Modern hematology emphasizes a multiparametric diagnostic approach and the basic parameters, beside history of the disease and clinical examination, are morphological, immunophenotypic and genetic evaluation. Flow cytometry plays an important role in diagnosis of a large group of hematological diseases. This article reviews the basic principles of flow cytometry and its use in hematology diagnosis, with emphasis on chronic lymphoproliferations.

by Dr Nataša Lazić

Introduction
In modern diagnostics, flow cytometry has an important place as one of the basic and irreplaceable tools for diagnosis, classification, monitoring and prediction of malignant hematological disease [1]. The extreme complexity of these diseases, on one hand, and the availability of the different therapeutic protocols for the different types of these diseases on the other, makes accurate and precise diagnosis imperative. Contributing to this is the fact that the World Health Organization (WHO), in the Classification of Tumours of Hemopoietic and Lymphoid Tissues, suggests a multiparametric approach in diagnosing these diseases; basic parameters required are morphological, immunophenotypic and genetic analysis for each entity of the disease, in addition to a detailed history of the disease and clinical examination [2, 3]. The clinical picture and cell morphology, as a well-known and traditionally-used means of examination, are insufficient in many cases; quite often, because of a similar clinical presentation and cell morphology, it is not possible to draw a diagnostic conclusion based on these findings or a wrong diagnosis may be reached in some cases.

Coulter’s principle of measuring the change in the electrical impedance of the individual cells flowing through the measuring cell, in the late 1940s, was the basis for construction of the first hematologic counter and later for the flow cytometer. Later inventions added new detection capabilities, such as light scatter and fluorescence detection. Fluorescent activated cell sorting (FACS) was invented in the late 1960s by Herzenberg, Bonner, Sweet and Hullet. Introduced as a commercial machine in the early 1970s, this is the class of instruments now commonly referred to as flow cytometer [4]. The invention of monoclonal antibodies by Milstein and colleagues in 1977 opened new perspective for flow cytometry. Further developments, especially in electronics, led to modern cytometers with multiple lasers, detectors, better performance characteristics, and the ability to measure larger amounts of data.

Flow cytometry principles
Flow cytometry is a powerful technology that simultaneously measures many aspects of single particles, usually cells. Any suspended particle or cell from 0.2–150 μm is suitable for analysis. However, it can also measure soluble molecules if trapped onto a particulate surface and bound by fluorochromes. Virtually any component or function of a cell can be measured if the fluorescent probe can be made to detect it.

Sample preparation should provide a homogeneous suspension of cells with monoclonal antibodies conjugated with fluorochromes of a different emission spectrum. Depending on the sample, it most often includes incubation, erythrocyte lysis, centrifugation, washing and fixation.

The cytometer needs to be adjusted to have the appropriate performance characteristics (linearity, sensitivity, CV, electronic and optical background noise, fluorescence detector efficiency, etc). This is achieved by adjusting voltages on the detectors and by spectral overlap compensation (Fig. 1).

The three main systems of flow cytometer are fluidics, optics and electronics (Fig. 2). Parameters measured include forward scatter (FSC) corresponding to cell size, side scatter (SSC) depending on internal complexity and fluorescence intensity for different fluorochromes.
Becoming more available in clinical laboratories, a wide range of clinical applications of flow cytometry are constantly expanding and the most common among them are in, for example, lymphoma and leukemia diagnosis, stem cell enumeration for transplantation, estimation of minimal residual disease, paroxysmal nocturnal hemoglobinuria diagnosis, immunodeficiencies, HIV infection.

Flow cytometry in hematology
Flow cytometric immunophenotyping enables examination of the phenotype of the separate cells in the suspension and summarizing of the results, which gives data about the presence or absence of antigen expression as well as the expression intensity [5]. Hence, an immunophenotypic pattern is obtained on the cell population of interest for the examined disease. Meanwhile, there are no separate antigens specific for the particular disease. Instead, their mutual relation is observed and analysed, which makes the analysis of the flow cytometry results very demanding and complex, but usually very useful and precise owing to the huge amount of data that can be collected from the cells [6]. Therefore, flow cytometry helps with determining the cell line, the degree of cell maturity, abnormal patterns of expression and provides a detailed immunophenotype of the pathological cell population [7]. From information on all the aforementioned factors, a diagnostic conclusion is drawn if there is a phenotype characteristic for some disease. In the case of an atypical phenotype, the disease is assigned to the appropriate group and additional tests should be done to gain a precise diagnosis (such as immunohistochemical, FISH, molecular tests).

CD markers (clusters of differentiation) are blood cell antigens that enable their characterization. CD nomenclature was developed and reviewed by HLDA (Human Leukocyte Differentiation Antigen) workshops started in 1982. There were 10 such workshops and the nomenclature now encompasses about 400 CD markers. Monoclonal antibodies against those antigens are used for immunophenotype characterization.

The antibody panel for the analysis of the sample to be tested by flow cytometry depends, to a large extent, on the available information of other findings made for that patient. According to the Bethesda Group recommendations from 2006, which were aimed at regulating a more systematic approach in this field (and are still valid today), before sending a sample to flow cytometry, a detailed history of the disease, clinical examination, microscopic examination of cell morphology, and other laboratory tests should be carried out, and based on this, diagnosis or differential diagnosis determined. In this way significant rationalization and cost reduction can be achieved [8].
Immunophenotype characterization for chronic lymphoproliferative disorders
For both of the two major groups of malignant hematologic diseases, those derived from mature and from immature cells, flow cytometry is of a great importance. Neoplasms of mature lymphoid cells, according to the WHO Classification, include chronic lymphoid leukemia and non-Hodgkin’s lymphoma. Their basic characteristic is that they have an immunophenotype similar to mature lymphoid cells and, accordingly, they show an absence of immaturity indicators (CD34, TdT). According to the origin, in relation to the cell line, they can be divided into T, B and NK neoplasms. [7]

Mature B-cell lymphoproliferations make up most of the malignant blood diseases: 90 % of the total lymphoid malignancies, according to WHO data. They present 4 % of the newly discovered carcinomas per year. As already known, the malignant cell derived from B-cell lineage in most cases imitates the normal B-cells stopped at a certain maturity level. The classification of this disease group mostly relies on this fact. The most common in this group are chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma (MCL), plasma cell leukemia [12]. Immunophenotype characterization in the diagnosis of B-cell chronic lymphoproliferative diseases is an irreplaceable method and, together with morphology, it presents the essential search that should be undertaken in the diagnosis of these diseases[2, 9]. Based on the finding of the immunophenotype characterization it is possible to discover aberrant expression patterns and establish the phenotypic characteristics related to particular diseases. The application of a scoring system as an additional tool is the result of a need for some standardization and quantification in the diagnosis of B-cell chronic lymphoproliferative diseases. In order to increase the precision of the scoring system, different studies with different CD markers are taken [10–12]. The most common scoring system of 5 points includes CD5, CD23, FMC7, CD79b and surface immunoglobulin chains with an accuracy of 96.6 % if a three-point cut-off is used [10].

