Prostatic Neoplasms: Diamandis EP

Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brünner N, Chan DW, Babaian R, Bast RC, Dowell B, Esteva FJ, Haglund C, Harbeck N, Hayes DF, Holten-Andersen M, Klee GG, Lamerz R, Looijenga LH, Molina R, Nielsen HJ, Rittenhouse H, Semjonow A, Shih IeM, Sibley P, Sölétormos G, Stephan C, Sokoll L, Hoffman BR, Diamandis EP, Anonymous00039. · Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Edinburgh, UK. · Clin Chem. · Pubmed #19042984 No free full text.

Abstract: BACKGROUND: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS: For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers.

Sardana G, Dowell B, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · Clin Chem. · Pubmed #18927246 No free full text.

Abstract: BACKGROUND: Early detection of prostate cancer (CaP), the most prevalent cancer and the second-leading cause of death in men, has proved difficult, and current detection methods are inadequate. Prostate-specific antigen (PSA) testing is a significant advance for early diagnosis of patients with CaP. CONTENT: PSA is produced almost exclusively in the prostate, and abnormalities of this organ are frequently associated with increased serum concentrations. Because of PSA's lack of specificity for CaP, however, many patients undergo unnecessary biopsies or treatments for benign or latent tumors, respectively. Thus, a more specific method of CaP detection is required to augment or replace screening with PSA. The focus recently has been on creating cost-effective assays for circulating protein biomarkers in the blood, but because of the heterogeneity of CaP, it has become clear that this effort will be a formidable challenge. Each marker will require proper validation to ensure clinical utility. Although much work has been done on variations of the PSA test (i.e., velocity, density, free vs bound, proisoforms) with limited usefulness, there are many emerging markers at various stages of development that show some promise for CaP diagnosis. These markers include kallikrein-related peptidase 2 (KLK2), early prostate cancer antigen (EPCA), PCA3, hepsin, prostate stem cell antigen, and alpha-methylacyl-CoA racemase (AMACR). We review biomarkers under investigation for the early diagnosis and management of prostate cancer. SUMMARY: It is hoped that the use of panels of markers can improve CaP diagnosis and prognosis and help predict the therapeutic response in CaP patients.

Stephan C, Jung K, Lein M, Diamandis EP. · Department of Urology, Charité - Universitätsmedizin Berlin, Berlin, Germany. · Eur J Cancer. · Pubmed #17689069 No free full text.

Abstract: Prostate cancer is the most common neoplasia of middle-aged men. Prostate specific antigen (PSA) is the first FDA-approved tumour marker for early detection of cancer and it is now in widespread clinical use. The discovery of different PSA molecular forms in serum (free PSA, PSA complexed with various protease inhibitors) in the early 1990s renewed clinical research to enhance the specificity of PSA. Also, the use of a homologous prostate-localised antigen, human glandular kallikrein 2 (KLK2) may further reduce the number of unnecessary prostate biopsies. More recently, promising data is emerging regarding molecular forms of free PSA (proPSA, BPSA, 'intact' PSA) and other members of the expanded human kallikrein family. These new findings may add substantial clinical information for early detection of prostate cancer.

Stephan C, Jung K, Diamandis EP, Rittenhouse HG, Lein M, Loening SA. · Department of Urology, University Hospital Charité, Humboldt University Berlin, Berlin, Germany · Urology. · Pubmed #11796270 No free full text.

This publication has no abstract.

Yousef GM, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. · Endocr Rev. · Pubmed #11294823 links to  free full text

Abstract: The human tissue kallikrein gene family was, until recently, thought to consist of only three genes. Two of these human kallikreins, prostate-specific antigen and human glandular kallikrein 2, are currently used as valuable biomarkers of prostatic carcinoma. More recently, new kallikrein-like genes have been discovered. It is now clear that the human tissue kallikrein gene family contains at least 15 genes. All genes share important similarities, including mapping at the same chromosomal locus (19q13.4), significant homology at both the nucleotide and protein level, and similar genomic organization. All genes encode for putative serine proteases and most of them are regulated by steroid hormones. Recent data suggest that at least a few of these kallikrein genes are connected to malignancy. In this review, we summarize the recently accumulated knowledge on the human tissue kallikrein gene family, including gene and protein structure, predicted enzymatic activities, tissue expression, hormonal regulation, and alternative splicing. We further describe the reported associations of the human kallikreins with various human diseases and identify future avenues for research.

Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Clin Chem. · Pubmed #10894830 links to  free full text

Abstract: BACKGROUND: Prostate-specific antigen (PSA) is a valuable prostatic cancer biomarker that is now widely used for population screening, diagnosis, and monitoring of patients with prostate cancer. Despite the voluminous literature on this biomarker, relatively few reports have addressed the issue of its physiological function and its connection to the pathogenesis and progression of prostate and other cancers. APPROACH: I here review literature dealing with PSA physiology and pathobiology and discuss reports that either suggest that PSA is a beneficial molecule with tumor suppressor activity or that PSA has deleterious effects in prostate, breast, and possibly other cancers. CONTENT: The present scientific literature on PSA physiology and pathobiology is confusing. A group of reports have suggested that PSA may act as a tumor suppressor, a negative regulator of cell growth, and an apoptotic molecule, whereas others suggest that PSA may, through its chymotrypsin-like activity, promote tumor progression and metastasis. SUMMARY: The physiological function of PSA is still not well understood. Because PSA is just one member of the human kallikrein gene family, it is possible that its biological functions are related to the activity of other related kallikreins. Only when the physiological functions of PSA and other kallikreins are elucidated will we be able to explain the currently apparently conflicting experimental data.

Black MH, Magklara A, Obiezu CV, Melegos DN, Diamandis EP. · Department of Pathology, Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5. · Clin Chem. · Pubmed #10351987 links to  free full text

Abstract: BACKGROUND: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). METHODS: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. RESULTS: Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations approximately 2. 5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1-34, with a median of 2.6. In seminal plasma, this ratio was 100-500. More than 94% of immunoreactive hK2 in serum was in the free form ( approximately 30 kDa); traces of hK2 complexed to alpha1-antichymotrypsin were present. CONCLUSIONS: The limit of detection of the method for hK2 measurement described here ( approximately 20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.

Pavlou M, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada. · Clin Chem. · Pubmed #19478024 No free full text.

This publication has no abstract.

Paliouras M, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, ON, Canada. · Biol Chem. · Pubmed #18627304 No free full text.

Abstract: The androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancer in vitro model. Using the cell line PC3(AR)(2), we stimulated cells with 5-alpha-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)(2) cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose an in vitro androgen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.

Sardana G, Jung K, Stephan C, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. · J Proteome Res. · Pubmed #18578523 No free full text.

Abstract: Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.

Rabien A, Fritzsche F, Jung M, Diamandis EP, Loening SA, Dietel M, Jung K, Stephan C, Kristiansen G. · Department of Urology, Charité-Universitatsmedizin Berlin, CCM, Berlin, Germany. · Tumour Biol. · Pubmed #18497543 No free full text.

Abstract: OBJECTIVE: Reliable prognostic tools for prostate cancer are still needed and KLK14, a young member of the growing family of human kallikrein-related peptidases, has been estimated to become a new significant marker. It is the aim of this study to analyze the clinical value of immunohistochemical expression of KLK14 in prostate cancer tissue samples. METHODS: Protein expression of KLK14 was assessed immunohistochemically in 186 tissue samples from radical prostatectomies. Areas of normal prostatic tissue, of prostatic epithelial neoplasia and of prostatic adenocarcinoma were checked in relation to clinicopathological parameters of the patients. Expression of KLK14 mRNA was quantified in 25 matches of normal and cancerous prostatic tissue, collected by laser capture microdissection. Real-time reverse transcriptase polymerase chain reaction analysis was used to supplement the immunohistochemical data. RESULTS: Expression of the KLK14 protein correlated with the pathological tumor status in prostate cancer and was associated with disease progression defined by prostate-specific antigen relapse in univariate Kaplan-Meier analysis. The multivariate Cox proportional hazards regression model proved KLK14 to be an independent prognostic factor in prostate cancer. CONCLUSION: In conclusion, we consider KLK14 to be a suitable prognostic marker for the detection of cases at risk of disease progression after radical prostatectomy.

Kulasingam V, Smith CR, Batruch I, Buckler A, Jeffery DA, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · J Proteome Res. · Pubmed #18186600 No free full text.

Abstract: While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms.

