Prostate cancer is the most common cancer in men and diagnosis involves a combination of assessments. Levels of the biomarker prostate-specific antigen (PSA) are commonly measured, but do not always equate to cancer status. Testing of PSA in combination with other biomarkers may help to improve diagnostic and prognostic accuracy, as well as minimizing overdiagnosis as well as unnecessary intervention. This article provides a summary of the information currently known about biomarkers showing promise for use in prostate cancer screening.
by Dr Alexandra Tabakin, Dr Sung Un Bang and Dr Isaac Y. Kim
Introduction
Prostate cancer is the most commonly diagnosed cancer type in the USA and second leading cause of cancer deaths in men. In 2018, there will be an estimated 164 690 new cases with an estimated 29 430 deaths [1]. Although many prostate cancers are indolent in nature and can be safely monitored with active surveillance, a significant proportion of patients will require intervention with surgery, radiation, or other therapies. One of the major challenges in treating prostate cancer is risk stratification and differentiating clinically significant prostate cancers in order to avoid overdiagnosis and overtreatment [2]. To do so, patients undergo risk stratification, which conventionally includes a combination of prostate-specific antigen (PSA) screening, digital rectal exam (DRE), trans-rectal ultrasonography (TRUS)-guided biopsy. Currently clinically insignificant prostate cancer, according to Epstein criteria, is defined as cancer with preoperative PSA <10ng/ml, clinical stage <T1c, Gleason score <6, PSA density <0.15, <2 positive biopsy cores, and <50% cancer involvement in any core [3–5]. However, as we learn more about the heterogeneity of prostate cancer tumours, various serum biomarkers have been investigated to improve diagnostic and prognostic accuracy and minimize treatment. In this review, we discuss various biomarkers and their utilization for the prediction of clinically significant prostate cancer.
Serum biomarkers
PSA
PSA, or prostate-specific antigen, is a serine protease released by the prostate. PSA gained notoriety in the 1980s when it was reported to have various uses including screening, monitoring disease progression, and detecting recurrence of the cancer. As PSA screening for prostate cancer increased, the incidence of prostate cancer also rose in the 1990s. However, PSA only has a 25–40% specificity rate for prostate cancer and can be elevated with infection, trauma, and benign prostatic hyperplasia (BPH) [6]. In fact, about 15% of men with a low level of PSA (<4.0 ng/ml) have prostate cancer [7].
Because the harms of biopsy and prostate cancer treatment may outweigh the benefits in some patients with clinically insignificant prostate cancer, efforts have been made to improve the validity of PSA in differentiating benign conditions and prostate cancer. One such effort is the use of free PSA; those with an elevated PSA and a lower serum percentage of free PSA (%f-PSA) are more likely to have BPH rather than prostate cancer [8]. The Prostate Health Index (PHI) formula incorporates serum total PSA, free PSA, and the [−2]proPSA to discriminate Gleason 3+4 and greater cancers with 90% sensitivity and 17% specificity [9]. 4K score is another novel test which is comprised of total PSA, free PSA, intact PSA, and human kallikrein-related peptidase 2 to detect clinically significant prostate cancer and discern those who would benefit from a prostate biopsy while preventing 30–58% of biopsies [10].
PAP
Prostatic acid phosphatase (PAP) was the first popularized prostate cancer serum biomarker, but was eventually replaced by PSA, as it is less sensitive in diagnosing prostate cancer and detecting recurrence. Elevated PAP level has been associated with a high risk of bone metastases [11], significantly shortened overall survival [12] as well as disease-free survival [13], and increased risk of biochemical recurrence [14]. Interestingly, recent studies have shown that PAP may be useful in detecting high-risk clinically significant prostate cancer patients; it is speculated that PAP may be associated with micro-metastatic disease prior to treatment, and therefore, predict response to treatment [15].
NLR, ANC, ALC
Inflammation and tissue microenvironment are important factors in cancer development. Although the temporal relationship is not well established, a lymphocyte-mediated immune response is thought to occur early in the development of prostate cancer. Neutrophil-to-lymphocyte ratio (NLR) compares the activity of both neutrophils and lymphocytes in the inflammatory response. NLR is a useful prognostic biomarker in mCRPC, where a higher score correlates less relative lymphocyte activity and a worse prognosis. When looking at localized low-risk prostate cancer, studies have shown that NLR is not associated with upstaging, upgrading, or biochemical recurrence. However, increased absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) were associated with upstaging and lower 5-year biochemical recurrence-free survival. More development on these markers may guide clinicians in re-stratifying patients who meet conventional criteria for low-risk prostate cancer [16].