In most cases of CLL, cell morphology is characteristic and typical for this disease. However, in a number of cases, flow cytometry has a huge and decisive significance for diagnosis (Fig. 3) [13]. CLL and MCL share many morphological and immunophenotypic features [14]. As a result of their partial overlap, a differential diagnosis of MCL is most considered when making a diagnosis of CLL. Because of the different therapeutic approach and prognoses of the diseases, their diagnostic differentiation is very important. For that purpose cyclin D1 testing is recommended [15, 16]. Unlike the other chronic lymphoproliferations, HCL cells do not match any stage of the normal lymphoid cells development. Morphologically typical HCL cells have fine, hair-like, cytoplasmic projections, which are sometimes difficult to find in the peripheral blood smear. Because of this and a very specific immunophenotype, flow cytometry is essential for HCL diagnosis [14, 17].

Advantages
The possibility of combining more antibodies in the same tube and analysing their interactions on the population of interest for the given disease is the greatest advantage of multiparametric flow cytometry, which involves simultaneously collecting and analysing a large amount of data from cells or particles.

Considerations
Comprehensive analysis involves considering possible causes of false-positive or false-negative results, thus avoiding an incomplete or incorrect interpretation of flow cytometry data (Fig. 4).

Other difficulties, such as non-standardized methods, particularly the issue of regulation in cytometry, different antibody panels, cut-off values, analysis subjectivity – recommended visual approach, result analysis complexity, report form, etc., are the subject of work by various associations dealing with cytometry in order to achieve harmonization in this area [13].

References
1. Paiva A, Alves GVA, Sales VSF, Silva ASJ, Silva DGKC, Alves E, Bahia F, Freitas RV, De Oliveira Paiva HD, Cavalcanti GB, Jr. Utility of flow cytometry immunophenotyping and hematological profile in chronic lymphoproliferative disorders. Blood 2017; 130: 5326 [poster abstract].
2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman J (eds). WHO classification of tumors of haematopoietic and lymphoid tissues. IARC 2008; Chapters 1, 8, 10. ISBN 978-9283224310.
3. Boyd SD, Natkunam Y, Allen JR, Warnke R. Selective immunophenotyping for diagnosis of B-cell neoplasms: immunohistochemistry and flow cytometry strategies and results. Appl Immunohistochem Mol Morphol 2013; 21: 116–131.
4. Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 2002; 48: 1819–1827.
5. Braylan RC. Impact of flow cytometry on the diagnosis and characterization of lymphomas, chronic lymphoproliferative disorders and plasma cell neoplasias. Cytometry A 2004; 58: 57–61.
6. Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology. Clin Chem 2000; 4: 1221–1229.
7. Craig FE, Foon FA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008; 111: 3941–3967.
8. Oberley MJ, Fitzgerald S, Yang DT, Morgan A, Johnson J, Leith C. Value-based flow testing of chronic lymphoproliferative disorders: a quality improvement project to develop an algorithm to streamline testing and reduce costs. Am J Clin Pathol 2014; 142: 411–418.
9. D’Arena G, Keating MJ, Carotenuto M. Chronic lymphoproliferative disorders: an integrated point of view for the differential diagnosis. Leuk Lymphoma 2000; 36: 225–237.
10. Matutes E, Wotherspoon A, Catovsky D. Differential diagnosis in chronic lymphocytic leukemia. Best Pract Res Clin Haematol 2007; 20: 367–384.
11. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994; 8: 1640–1645.
12. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997; 108: 378–382.
13. Rawstron AC, at al. Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: an European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project. Cytometry B Clin Cytom 2018; 9: 121–128.
14. Asaad NY, Abd El-Wahed MM, Dawoud MM. Diagnosis and prognosis of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) and Mantle cell lymphoma (MCL). J Egypt Natl Canc Inst 2005; 17: 279–290.
15. Matutes E, Polliack A. Morphological and immunophenotypic features of chronic lymphocytic leukemia. Rev Clin Exp Hematol 2000; 4: 22–47.
16. Vose JM. Mantle cell lymphoma; update on diagnosis, risk stratification and clinical management. Am J Hematol 2015; 90: 739–745.
17. Bacal NS, Mantovani E, Grossl S, Nozawa ST, Kanayama RH, Brito ACM, Albers CEM, de Campos Guerra JC, Mangueira CLP. Flow cytometry: immunophenotyping in 48 hairy cell leukemia cases and relevance of fluorescence intensity in CDs expression for diagnosis. Einstein 2007; 5: 123–128.

The authors
Nataša Lazić MD
Institute for Clinical Laboratory Diagnostics, University Clinical Centre of the Republika Srpska, Republika Srpska, Bosnia and Herzegovina

*Corresponding author
E-mail: natasa.lazic.bl@gmail.com

Flow cytometry has traditionally been used to identify hemato-lymphoid neoplasms. However, the flow cytometry laboratories that deal with tissues would often receive samples that have an epithelial neoplasm. In our laboratory, we use flow cytometry to identify cells with epithelial differentiation using Ber-EP4 antibody that targets CD326 (EpCAM). We have formulated an algorithm-based approach for the application of this marker. This approach has been elaborated in this article.

by Dr Pranav Dorwal and Dr Helen Moore

Introduction
The use of flow cytometry in the laboratory has traditionally been applied for diagnosing lymphomas and leukemias. The biggest advantage that flow cytometry has over histopathology is a much quicker turn-around-time, as most of the samples are fresh and can be processed right away, unlike a histopathology sample which needs to undergo fixation and processing before it is ready to be examined. Any additional testing on flow cytometry samples can be performed instantly, whereas the same usually requires another day in the histopathology lab. Many of the lymph node malignancies (primary lymphomas versus metastatic involvement) can appear undifferentiated. In these cases, the histopathologist needs the help of a plethora of immunohistochemical markers to reach a diagnosis. The ability to identify samples where non-hematological malignancies are present can be helpful for the treating physician as well as the reporting histopathologist, who can then test with a more dedicated panel. The ultimate aim of this testing is to get an early diagnosis so that patient’s treatment is not delayed.