Maki J, Robinson K, Reguly B, Alexander J, Wittock R, Aguirre A, Diamandis EP, Escott N, Skehan A, Prowse O, Thayer RE, Froberg MK, Wilson MJ, Maragh S, Jakupciak JP, Wagner PD, Srivastava S, Dakubo GD, Parr RL. · Genesis Genomics, Thunder Bay, Canada. · Am J Clin Pathol. · Pubmed #18089489 links to  free full text

Abstract: We report the usefulness of a 3.4-kb mitochondrial genome deletion (3.4 mtdelta) for molecular definition of benign, malignant, and proximal to malignant (PTM) prostate needle biopsy specimens. The 3.4 mtdelta was identified through long-extension polymerase chain reaction (PCR) analysis of frozen prostate cancer samples. A quantitative PCR assay was developed to measure the levels of the 3.4 mtdelta in clinical samples. For normalization, amplifications of a nuclear target and total mitochondrial DNA were included. Cycle threshold data from these targets were used to calculate a score for each biopsy sample. In a pilot study of 38 benign, 29 malignant, and 41 PTM biopsy specimens, the difference between benign and malignant core biopsy specimens was well differentiated (P & .0001), with PTM indistinguishable from malignant samples (P = .833). Results of a larger study were identical. In comparison with histopathologic examination for benign and malignant samples, the sensitivity and specificity were 80% and 71%, respectively, and the area under a receiver operating characteristic (ROC) curve was 0.83 for the deletion. In a blinded external validation study, the sensitivity and specificity were 83% and 79%, and the area under the ROC curve was 0.87. The 3.4 mtdelta may be useful in defining malignant, benign, and PTM prostate tissues.

Memari N, Diamandis EP, Earle T, Campbell A, Van Dekken H, Van der Kwast TH. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Prostate. · Pubmed #17654496 No free full text.

Abstract: BACKGROUND: Human tissue kallikrein-related peptidases (genes, KLKs; proteins, KLKs) are a subgroup of serine proteases present in a variety of tissues and biological fluids. A number of human tissue KLKs are established or candidate serologic biomarkers for prostate cancer. Human kallikrein-related peptidase 12 (KLK12, KLK12), recently identified in our laboratory, is a novel member of the KLK gene family. Here, we report generation of antibodies against the full-length recombinant KLK12 (classical form) and the immunohistological localization of this KLK in normal and malignant prostate tissues. METHODS: The mature form of KLK12 cDNA was amplified using PCR and cloned into a plasmid vector for protein production in E. coli. Following identification by mass spectroscopy, recombinant KLK12 was purified and used as immunogen in rabbits. Anti- KLK12 antibody was used for immunostaining of paraffin-embedded sections of human prostate tissue. Immunoexpression of KLK12 in benign and malignant prostate tissue was evaluated using a prostate cancer tissue array. RESULTS: Anti-KLK12 antibody showed a predominantly apical and membranous staining of the luminal cells of the normal prostate in contrast with the predominantly diffuse cytoplasmic staining observed in both prostatic intra-epithelial neoplasia and adenocarcinomas. This was occasionally associated with an intense granular supranuclear staining. More than 95% of the prostate cancers on the tissue microarray were KLK12 positive. CONCLUSION: Higher levels of KLK12 in malignant prostatic glands, and the shift in subcellular localization of KLK12 in prostate cancer observed in this study point to the potential role of this kallikrein during prostate carcinogenesis.

Rabien A, Kristiansen G, Diamandis EP, Jung K, Stephan C. · Klinik für Urologie, Charité--Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin. · Urologe A. · Pubmed #17605120 No free full text.

This publication has no abstract.

Stephan C, Xu C, Cammann H, Graefen M, Haese A, Huland H, Semjonow A, Diamandis EP, Remzi M, Djavan B, Wildhagen MF, Blijenberg BG, Finne P, Stenman UH, Jung K, Meyer HA. · Department of Urology, Charité-Universitätsmedizin Berlin, CCM, Berlin, Germany. · World J Urol. · Pubmed #17333205 No free full text.

Abstract: Use of percent free PSA (%fPSA) and artificial neural networks (ANNs) can eliminate unnecessary prostate biopsies. In a total of 4,480 patients from five centers with PSA concentrations in the range of 2-10 ng/ml an IMMULITE PSA-based ANN (iANN) was compared with other PSA assay-adapted ANNs (nANNs) to investigate the impact of different PSA assays. ANN data were generated with PSA, fPSA (assays from Abbott, Beckman, DPC, Roche or Wallac), age, prostate volume, and DRE status. In 15 different ROC analyses, the area under the curve (AUC) in the PSA ranges 2-4, 2-10, and 4-10 ng/ml for the nANN was always significantly larger than the AUC for %fPSA or PSA. The nANN and logistic regression models mostly also performed better than the iANN. Therefore, for each patient population, PSA assay-specific ANNs should be used to optimize the ANN outcome in order to reduce the number of unnecessary biopsies.

