The Role of Molecular Biology in Detection and Monitoring of Prostate Cancer

Summary

The study of molecular markers in various types of human carcinomas, as well as in carcinoma of prostate, is focused on genes responsible for the formation of carcinoma. Mutation, amplification or other changes in these genes or in their protein products are being examined and compared with traditional prognostic markers. These genes can be characterized as oncogenes, tumor suppressor genes or genes for other significant cell functions. However, studies are often limited by heterogenity and multifocality of tumors, especially in prostate cancer. In this review, we offer a survey of some of the most frequent diagnostic and prognostic parameters of molecular biology research in relation to prostate cancer.

Key Words:
prostate cancer – molecular biology – molecular markers

Prostate cancer has become one of the most frequently diagnosed tumors in men and is one of the leading causes of death in men over the age 50 (3). In most cases, it is represented by acinar (conventional) adenocarcinoma (11). Prostatic adenocarcinoma is a heterogenous group of neoplasms with a broad spectrum of pathologic and molecular characteristics and clinical behaviors (15). Standardly used diagnostic and prognostic parameters of prostate cancer have too many limitations and seem to be insufficient in the prediction of the behavior of this disease in many cases. The tendentions to impose molecular-genetic methods grow more and more stronger (18). The target of the molecular biology research is to find more persuasive markers of biological character of prostate gland carcinoma in individual patients that would be essential for the follow-up determination of adequate strategy and agressivity of the treatment. The purpose of this article is to review some novel molecular agents that play a role in the diagnosis and monitoring of prostate cancer. 

PSA/PSM EXPRESSING CELLS AND THE RT-PCR REACTION 

The serum biomarkers – prostatic acid phosphatase and especially prostate-specific antigen (PSA), which is a glycoprotein, have both been widely used for diagnosis and clinical monitoring of patients with prostate cancer (8). However, they are not accurate enough to predict occult invasion or metastatic disease, which are diagnosed at the time of radical surgery in almost half of the patients believed to have localized disease. Detection of circulating PSA-positive malignant cells by amplification of PSA mRNA has opened new expectations and proved to be useful to identify cancer cells in lymph nodes. To better identify early extra-prostatic spread of cancer, blood samples of patients with clinically localized or locally advanced disease are being assessed (4, 19). Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) was introduced to improve the limit of PSA mRNA detection (7). Besides, prostate-specific membrane antigen (PSM), an integral transmembrane glycoprotein, is another promising prostate-specific marker, and RT-PCR reaction is used not only to detect PSA- but also PSM-expressing cells in peripheral circulation (14).

The circulating tumor cells in patients with prostatic carcinoma are originally epithelial prostatic cells that are characterized by very specific expression of genes for prostate specific antigen (PSA) and for prostate-specific membrane antigen (PSM) (2). Normal immunological reaction of human organism does not anticipate the survival of normal, non-malignant prostatic cells in blood and their presence in circulation is regarded as one of the first steps in the cascade of metastasing process (6). Circulating prostate cells in cancer patients can be detected by RT-PCR assay for PSA and PSM mRNA. RT-PCR is an extremely sensitive method; experimental data indicate that RT-PCR can detect a single PSA/PSM - expressing prostate cancer cell in up to 100 million other ambient cells in vitro (21, 9). Using this method, one PSA/PSM positive cell can be detected in 5 ml of peripheral blood. The presence of 1000 PSA/PSM expressing cells in peripheral circulation eventuates in positive RT-PCR (2). Hence, the sensitivity of this method is high enough to detect smaller amount of circulating tumour cells than is needed for the creation of metastasis (approximately 10 000 circulating tumour cells in peripheral blood). The result of the RT-PCR method in vivo thus depends on the ongoing process of the dissemination of tumour cells in circulation and on the efficiency of the immunological processes of human organism to eliminate them (17).

Detection of circulating tumour cells in peripheral blood of patients with prostate cancer by RT-PCR reaction seems to have a better predicting value than monitoring the serum level of PSA in many cases. Several researches have been done up to this time that have proved high sensitivity of RT-PCR reaction. The results of RT-PCR reaction often showed a good correlation with the clinical status of the disease. RT-PCR positive patients were at higher risk for recurrence of prostate cancer after radical prostatectomy. Moreover, RT-PCR for PSM seems to be more relevant marker of the progression of the disease (18). We assume that RT-PCR reaction can be considered early staging modality for radical prostatectomy candidates. Circulating tumor cells continue to provide important prognostic information and will likely become an important aspect of future clinical decision-making (17).

TUMOR SUPPRESSORS AND ONCOGENES 

Genes involved in cancer generation are usually tumor suppressors and oncogenes. Progressive genetic alterations in these genes are involved in the mechanisms of tumorigenesis. In prostate cancer, additionally several chromosomal loci that should harbor mutated genes have been proposed. Some genes have been found altered in prostate cancer, such as PTEN, TP53, AR, RNASEL (HPC1), ELAC2 (HPC2), CDKN2A and MSR1, and those can be natural targets for new strategies of treatment. Gene therapy has namely been suggested to be suitable for prostate cancer treatment (1). We point at some of the genes and transcription factors involved in the tumorigenesis of prostate cancer.

Protooncogen c-myc

Protooncogen c-myc is a nuclear transcription factor that is important for the regulation of cell growth, cell division and apoptosis by regulating a multiple number of genes. It is a member of initiating complex in the regulation of cell cycle (together with c-fos and c-jun). It plays a big role in the progression of the disease and its amplification is characterized for more aggressive forms of cancers (5). It can be detected using fluorescence in situ hybridization (FISH).