A large number of markers have been used to identify epithelial differentiation in tumours by immunohistochemistry (IHC), including cytokeratin (CK), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125) as well as epitopes recognized by the antibodies LeuM1 (anti-CD15 antibody), and MOC-31 and Ber-EP4 antibodies, both of which recognize epitopes on EpCAM (the epithelial cell adhesion molecule). However, most of these are not available for use by flow cytometry. EpCAM (also known as CD326) was first discovered in 1979 and at that time thought to be specific for colonic carcinoma [1]. Ber-EP4 is, therefore, an anti-CD326 antibody which binds to a cell membrane glycoprotein on human epithelia. There is a comprehensive list of tumours that are Ber-EP4 positive, as described by Went et al. and Spizzo et al. [2, 3]. The traditional use of Ber-EP4 in histopathology has been limited essentially for differentiation between adenocarcinoma and malignant mesothelioma [4]. This could be due to the fact that other epithelial markers (such as CK) are expressed more often than the CD326 (EpCAM) in epithelial malignancies and thus are more helpful in lineage determination.

We use an algorithmic approach to decide the flow cytometry panel to be applied (Fig. 1). When the clinical details or radiological findings are indicative of a non-hematopoietic malignancy, we apply the CD326 panel. This panel is composed of CD326, CD56 and CD45. CD56 was included in the panel to identify myeloma cells (which may be present in the CD45-negative region) and cells with neuroendocrine differentiation. If, on analysis, there is no CD45-negative population and the sample is composed of predominantly lymphoid cells, a lymphoid screening panel is then used. Samples that are received with diagnosis of suspected lymphoma are initially processed with a routine lymphoid screening panel. In these cases, Ber-EP4 antibody is tested only if large numbers of CD45-negative events are identified.

Method for Ber-EP4 testing
The tissue and fine-needle aspirate (FNA) samples are received fresh in RPMI medium. The tissues are placed on the metal sieve and ground using a glass pestle to form a cell suspension using 2 % PBS-FCS. This suspension is subsequently filtered, which is then washed and lysed. The cell count is ascertained by the cell counter only in cases of larger tissues, where we may have to dilute the sample to adjust the cell count to approximately 10×10⁹/L. FNA and core biopsies are usually paucicellular and do not need a cell count.

The sample is stained with 5 µl of CD45-PC5 [Immunotech SAS (Beckman Coulter)], 20 µl of CD56-PE (Immunotech SAS) and 10 µl of monoclonal mouse anti-human epithelial antigen-FITC conjugated antibody (Clone: Ber-EP4) (Dako Denmark A/S). The sample is then incubated at 4 °C for 30 minutes, followed by a washing step and is ready to be run on the flow cytometer (Beckman Coulter Life Sciences). A total of 10 000 events are acquired with the time threshold set at 300 seconds for the acquisition.

Flow cytometric analysis
The flow cytometric analysis is performed using Navios and Kaluza softwares (Beckman Coulter Life Sciences). The various populations of interest are gated with the focus on identifying the expression of CD326 (with or without CD56) in the CD45-negative population.

Discussion
In our experience of testing for CD326 by flow cytometry, we have been able to comment on the presence or absence of CD326 expression in CD45-negative populations (Figs 2(a, b) and 3). The various carcinomas where we have identified CD326 positivity are: adenocarcinoma, small cell carcinoma, Merkel cell carcinoma, renal cell carcinoma, squamous cell carcinoma, prostate carcinoma, germ cell tumour of testis, and myxoma. We have observed that the expression of CD326 in melanomas can be variable, but they more frequently express CD56. The co-expression of CD326 and CD56 usually indicates a neuroendocrine tumour. Our concordance rate with histopathology using CD326 testing was found to be 97.6 %, which we have published previously [5].

CD326 expression has also been reported to be a prognostic marker with poor outcomes in epithelial ovarian and gall bladder carcinomas [6, 7]. Another important role of this testing could be application in decision making for use of monoclonal antibodies for targeted therapy. The first EpCAM targeting antibody, Catumaxomab (trade name Removab, Fresenius Biotech GmbH) received European market approval in EpCAM-positive carcinomas for the treatment of malignant ascites. Another modification that could be useful in diagnosing epithelial malignancies is to apply Ki67 testing using flow cytometry. This could be done in the same tube as CD326, and thus more information could be obtained with the same amount of sample [8].

There has been considerable data describing the use of the Ber-EP4 antibody in malignant effusions [9–11]. The literature mentions that the presence of epithelial cells in the body fluid should raise the suspicion of metastatic epithelial malignancy, as the reactive body fluids may be composed of lymphocytes and reactive mesothelial cells in varying proportions. There have been multiple studies in the past where flow cytometric CD326 testing has been applied for identifying epithelial cells in body fluid effusions. We have found that our results have a very good concordance with histopathology results. This is in keeping with the findings of Davidson et al., although their study looked at the detection of malignant cells in effusions [12].

The disadvantage of using Ber-EP4 for identifying epithelial differentiation is that there are many epithelial malignancies that do not express CD326 (EpCAM). As mentioned earlier, the use of a broader antibody like cytokeratin (pan-CK) may solve this problem. But unfortunately, such an antibody is not currently available for clinical use by flow cytometry, to the best of our knowledge. Meanwhile, Ber-EP4 should give us the answer in most of the cases. Another disadvantage is that CD326 will be negative in cases of neoplasms of mesenchymal origin, such as sarcomas.
Most flow cytometry laboratories across the world will liaise with histopathology departments for the diagnosis of non-Hodgkin lymphomas. The use of Ber-EP4-testing flow cytometry may play an important role even in epithelial malignancies. The antibody used by us is a CE-marked antibody for in vitro diagnostics and, thus, requires a limited verification process. We followed the method recommended by the manufacturer. The rapid turn-around-time of flow cytometry results makes it a useful screening tool. Our experience shows that flow cytometric testing for CD326 (EpCAM) can be a useful method for diagnosing non-lymphoid malignancies that are poorly differentiated. We suggest that this method would be more useful if the protocol for its application is set up in consultation with the histopathology department, along with setting up a channel of bilateral communication. The histopathologist, based on the flow cytometry information provided, can then set up a more directed immunohistochemical panel. We would like to emphasize at this stage that the aim of the flow cytometric CD326 testing is not to formally diagnose carcinomas, but to highlight the presence of epithelial cells which may lead to the diagnosis of carcinoma. Final classification obviously remains the role of the histopathologist.