Sardana G, Marshall J, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. · Clin Chem. · Pubmed #17259234 links to  free full text

Abstract: OBJECTIVE: Prostate-specific antigen measurement, widely used for early detection of prostate cancer (CaP), suffers from low specificity. Additional tumor markers are needed for the early detection of clinically relevant CaP. Our objective was to perform a qualitative proteomic analysis of conditioned medium (CM) from the CaP cell line PC3(AR)(6). METHODS: We used a roller bottle culture system to culture the PC3(AR)(6) cell line in chemically defined serum-free medium for 14 days. By using strong anion-exchange chromatography, we fractionated the CM and trypsinized the fractions. The tryptic peptides were further fractionated by reversed-phase C-18 chromatography before being subjected to electrospray ionization tandem mass spectrometry. We used MASCOT software to search the mass spectra generated and organized identified proteins based on their genome ontology classification of cellular location. We used an immunoassay to measure a newly identified secreted protein, Mac-2BP, and kallikreins 5, 6, and 11 in serum samples from CaP patients and healthy men. RESULTS: We classified 262 proteins according to cellular location; the sample was found to contain a significant proportion of secreted (23%) and membrane (16%) proteins. In a proportion of cancer patients compared with healthy men, we determined by ELISA that serum concentrations of a novel candidate biomarker Mac-2BP were increased. CONCLUSIONS: These identified proteins, and possibly many others found in the CM, may have utility as novel CaP biomarkers.

Borgoño CA, Michael IP, Shaw JL, Luo LY, Ghosh MC, Soosaipillai A, Grass L, Katsaros D, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. · J Biol Chem. · Pubmed #17110383 links to  free full text

Abstract: Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.

Parr RL, Dakubo GD, Crandall KA, Maki J, Reguly B, Aguirre A, Wittock R, Robinson K, Alexander JS, Birch-Machin MA, Abdel-Malak M, Froberg MK, Diamandis EP, Thayer RE. · Genesis Genomics Inc., 1294 Balmoral St., Thunder Bay, Ontario, Canada, P7B 5Z5. · J Mol Diagn. · Pubmed #16825503 links to  free full text

Abstract: Studies of somatic mitochondrial DNA mutations have become an important aspect of cancer research because these mutations might have functional significance and/or serve as a biosensor for tumor detection. Here we report somatic mitochondrial DNA mutations from three specific tissue types (tumor, adjacent benign, and distant benign) recovered from 24 prostatectomy samples. Needle biopsy tissue from 12 individuals referred for prostate biopsy, yet histologically benign (symptomatic benign), were used as among individual control samples. We also sampled blood (germplasm tissue) from each patient to serve as within individual controls relative to the somatic tissues sampled (malignant, adjacent, and distant benign). Complete mitochondrial genome sequencing was attempted on each sample. In contrast to both control groups [within patient (blood) and among patient (symptomatic benign)], all of the tissue types recovered from the malignant group harbored significantly different mitochondrial DNA (mtDNA) mutations. We conclude that mitochondrial genome mutations are an early indicator of malignant transformation in prostate tissue. These mutations occur well before changes in tissue histo-pathology, indicative of prostate cancer, are evident to the pathologist.

Stephan C, Meyer HA, Cammann H, Nakamura T, Diamandis EP, Jung K. · Department of Urology, University Hospital Charité, D-10098 Berlin, Germany. · Biol Chem. · Pubmed #16800743 No free full text.

Abstract: Human kallikrein 11 (hK11) was evaluated in a percentage free PSA-based artificial neural network (ANN) to reduce unnecessary prostate biopsies. Serum samples from 357 patients with (n=132) and without (n=225) prostate cancer (PCa) were analyzed and ANN models were constructed and compared to all parameters. The discriminatory power of hK11 was lower than that of PSA, but receiver operator characteristic (ROC) analyses demonstrated significantly larger areas under the curves for the ANN compared to all other parameters. ANNs with hK11 may lead to a further reduction in unnecessary prostate biopsies, especially when analyzing patients with less than 15% free PSA.

Pampalakis G, Diamandis EP, Sotiropoulou G. · Department of Pharmacy, School of Health Sciences, University of Patras, GR-26500 Rion-Patras, Greece, and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada. · Biol Chem. · Pubmed #16800742 No free full text.

Abstract: The tissue kallikrein gene family consists of 15 genes tandemly arranged on human chromosome 19q13.4. Most kallikrein genes are characterized by aberrant expression patterns in various human cancers, a feature that makes them ideal cancer biomarkers. In the present study, we investigated the effect of the epigenetic drug compound 5-aza-2'-deoxycytidine on the expression of downregulated kallikrein genes in prostate, breast, and ovarian cancer cell lines. Reactivation of multiple kallikrein genes was observed, although some of these genes do not contain CpG islands in their genomic sequence. Epigenetic regulation provides a new mechanism for the pharmacological modulation of kallikreins in human cancers with putative therapeutic implications.