Androgen receptor gene

Testosterone, the male hormone, directly stimulates the growth of both normal prostate tissue and prostate cancer cells. Not surprisingly, therefore, this hormone is thought to be involved in the development and growth of prostate cancer. Androgen receptor gene plays a key role especially in patients with advanced prostate cancer. Amplification of AR gene is often noticed in recurrent cancers of prostate (10).

The expression level of the androgen receptor (AR) gene in androgen-dependent and -independent prostate cancer was determined by using real-time quantitative RT-PCR assay. A study by Linja et al. showed that in hormone refractory tumors AR gene amplification was detected by fluorescence in situ hybridization (31 %). Androgen-independent tumors with gene amplification expressed, on average, a 2-fold higher level of AR than the refractory tumors without the gene amplification. The findings demonstrate that AR is highly expressed in androgen-independent prostate cancer, suggesting that the AR signaling pathway is important in the progression of prostate cancer during endocrine treatment (13).

Transcription factors ERG and ETV1

Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. Tomlins et al. used bioinformatics’ approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors, ERG and ETV1, were identified as outliers in prostate cancer. They identified recurrent gene fusions of the 5’untranslated region of TMPRSS2 to ERG or ETV1 in prostate cancer tissues with outlier expression. Using fluorescence in situ hybridization, it was revealed that 23 of 29 prostate cancer samples harbor rearrangements in ERG or ETV1. Cell line experiments suggest that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer (20).

PTEN / MMAC1 gene

Recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. In 2001, Magee examined PTEN/MMAC1 in advanced prostate cancer for gene mutation or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1 – specific antibody (16). Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and RT-PCR reaction of mRNA. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblotting analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. Besides, its homology to tensin was observed.

The results of the study demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer. The frequent loss of PTEN/MMAC1 expression suggests that inactivating the function of PTEN/MMAC1 is a critical step in the development or progression of prostate cancer. Because the xenografts and patient material used in the study were from patients with advanced stage disease, it is not yet clear if PTEN/MMAC1 inactivation occurs early or late in disease progression and further studies are needed. Nevertheless, the cytoplasmic location of the protein and its homology to tensin provide an important starting point in the search for physiologically relevant substrates. The ability to restore PTEN/MMAC1 expression may have important clinical implications (22).

Gene for hepsin

Many of the genetic changes that coincide with prostate cancer progression remain enigmatic. Magee et al. addressed this problem by characterizing the expression profiles of several benign and malignant human prostate samples. In their research, several genes that are differentially expressed between benign and malignant glands were identified. One gene that was overexpressed encodes the serine protease hepsin. They used an independent sample set to confirm that hepsin is overexpressed in prostate tumors, and in situ hybridization demonstrated that hepsin is specifically overexpressed in the carcinoma cells themselves. These facts, together with the molecular properties of hepsin, make it an ideal target for prostate cancer therapy (16).

PCA3 AND EPCA-2

Relatively new trends of molecular biology in seeking for prognostic parameters of prostate cancer are focused on oncomarkers PCA3 (prostate cancer gene 3) and EPCA-2 (early prostate cancer antigene 2). PCA3 is a prostate specific, nonprotein coding RNA that is over-expressed in prostate cancer. PCA3 test is realized on a sample of urine. Recent studies showed the diagnostic potential of a urine based PCA3 for predicting biopsy outcome. It seems to be a highly specific marker for prostatic carcinoma. Unlike PSA antigen, its levels do not increase in patients with benign prostatic hyperplasia or prostatitis. Also its sensitivity is high; patients with locally advanced prostatic carcinoma have significantly higher PCA3 score than patients without extracapsular extension.

In a study realized by Whitman et al. post-digital rectal examination urine specimens were obtained from 72 men with prostate cancer before radical prostatectomy. PCA3 and PSA mRNA were measured. The ratio of PCA3 to PSA mRNA was recorded as a PCA3 score and correlated with data on each prostate specimen. Patients with extracapsular extension had a significantly higher median PCA 3 score than patients without extrapasular extension. PCA3 score significantly correlated with total tumor volume. At a cutoff PCA3 score of 47 extracapsular extension was predicted with 94 % specificity and an 80% positive predictive value. When combined with serum PSA and biopsy Gleason score, its predicting value of extracapsular extension was even higher. PCA3 detected in the postdigital rectal examination urine of patients with prostate cancer correlated with pathological findings. It seems that PCA3 can provide a relevant prognostic information (15).

Great expectations are dedicated also to EPCA test. EPCA-2 is a new serum marker, highly specific for the prostate cancer. Similarly as PCA3 test, EPCA-2 test has high sensitivity and specificity and accurately differentiates between men with organ-confined and non-organ confined disease (12). However, the way how this marker, with its relation to cell nucleus, gets from intracellular environment to circulation is not clear, yet. Apoptosis and its consequent release is assumed. Research of PCA3 and EPCA-2 continues, and it is supposed that it will have a significant impact on future diagnosis and monitoring of prostate cancer (12, 15).

CONCLUSION 

This review focused on some new findings in the pathology and molecular biology of prostate cancer. The study of molecular markers of prostate cancer are only at the beginning. However, seeking for new parameters able to better predict biological behavior of the tumor is the only way leading to improved and individualized approach to the treatment of particular patients. Molecular  biology plays a big role in this. 

Correspondence to:

MUDr. Anežka ZUMMEROVÁ

Department of pathology and forensic medicine,

Health Care Surveillance Authority

Antolská 11, SK-851 07 Bratislava, Slovakia

Tel: 00421 903 960 836

Fax: 00421 2 6353 1990

E-mail:anezkazummerova@yahoo.com, anezkazummerova@gmail.com