References
1. Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 2012; 38(1): 68–75.
2. Went PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum Pathol 2004; 35(1): 122–128.
3. Spizzo G, Fong D, Wurm M, Ensinger C, Obrist P, Hofer C, Mazzoleni G, Gastl G, Went P. EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J Clin Pathol 2011; 64(5): 415–420.
4. Sheibani K, Shin SS, Kezirian J, Weiss LM. Ber-EP4 antibody as a discriminant in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. Am J Surg Pathol 1991; 15(8): 779–784.
5. Dorwal P, Moore H, Stewart P, Harrison B, Monaghan J. CD326 (EpCAM) testing by flow cytometric BerEP4 antibody is a useful and rapid adjunct to histopathology. Cytometry B Clin Cytom 2017; doi: 10.1002/cyto.b.21543.
6. Spizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol 2006; 103(2): 483–488.
7. Varga M, Obrist P, Schneeberger S, Mühlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Schäfer G, et al. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res 2004; 10(9): 3131–3136.
8. Sikora J, Dworacki G, Zeromski J. DNA ploidy, S-phase, and Ki-67 antigen expression in the evaluation of cell content of pleural effusions. Lung 1996; 174: 303-313.
9. Pillai V, Cibas ES, Dorfman DM. A simplified flow cytometric immunophenotyping procedure for the diagnosis of effusions caused by epithelial malignancies. A J Clin Pathol 2013; 139(5): 672–681.
10. Krishan A, Ganjei‐Azar P, Hamelik R, Sharma D, Reis I, Nadji M. Flow immunocytochemistry of marker expression in cells from body cavity fluids. Cytometry A 2010; 77(2): 132–143.
11. Risberg B, Davidson B, Dong HP, Nesland JM, Berner A. Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings. J Clin Pathol 2000; 53(7): 513–517.
12. Davidson B, Dong HP, Berner A, Christensen J, Nielsen S, Johansen P, Bryne M, Asschenfeldt P, Risberg B. Detection of malignant epithelial cells in effusions using flow cytometric immunophenotyping. Am J Clin Pathol 2002; 118(1): 85–92.

The authors
Pranav Dorwal* MBBS, DCP, DNB; Helen Moore MBChB, FRACP, FRCPA
Waikato Hospital, Pembroke St, Hamilton 3204, New Zealand

*Corresponding author
E-mail: Pranav.dorwal@waikatodhb.health.nz

Vitamin D status is currently assessed by measurements of total 25-hydroxyvitamin D [25(OH)D]. However, over 99% of circulating 25(OH)D is bound to protein, vitamin D binding protein in particular. The free hormone hypothesis stipulates that only the free form crosses the cell membrane to exert biologic action. Measurement of free 25(OH)D is now available.

by Professor Daniel D Bikle

Introduction
Circulating levels of 25-hydroxyvitamin D [25(OH)D] are the most commonly used marker for the assessment of vitamin D nutritional status. This is because its concentration in blood is higher than all other vitamin D metabolites, making it easier to measure, and because its conversion from vitamin D is substrate dependent with minimal regulation. However, 25(OH)D is not the most biologically active metabolite of vitamin D. Instead 25(OH)D must be further metabolized to 1,25 dihydroxyvitamin D [1,25(OH)2D] for vitamin D to achieve its full biologic potential. 1,25(OH)2D is the ligand for a nuclear transcription factor, the vitamin D receptor (VDR), that mediates the genomic and at least some of the nongenomic actions of vitamin D within the cell. Nearly all, if not all, cells express the VDR at some stage in their development or activation. As the appreciation that vitamin D and its metabolites affect numerous physiologic processes and not just bone and mineral metabolism, and that these physiologic processes may have different requirements for these vitamin D metabolites, interest in determining optimal levels of the vitamin D metabolites to effect these different biologic processes has grown. Complicating this determination is the fact that all the vitamin D metabolites circulate in blood tightly bound to proteins, of which the vitamin D binding protein (DBP) plays the major role. For most cells, these binding proteins limit the flux of the vitamin D metabolites from blood into the cell where they exert their biologic activity. This raises the issue of what should we measure to determine vitamin D status: the total levels of these metabolites or their free levels?

The free hormone hypothesis: why measure free 25(OH)D
The free hormone hypothesis postulates that only the non-bound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert their biologic effects. This hypothesis applies to steroid hormones, thyroid hormone and vitamin D. For the vitamin D metabolites this hypothesis needs to be qualified in that some tissues, kidney and parathyroid glands in particular, express a transport system, the megalin/cubilin complex, that enables 25(OH)D bound to DBP to be transported into these cells. However, for cells lacking this complex the free fraction is felt to be the fraction capable of entering these cells. In serum samples from normal individuals, ~85% of circulating vitamin D metabolites are bound to DBP, whereas albumin with its substantially lower binding affinity binds only ~15% of these metabolites despite its 10-fold higher concentration than DBP. Approximately 0.4% of total 1,25(OH)2D and 0.02–0.03% of total 25(OH)D is free in serum from normal non-pregnant individuals. The fraction of ‘bioavailable’ vitamin D metabolites is composed of the fraction of the free vitamin D and the fraction bound to albumin, thus measuring around 15% in normal individuals. At this point there is little evidence that the albumin fraction is truly bioavailable. A simple strategy might be to estimate the free concentration based on measurements of DBP and total 25(OH)D with known binding constants of DBP for 25(OH)D. This has in fact been done, but as subsequent research has documented, this relationship is affected by numerous clinical conditions and the different DBP variants with different affinities for 25(OH)D.