Urine biomarkers
PCA3
Urinary prostate cancer antigen 3 (PCA3) is among the most promising non-invasive biomarker among non-PSA based tests, as it is overexpressed in over 95% of prostate cancers [6]. PCA3 is a long noncoding RNA collected from shed prostate cells during urination. PCA3 is a more specific biomarker for prostate cancer because, unlike PSA, PCA3 levels are not influenced by prostate size, BPH, prostatitis, or the use of 5α-reductase inhibitors [17]. At the genetic level, PCA3 may help distinguish between prostate cancer and high-grade prostatic intraepithelial neoplasia (HG-PIN), as PCA3 is seldom expressed in HG-PIN [18]. Several studies have demonstrated that a higher PCA3 score correlates with clinically significant prostate cancer and larger tumour volume, therefore potentially aiding in selecting patients for active surveillance [19]. In 2012, the FDA approved the PROGENSA PCA3 assay predict men who would benefit from a repeat biopsy in those with a previous negative prostate biopsy [6]. In a recent meta-analysis of 46 clinical trials, the sensitivity and specificity of PROGENSA PCA3 was 65% and 73%, respectively [20].
Genetic mutations
TMPRSS2::ERG
TMPRSS2::ERG, or T2:ERG, gene fusions constitute 90% of gene fusions implicated in prostate cancer, as well as half of all prostate cancers [21, 22]. When the androgen responsive regulatory element TMPRSS2 and the gene for the transcription factor ERG are fused together, androgen-driven genes are overexpressed and tumorigenesis occurs [21]. When used alone, urinary T2:ERG RNA has a reported 86% specificity and 45% sensitivity in prostate cancer detection. However, when used in conjunction with PCA3, specificity and sensitivity improve to 90% and 80%, respectively [6, 23]. Moreover, this test may prevent up to 42% of unnecessary biopsies, limiting healthcare costs [24].
PTEN
Loss of the tumour-suppressor gene, PTEN, or phosphatase and tensin homologue, leading to activation of the PI3K/AKT/mTOR signalling has been found in both early stage and castrate-resistant prostate cancers. Preclinical data show that PI3K pathway activation is related to resistance to androgen deprivation, leading to disease progression and poor response to treatment. In mouse models, conditional deletion of PTEN initially led to the development of prostate hyperplasia and later on invasive and metastatic prostate cancers likely by modulating the p110β catalytic subunit of PI3K. In addition, ablation of p110β hindered AKT signalling and reduced tumorigenesis [25]. In a cohort of 77 men, loss of PTEN at initial prostate biopsy was predictive of the development of castrate-resistant prostate cancer, response to androgen deprivation therapy, and prostate-cancer-specific mortality [26]. Additionally, decreased PTEN expression has been associated with increased risk of biochemical and clinical recurrence after prostatectomy [27]. Therefore, the association between castrate-resistant prostate cancer and PTEN loss as well as PI3K/AKT/mTOR pathway activation suggests that PTEN may have prognostic value in prostate cancer risk stratification.
CHD1
The CHD1 gene, encoding chromodomain helicase DNA-binding protein 1, is commonly deleted in 10–26% of all prostate cancers. The loss of CHD1 affects the ability to repair DNA double-strand breaks via homologous recombination. CHD1 deletion has generally been associated with a poor prognosis. However, studies have demonstrated tumours with CHD1 deletions to be sensitive to both PARP inhibitors and carboplatin, both in vitro and in vivo, suggesting that future research may be able to identify to response to treatment based off of tumour genotypes [28].
Circulating biologics
Circulating tumour cells
Circulating tumour cells (CTCs) are defined as cells that leave the site of a primary cancer, travel through the bloodstream, and settle at other sites in the body where they grow into new tumours, or metastases [29]. In 2004, CellSearch Circulating Tumor Cell System was approved by the FDA as an assay for enumerating CTCs. In recent studies, CTC count, deemed the ‘liquid biopsy’, has been shown to have a role in predicting overall survival and response to treatment, risk stratification, and detecting metastases. In addition, using CTCs to detect biomarkers, such as ERG, PTEN, and AR may allow for personalized treatments based on tumour genomes [9, 30].