Stephan C, Jung K, Nakamura T, Yousef GM, Kristiansen G, Diamandis EP. · Department of Urology, University Hospital Charité Berlin, Berlin, Germany. · Int J Urol. · Pubmed #16643616 No free full text.

Abstract: BACKGROUND: Human glandular kallikrein (hK2) has been shown to add important information regarding the early detection and staging of prostate cancer. Preliminary analysis pointed out that hK2 may discriminate between pT2 and pT3 tumors, and that hK2 may predict Gleason grade 4/5 cancer volume, better than prostate-specific antigen (PSA) or percent free PSA (%fPSA). We investigated the role of hK2 serum values for predicting pathological stage, grade and Gleason score. METHODS: Prostate-specific antigen, free PSA and hK2 were measured on 222 untreated prostate cancer patients who had received radical prostatectomy at the Charité Hospital, Berlin, Germany. Pathological work up revealed pT2-cancer in 111 patients and pT3-cancer in 111 patients. Grade 2 was found in 118 patients whereas grade 3 tumors were found in 104 patients. RESULTS: For pT2 and pT3 patients, the %fPSA (P=0.006), the ratios hK2/fPSA (P=0.08) and hK2xtPSA/fPSA (P=0.002) were all significant different whereas hK2 (P=0.143) and PSA (P=0.1) did not differ. Between grade 2 and grade 3 tumors, the hK2 alone (P=0.27), the %fPSA (P=0.13), the ratios hK2/fPSA (P=0.94) and hK2xtPSA/fPSA (P=0.12) did not separate, whereas PSA (P=0.039) showed a difference. The same relationships were found between the two groups in Gleason score<7 and >or=7. Neither the hK2 ratio, nor % fPSA was different. CONCLUSION: Human glandular kallikrein was not different between pT2 and pT3, nor between G2 versus G3 or Gleason scores<7 and >or=7 prostate cancer. Together with %fPSA, hK2 may only help to distinguish preoperatively between pT2 and pT3 prostate cancer but cannot add further information.

Oikonomopoulou K, Scorilas A, Michael IP, Grass L, Soosaipillai A, Rosen B, Murphy J, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. · Tumour Biol. · Pubmed #16557045 No free full text.

Abstract: BACKGROUND: Kallikreins are a family of secreted serine proteases, encoded by 15 genes which all localize in tandem on chromosome 19q13.4. Several members of this family have been previously associated with ovarian cancer. Kallikreins 6 (KLK6) and 10 (KLK10) are elevated in tumour cells, serum and ascites fluid of ovarian cancer patients and correlate with disease prognosis. Other kallikreins that have been related to ovarian cancer include KLK4, 5, 7, 8, 9, 11, 13, 14 and 15. We hypothesized that KLK6 and KLK10 can be utilized to monitor dissemination of ovarian cancer cells in blood and ascites fluid of ovarian cancer patients. METHODS: RNA was isolated by immunomagnetic separation of cancer cells and was amplified by RT-PCR. RESULTS: Screening for disseminated cancer cells in blood from 24 ovarian cancer patients, with RT-PCR for KLK6 mRNA, resulted in 75% positivity; however, this was not different from the positivity of normal controls. By utilizing KLK10 as a marker, the positivity of patients was 40% versus 20% of controls. Screening of ascites fluid of ovarian cancer patients revealed 90% positivity for KLK6 and KLK10 mRNA compared with 33% for other cancer types. Significant correlations were identified among mRNA of KLK4, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 in cancer cells isolated from ascites fluid. CONCLUSION: Kallikrein expression by ovarian cancer cells is not specific enough for detecting disseminated disease. Kallikrein expression may have some value for differentiating ovarian cancer from other types of cancer or from non-malignant diseases that lead to ascites accumulation.

Michael IP, Pampalakis G, Mikolajczyk SD, Malm J, Sotiropoulou G, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. · J Biol Chem. · Pubmed #16517595 links to  free full text

Abstract: Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsin-like activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.

Luo LY, Shan SJ, Elliott MB, Soosaipillai A, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Clin Cancer Res. · Pubmed #16467084 links to  free full text

Abstract: PURPOSE: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma. EXPERIMENTAL DESIGN: hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry. RESULTS: hK11 is present in seminal plasma at concentrations ranging from 2 to 37 microg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of approximately 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of approximately 20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, alpha1-antichymotrypsin, alpha2-antiplasmin, and alpha1-antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg156. CONCLUSION: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.