DBP
DBP is a 51–58 kDa multifunctional serum glycoprotein synthesized primarily in the liver. Initially, isoelectric focusing migration patterns identified phenotypic variants termed Group-Specific Component (Gc), the most common of which are Gc1f, Gcs and Gc2. Two common missense point mutations (SNPs) in exon 11 of the DBP gene, rs7041 (G/T single-nucleotide variation) and rs4588 (an A/C single-nucleotide variation), result in the three most common isoforms with amino acid changes at positions 416 and 420: Gc1f (Asp416, Thr420), Gc1s (Glu 416, Thr420), and Gc2 (Asp416, Lys420). Gc2 is the least abundant and Gc1f the most abundant. The distribution of the Gc alleles varies by race. Black and Asian populations are more likely to carry the Gc1f form, whereas the Gc2 form is rare, whereas Whites more frequently express the Gc1s and the Gc2 alleles. Although affinities of these DBP variants for 25(OH)D appear to vary, the rank order remains controversial, and their contribution of total 25(OH)D levels and the relationship between free and total 25(OH)D is modest in comparison to differences influenced by clinical condition. In the absence of disease or pregnancy, DBP levels are relatively constant over time in adults. That said, various substances in the blood such as polyunsaturated fatty acids may alter the affinity of DBP for the vitamin D metabolites, as can various clinical conditions. Liver disease leads to reduced levels of DBP, as do protein-losing nephropathies and acute illness (DBP is an acute phase reactant), whereas DBP levels are elevated during the latter stages of pregnancy. Moreover, various clinical conditions appear to shift the relationship between free and total 25(OH)D seemingly independent of DBP levels or DBP haplotypes. Thus, the measurement of total 25(OH)D may not provide the best assessment of vitamin D status. Calculation of free 25(OH)D from DBP and total 25(OH)D measurements using affinity constants obtained by measurements in normal sera may be inaccurate, at least in some clinical situations. Therefore, direct measurement of free 25(OH)D would appear to offer information about vitamin D nutritional status that at least complements that of total 25(OH)D.

The free 25(OH)D assay
The original free 25(OH)D assay employed centrifugal ultrafiltration. This was a labour- and reagent-intensive assay suitable only for a dedicated research laboratory. However, it sufficed to determine free 25(OH)D levels in a number of patient groups including cirrhotics and pregnant women, providing proof of concept that the free 25(OH)D measurement would add to the assessment of vitamin D nutritional status. This assay has subsequently been superseded by a much simpler method capable of high throughput.

A two-step ELISA that directly measures free 25(OH)D levels was recently developed by Future Diagnostics Solutions using monoclonal antibodies from DIAsource Immunoassays. In the first incubation step, an anti-25(OH)D monoclonal antibody immobilized on a microtitre plate binds the free 25(OH)D in the serum sample. The serum is removed and biotinylated 25(OH)D in a known amount is added to react with the unoccupied binding sites on the monoclonal antibody attached to the plate. The non-bound biotinylated 25(OH)D is then removed followed by the addition of streptavidin peroxidase conjugate and the substrate 3,3ʹ,5,5ʹ-Tetramethylbenzidine (TMB). The bound streptavidin peroxidase can be quantified by measuring the absorbance at 450 nm generated in the reaction. The intensity is inversely proportional to the level of free 25(OH)D. The limit of detection is 2.8 pg/mL. The antibody in the current assay does not recognize 25(OH)D₂ as well as 25(OH)D₃ (77% of the 25(OH)D₃ value), and so it underestimates the free 25(OH)D₂. However, under most situations where the predominant vitamin D metabolite is 25(OH)D₃ this issue is not a major concern. The data for both normal subjects and those with different DBP levels (cirrhotics, pregnant women) compare quite well to those obtained from similar populations using the centrifugal ultrafiltration assay.

Clinical implications
In a study currently under review for publication we compiled data from over 1600 individuals in whom free 25(OH)D had been measured by this ELISA. The samples included sera from both normal subjects and those with a variety of clinical conditions and a variety of DBP alleles. In the nearly 1000 normal and community dwelling outpatient subjects the normal range for free 25(OH)D was established at 4.3±1.9 pg/mL with a mean total 25(OH)D of 21.9±9.9 ng/mL, providing a percent free 25(OH)D of 0.02%. These results are essentially identical to those reported by the author using centrifugal ultrafiltration 30 years ago. As expected, clinical conditions affecting DBP values made a big difference. Liver disease resulted in lower DBP levels and higher percentage free 25(OH)D resulting in the population of cirrhotics studied having among the highest free 25(OH)D despite the lowest total 25(OH)D. Nursing home patients also had unexpectedly high free 25(OH)D, higher than that of the cirrhotics, with only modest reductions in DBP levels. Pregnancy (third trimester), however, resulted in increased DBP levels and the lowest free 25(OH)D levels, although the free fraction was not lower than that of the normal subjects. Overall, these results indicate that the free fraction is altered by the clinical situation not only in terms of altered DBP levels but in the relationship between total and free 25(OH)D for any given DBP level. Therefore, it is recommended that the free 25(OH)D level needs to be measured directly if the free level is thought to have particular relevance to the clinical situation that cannot be captured by measuring total 25(OH)D.

At this point it is not yet clear whether the determination of free 25(OH)D is a better marker of vitamin D nutritional status and biologic action than the determination of total 25(OH)D. Using a convenient marker such as parathyroid hormone (PTH), much as we use thyroid-stimulating hormone (TSH) as a marker of thyroid status, is problematic. First of all PTH levels are controlled by calcium as well as
vitamin D. Second, regulation of PTH secretion is mediated primarily by the 1,25(OH)₂D produced within the gland itself (much as TSH secretion is controlled by triiodothyronine (T3) produced within the pituitary). Third, the parathyroid gland has the megalin/cubilin transport system to enable 25(OH)D bound to DBP to enter the cells, obviating any advantage free 25(OH)D might have in cell uptake. However, several studies have demonstrated a stronger correlation between free 25(OH)D and bone markers than that observed with total 25(OH)D. But at this point, determining the role that free 25(OH)D measurements play in the assessment of vitamin D nutrition and action requires further investigation.

