in Figure 1.

Figure 1. P-Mark project design

Organisational structure of P-Mark
The P-Mark consortium is composed of top European research laboratories in the field of (urological) oncological marker development and SMEs providing pivotal state-of-the-art technologies. The following nine participants (principal investigator(s) in brackets) contribute to P-Mark:
1. Erasmus MC, Rotterdam,The Netherlands (Prof. Chris Bangma, Prof. Fritz Schröder and Dr. Theo Luider)
2. Lunds universitet, Malmö, Sweden (Prof. Hans Lilja and Dr. Anders Bjartell)
3. University Medical Centre Nijmegen, The Netherlands (Prof. Jack Schalken)
4. University of Sheffield, United Kingdom (Prof. Freddie Hamdy)
5. University of Helsinki, Finland (Prof. Ulf-Håkan Stenman)
6. University of Turku, Finland (Prof. Kim Pettersson)
7. Rijksuniversiteit Groningen, The Netherlands (Prof. Rainer Bischoff)
8. Innotrac Diagnostics Oy, Turku, Finland (Dr. Harri Takalo)
9. CanAg Diagnostics AB, Göteborg, Sweden (Dr. Olle Nilsson)

P-Mark is coordinated by Prof. dr. Chris Bangma (Erasmus MC, Rotterdam, The Netherlands). For the day-to-day management of the project, a management office located at the same institute will assist the coordinator. All important project decisions will be taken by the General Assembly, which is composed of one representative for each participant. A Scientific Advisory Board consisting of experts covering the fields of proteomics, biostatistics, clinical urology and marker commercialisation, will advice the General Assembly. An overview of the organisational structure of P-Mark is presented in Figure 2.

Figure 2. Organisational structure of P-Mark