Bibliography
1. Bikle DD. Vitamin D Assays. Front Horm Res 2018; 50: 14–30.
2. Malstroem S, Rejmark L, et al. Current assays to determine free 25-hydroxyvitamin D in serum. J AOAC Internl 2017; 100: 1323–1327.
3. Bikle D, Bouillon R, et al. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? J Steroid Biochem Mol Biol 2017; 173: 1054–1116.
4. Bikle DD, Malmstroem S, Schwartz J. Current controversies: are free vitamin metabolite levels a more accurate assessment of vitamin D status than total levels? Endo Clinics NA 2017; 46: 901–918.
5. Lai JC, Bikle DD, et al. Total 25(OH) vitamin D, free 25(OH) vitamin D, and markers of bone turnover in cirrhotics with and without synthetic dysfunction.  Liver Int 2015; 35: 2294–2300.
6. Schwartz JB,  Lai J, et al. A comparison of direct and calculated free 25-OH vitamin D levels in clinical populations. J Clin Endocrinol Metab 2014; 99: 1631–1637.

The author
Daniel D Bikle MD, PhD
VA Medical Center and University of
California San Francisco, San Francisco,
CA 94158, USA

E-mail: Daniel.bikle@ucsf.edu

CTC-derived AR-V7 detection as a prognostic and predictive biomarker in advanced prostate cancer
^{Bastos DA, Antonarakis ES. Expert Rev Mol Diagn 2018; 18(2): 155–163}

Prostate cancer is a highly heterogeneous disease, with remarkably different prognosis across all stages. Increased circulating tumour cell (CTC) count (≥5) using the CellSearch assay has been identified as one of the markers that can be used to predict survival, with added value beyond currently available prognostic factors. Recently, androgen receptor splice variant 7 (AR-V7) detection has been associated with worse outcomes for patients with castration-resistant prostate cancer (CRPC) treated with novel androgen receptor-signalling (ARS) inhibitors such as abiraterone and enzalutamide but not taxane chemotherapies. Areas covered: In this manuscript, the authors review the available biomarkers in CRPC and discuss emerging data on the value of CTC-derived AR-V7 status to assess prognosis and its potential role to guide treatment selection for patients with advanced prostate cancer. Expert commentary: Current evidence supports AR-V7 status as a prognostic biomarker and also as a potential predictive biomarker for patients with mCRPC. The authors expect that the incorporation of AR-V7 status and other biomarkers (e.g. AR mutations) in the sequential assessment of patients with advanced prostate cancer will lead to a more rational use of available and future therapies, with significant improvements in outcomes for our patients.

Defining a cohort of men who may not require repeat prostate biopsy based on PCA3 score and MRI: The dual negative effect
^{Perlis N, Al-Kasab T, Ahmad A, Goldberg E, Fadak K, Sayid R, et al. J Urol 2017; doi: 10.1016/j.juro.2017.11.07}

PURPOSE: Prostate cancer over diagnosis and overtreatment are concerns for clinicians and policy makers. Multiparametric magnetic resonance imaging and the PCA3 (prostate cancer antigen 3) urine test select for clinically significant cases. We explored how well the tests performed together in with previous biopsies.

MATERIALS AND METHODS: In accordance with ethics committee approval we collected clinicopathological data on all patients in whom a PCA3 test from was done 2011 to June 2016. This included patients on active surveillance for low-risk prostate cancer and those without prostate cancer who had previous negative biopsies and suspicion of occult disease. We explored whether age, prostate-specific antigen, PCA3 score, multiparametric magnetic resonance imaging, digital rectal examination, family history and prostate size would predict clinically significant prostate cancer on repeat biopsy. The negative predictive value of multiparametric magnetic resonance imaging and PCA3 score was calculated.

RESULTS: A total of 470 patients were included in study. The PCA3 score was abnormal at 35 or greater in 32.5 % of cases. In the multivariate model including 154 men only age (OR 1.08, 95 % CI 1.01–1.16), multiparametric magnetic resonance imaging PI-RADS™ (Prostate Imaging-Reporting and Data System) score 4 (OR 16.6, 95 % CI 3.9–70.0) or 5 (OR 28.3, 95 % CI 5.7–138) and PCA3 score (OR 2.9, 95 % CI 1.0–8.8) predicted clinically significant cancer on biopsy. No patient with negative multiparametric magnetic resonance imaging and a normal PCA3 score had clinically significant prostate cancer on biopsy for a negative predictive value of 100 % (p<0.0001).

CONCLUSIONS: In patients with dual negative tests (multiparametric magnetic resonance imaging and PCA3 score) clinically significant prostate cancer was never found on biopsy, which may be unnecessary in this group. This study was limited by its retrospective design, selection bias and lack of cost-effectiveness data.

Quantitative mass spectrometry-based proteomic profiling for precision medicine in prostate cancer
^{Flores-Morales A, Iglesias-Gato D. Front Oncol 2017; 7: 267}

Prostate cancer (PCa) is one of the most frequently diagnosed cancer among men in the western societies. Many PCa patients bear tumours that will not threat their lives if left untreated or if treatment is delayed. Our inability for early identification of these patients has resulted in massive overtreatment. Therefore, there is a great need of finding biomarkers for patient stratification according to prognostic risk; as well as there is a need for novel targets that can allow the development of effective treatments for patients that progress to castration-resistant PCa. Most biomarkers in cancer are proteins, including the widely-used prostate-specific antigen (PSA). Recent developments in mass spectrometry allow the identification and quantification of thousands of proteins and posttranslational modifications from small amounts of biological material, including formalin-fixed paraffin-embedded tissues, and biological fluids. Novel diagnostic and prognostic biomarkers have been identified in tissue, blood, urine, and seminal plasma of PCa patients, and new insights in the ethology and progression of this disease have been achieved using this technology. In this review, we summarize these findings and discuss the potential of this technology to pave the way toward the clinical implementation of precision medicine in PCa.

Biomarkers for prostate biopsy and risk stratification of newly diagnosed prostate cancer patients
^{Loeb S. Urol Pract 2017; 4(4): 315–321}

INTRODUCTION: Many new markers are now available as an aid for decisions about prostate biopsy for men without prostate cancer, and/or to improve risk stratification for men with newly diagnosed prostate cancer.

METHODS: A literature review was performed on currently available markers for use in decisions about prostate biopsy and initial prostate cancer treatment.

RESULTS: Although total prostate-specific antigen cutoffs were traditionally used for biopsy decisions, PSA elevations are not specific. Repeating the PSA test, and adjusting for factors like age, prostate volume and changes over time can increase specificity for biopsy decisions. The Prostate Health Index (phi) and 4K Score are new PSA-based markers that can be offered as second-line tests to decide on initial or repeat prostate biopsy. The PCA3 urine test and ConfirmMDx tissue test are additional options for repeat biopsy decisions. For men with newly diagnosed prostate cancer, genomic tests are available to refine risk classification and may influence treatment decisions.

CONCLUSIONS: Numerous secondary testing options are now available that can be offered to patients deciding whether to undergo prostate biopsy and those with newly diagnosed prostate cancer.

Bidirectional electrochemiluminescence color switch: an application in detecting multimarkers of prostate cancer
^{Wang YZ, Ji SY, Xu HY, Zhao W, Xu JJ, Chen HY. Anal Chem 2018; doi: 10.1021/acs.analchem.8b00014}

A selective excitation of [Ir(df-ppy)₂(pic)] and [Ru(bpy)3]²⁺ through tuning the electrode potential is reported in this work. Bidirectional colour change from blue-green to red could be observed along with increase and decrease of the potential, which was ascribed to the dual-potential excitation property of [Ir(df-ppy)₂(pic)]. Similar to the three-electrode system, selective excitation of ECL could be achieved at the anode of the bipolar electrode (BPE). Both increase and decrease of the faradic reactions at the cathode of the BPE could induce ECL reporting colour at the other pole switched from blue-green to red. We applied a closed BPE device for the bioanalysis of multicolour ECL since the organic solvent containing electrochemiluminophores could be separated from the bioanalytes. On the basis of BPE arrays coupled with the ECL switch, the detection of three biomarkers of prostate cancer, PSA, microRNA-141, and sarcosine were integrated in a same device. The cutoff values of the biomarkers could be recognized directly by the naked eye. Such a device holds great potential in the early diagnosis of prostate cancer.

Molecular biomarkers in the clinical management of prostate cancer
^{Udager AM, Tomlins SA. Cold Spring Harb Perspect Med 2018; doi: 10.1101/cshperspect.a030601}

Prostate cancer, one of the most common non-cutaneous malignancies in men, is a heterogeneous disease with variable clinical outcome. Although the majority of patients harbour indolent tumours that are essentially cured by local therapy, subsets of patients present with aggressive disease or recur/progress after primary treatment. With this in mind, modern clinical approaches to prostate cancer emphasize the need to reduce overdiagnosis and overtreatment via personalized medicine. Advances in our understanding of prostate cancer pathogenesis, coupled with recent technologic innovations, have facilitated the development and validation of numerous molecular biomarkers, representing a range of macromolecules assayed from a variety of patient sample types, to help guide the clinical management of prostate cancer, including early detection, diagnosis, prognostication, and targeted therapeutic selection. Herein, we review the current state of the art regarding prostate cancer molecular biomarkers, emphasizing those with demonstrated utility in clinical practice.

Genomic markers in prostate cancer decision making
^{Cucchiara V, Cooperberg MR, Dall’Era M, Lin DW, Montorsi F, Schalken JA, et al. Eur Urol. 2017; doi: 10.1016/j.eururo.2017.10.036}

CONTEXT: Although the widespread use of prostate-specific antigen (PSA) has led to an early detection of prostate cancer (PCa) and a reduction of metastatic disease at diagnosis, PSA remains one of the most controversial biomarkers due to its limited specificity. As part of emerging efforts to improve both detection and management decision making, a number of new genomic tools have recently been developed.

OBJECTIVE: This review summarizes the ability of genomic biomarkers to recognize men at high risk of developing PCa, discriminate clinically insignificant and aggressive tumours, and facilitate the selection of therapies in patients with advanced disease.

EVIDENCE ACQUISITION: A PubMed-based literature search was conducted up to May 2017. The most recent and relevant original articles and clinical trials that have provided indispensable information to guide treatment decisions were selected.

EVIDENCE SYNTHESIS: Genome-wide association studies have identified several genetic polymorphisms and inherited variants associated with PCa susceptibility. Moreover, the urine-based assays SelectMDx, Mi-Prostate Score, and ExoDx have provided new insights into the identification of patients who may benefit from prostate biopsy. In men with previous negative pathological findings, Prostate Cancer Antigen 3 and ConfirmMDx predicted the outcome of subsequent biopsy. Commercially available tools (Decipher, Oncotype DX, and Prolaris) improved PCa risk stratification, identifying men at the highest risk of adverse outcome. Furthermore, other biomarkers could assist in treatment selection in castration-resistant PCa. AR-V7 expression predicts resistance to abiraterone/enzalutamide, while poly(ADP-ribose) polymerase-1 inhibitor and platinum-based chemotherapy could be indicated in metastatic patients who are carriers of mutations in DNA mismatch repair genes.

CONCLUSIONS: Introduction of genomic biomarkers has dramatically improved the detection, prognosis, and risk evaluation of PCa. Despite the progress made in discovering suitable biomarker candidates, few have been used in a clinical setting. Large-scale and multi-institutional studies are required to validate the efficacy and cost utility of these new technologies.

PATIENT SUMMARY: Prostate cancer is a heterogeneous disease with a wide variability. Genomic biomarkers in combination with clinical and pathological variables are useful tools to reduce the number of unnecessary biopsies, stratify low-risk from high-risk tumours, and guide personalized treatment decisions.

The use of biomarkers in prostate cancer screening and treatment
^{Ashley VA, Joseph MB, Kamlesh KY, Shalini SY, Ashutosh KT, Joseph R. Rev Urol 2017; 19(4): 221–234}

Prostate cancer screening and diagnosis has been guided by prostate-specific antigen levels for the past 25 years, but with the most recent US Preventive Services Task Force screening recommendations, as well as concerns regarding overdiagnosis and overtreatment, a new wave of prostate cancer biomarkers has recently emerged. These assays allow the testing of urine, serum, or prostate tissue for molecular signs of prostate cancer, and provide information regarding both diagnosis and prognosis. In this review, we discuss 12 commercially available biomarker assays approved for the diagnosis and treatment of prostate cancer. The results of clinical validation studies and clinical decision-making studies are presented. This information is designed to assist urologists in making clinical decisions with respect to ordering and interpreting these tests for different patients. There are numerous fluid and biopsy-based genomic tests available for prostate cancer patients that provide the physician and patient with different information about risk of future disease and treatment outcomes. It is important that providers be able to recommend the appropriate test for each individual patient; this decision is based on tissue availability and prognostic information desired. Future studies will continue to emphasize the important role of genomic biomarkers in making individualized treatment decisions for prostate cancer patients.

A four-kallikrein panel and β-microseminoprotein in predicting high-grade prostate cancer on biopsy: an independent replication from the Finnish Section of the European Randomized Study of Screening for Prostate Cancer
^{Assel M, Sjöblom L, Murtola TJ, Talala K, Kujala P, Stenman UH, et al. Eur Urol Focus 2017; doi: 10.1016/j.euf.2017.11.002}

BACKGROUND: A panel of four kallikrein markers (total, free, and intact prostate-specific antigen [PSA] and human kallikrein-related peptidase 2 [hK2]) improves predictive accuracy for Gleason score ≥7 (high-grade) prostate cancer among men biopsied for elevated PSA. A four-kallikrein panel model was originally developed and validated by the Dutch centre of the European Randomized Study of Screening for Prostate Cancer (ERSPC). The kallikrein panel is now commercially available as 4Kscore™.

OBJECTIVE: To assess whether these findings could be replicated among participants in the Finnish section of ERSPC (FinRSPC) and whether β-microseminoprotein (MSP), a candidate prostate cancer biomarker, adds predictive value.

DESIGN, SETTING, AND PARTICIPANTS: Among 4861 biopsied screening-positive participants in the first three screening rounds of FinRSPC, a case-control subset was selected that included 1632 biopsy-positive cases matched by age at biopsy to biopsy-negative controls.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The predictive accuracy of prespecified prediction models was compared with biopsy outcomes.

RESULTS AND LIMITATIONS: Among men with PSA of 4.0–25 ng/ml, 1111 had prostate cancer, 318 of whom had high-grade disease. Total PSA and age predicted high-grade cancer with an area under the curve of 0.648 (95 % confidence interval [CI] 0.614–0.681) and the four-kallikrein panel increased discrimination to 0.746 (95 % CI 0.717–0.774). Adding MSP to the four-kallikrein panel led to a significant (Wald test; p=0.015) but small increase (0.003) in discrimination. Limitations include a risk of verification bias among men with PSA of 3.0–3.99 ng/ml and the absence of digital rectal examination results.
CONCLUSIONS: These findings provide additional evidence that kallikrein markers can be used to inform biopsy decision making. Further studies are needed to define the role of MSP.

PATIENT SUMMARY: Four kallikrein markers and β-microseminoprotein in blood improve discrimination of high-grade prostate cancer at biopsy in men with elevated prostate-specific antigen.

Combinations of elevated tissue miRNA-17-92 cluster expression and serum prostate-specific
antigen as potential diagnostic biomarkers for prostate cancer
^{Feng S, Qian X, Li H, Zhang X. Oncol Lett 2017; 14(6): 6943–6949}

The aim of the present study was to investigate the effectiveness of the miR-17-92 cluster as a disease progression marker in prostate cancer (PCa). Reverse transcription-quantitative polymerase chain reaction analysis was used to detect the microRNA (miR)-17-92 cluster expression levels in tissues from patients with PCa or benign prostatic hyperplasia (BPH), in addition to in PCa and BPH cell lines. Spearman correlation was used for comparison and estimation of correlations between miRNA expression levels and clinicopathological characteristics such as the Gleason score and prostate-specific antigen (PSA). Receiver operating curve (ROC) analysis was performed for evaluation of specificity and sensitivity of miR-17-92 cluster expression levels for discriminating patients with PCa from patients with BPH. Kaplan-Meier analysis was plotted to investigate the predictive potential of miR-17-92 cluster for PCa biochemical recurrence. Expression of the majority of miRNAs in the miR-17-92 cluster was identified to be significantly increased in PCa tissues and cell lines. Bivariate correlation analysis indicated that the high expression of unregulated miRNAs was positively correlated with Gleason grade, but had no significant association with PSA. ROC curves demonstrated that high expression of miR-17-92 cluster predicted a higher diagnostic accuracy compared with PSA. Improved discriminating quotients were observed when combinations of unregulated miRNAs with PSA were used. Survival analysis confirmed a high combined miRNA score of miR-17-92 cluster was associated with shorter biochemical recurrence interval. miR-17-92 cluster could be a potential diagnostic and prognostic biomarker for PCa, and the combination of the miR-17-92 cluster and serum PSA may enhance the accuracy for diagnosis of PCa.


Prostate-specific antigen screening impacts on biochemical recurrence in patients with clinically localized prostate cancer
^{Hashimoto T, Ohori M, Shimodaira K, Kaburaki N, Hirasawa Y, Satake N, Gondo T, Nakagami Y, Namiki K, Ohno Y. Int J Urol 2018; doi: 10.1111/iju.13563}

OBJECTIVE: To clarify the impact of prostate-specific antigen screening on surgical outcomes of prostate cancer.

METHODS: Patients who underwent radical prostatectomy were divided into two groups according to prostate-specific antigen testing opportunity (group 1, prostate-specific antigen screening; group 2, non-prostate-specific antigen screening). Perioperative clinical characteristics were compared using the Wilcoxon rank-sum and χ2 -tests. Cox proportional hazards models were used to identify independent predictors of postoperative biochemical recurrence-free survival.

RESULTS: In total, 798 patients (63.2 %) and 464 patients (36.8 %) were categorized into groups 1 and 2, respectively. Group 2 patients were more likely to have a higher prostate-specific antigen level and age at diagnosis and larger prostate volume. Clinical T stage, percentage of positive cores and pathological Gleason score did not differ between the groups. The 5-year biochemical recurrence-free survival rate was 83.9 % for group 1 and 71.0 % for group 2 (p<0.001). On multivariate analysis, prostate-specific antigen testing opportunity (hazard ratio 2.530; p<0.001) was an independent predictive factor for biochemical recurrence after surgery, as well as pathological T stage, pathological Gleason score, positive surgical margin and lymphovascular invasion. Additional analyses showed that prostate-specific antigen screening had a greater impact on biochemical recurrence in a younger patients, patients with a high prostate-specific antigen level, large prostate volume and D’Amico high risk, and patients meeting the exclusion criteria of the Prostate Cancer Research International Active Surveillance study.

CONCLUSIONS: Detection by screening results in favourable outcomes after surgery. Prostate-specific antigen screening might contribute to reducing biochemical recurrence in patients with localized prostate cancer.