Circulating exosomes
Circulating prostate cancer-related exosomes are double-membrane vesicles carrying RNA and pro-oncogenic molecules, which induce malignant transformation in normal cells. ExoDx prostate Intelliscore urine exosome assay (developed by Exosome Diagnostics Inc.) detects exosomal RNA expression of ERG, PCA3 and SPDEF (SAM pointed domain containing ETS transcription factor) in voided urine samples. A score is generated that is able to predict high-grade prostate cancer (Gleason >7) with a negative predictive value of 91%. This assay may be useful in discriminating clinically significant prostate cancer and reducing the number of unnecessary biopsies [31].
Conclusion
The emergence and widespread usage of PSA as a routine screening test for prostate cancer allows for early detection and risk stratification. Testing for PSA in combination with novel biomarkers such as PCA3, TMPRSS2::ERG, PTEN, and others may improve our diagnostic abilities, given the heterogeneity of prostate cancers and their treatments. There are many challenges in establishing useful biomarkers including creating affordable and accessible testing, determining cut-offs, and determining accuracy. As the clinical utility of these biomarkers is further defined, we hope to better risk stratify patients and select appropriate treatments early on in diagnosis. Further research efforts should focus on the synergism between the Epstein criteria, biomarker utility, and how to best bring these tools from bench to bedside.
References
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15. Faiena I, Kim S, Farber N, Kwon YS, Shinder B, Patel N, et al. Predicting clinically significant prostate cancer based on pre-operative patient profile and serum biomarkers. Oncotarget 2017; 8(65): 109783–109790.
16. Kwon YS, Han CS, Yu JW, Kim S, Modi P, Davis R, et al. Neutrophil and lymphocyte counts as clinical markers for stratifying low-risk prostate cancer. Clin Genitourin Cancer 2016; 14(1): e1–8.
17. Roobol MJ, Schröder FH, van Leeuwen P, Wolters T, van den Bergh RCN, van Leenders GJLH, et al. Performance of the prostate cancer antigen 3 (PCA3) gene and prostate-specific antigen in prescreened men: exploring the value of PCA3 for a first-line diagnostic test. Eur Urol 2010; 58(4): 475–481.
18. Wei W, Leng J, Shao H, Wang W. High PCA3 scores in urine correlate with poor-prognosis factors in prostate cancer patients. Int J Clin Exp Med 2015; 8(9): 16606–16612.
19. Ploussard G, Epstein JI, Montironi R, Carroll PR, Wirth M, Grimm M-O, et al. The contemporary concept of significant versus insignificant prostate cancer. Eur Urol 2011; 60(2): 291–303.
20. Cui Y, Cao W, Li Q, Shen H, Liu C, Deng J, et al. Evaluation of prostate cancer antigen 3 for detecting prostate cancer: a systematic review and meta-analysis. Sci Rep 2016; 6: 25776.
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The authors
Alexandra Tabakin MD,
Sung Un Bang MD, Isaac Yi Kim MD PhD
Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey and Division of Urology, Rutgers Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA
*Corresponding author
E-mail: kimiy@cinj.rutgers.edu
A Technology Evolution in Critical Care Testing
, /in Featured Articles /by 3wmediaSTA-NeoPTimal – The new PT reagent
, /in Featured Articles /by 3wmediaTowards a diagnostic test for Parkinson’s disease
, /in Featured Articles /by 3wmediaParkinson’s disease (PD) is a chronic neurological disorder affecting one in 100 people over the age of 60, with estimates suggesting that approximately 5 million people are suffering from the condition worldwide. PD develops when the dopamine-producing neurons of the substantia nigra part of the brain are lost over time. Dopamine is needed for the coordination of movement, the loss of which is therefore responsible for the appearance of the main PD symptoms of stiffness, tremor and slowness. There is no cure for PD and treatment is aimed at managing symptoms, with medication being effective only in the short term. Currently there is no clinical test for PD, but diagnosis is based on medical history and assessment of simple physical tasks. Additionally, most instances of PD are idiopathic, with the risks from genetic and/or environmental causes being very low, except in certain rare cases. Hence, diagnosis, particularly in the early stages of symptoms, can be difficult and inconclusive. Additionally, as with many of the neurodegenerative conditions, physical symptoms only become apparent late in the development of the condition – after the loss of 80% of dopamine. However, the help of a woman with a remarkable sense of smell is bringing the creation of a definitive clinical test for PD closer. Joy Milne is a retired nurse from Perth, Scotland, whose husband Les, a consultant anesthetist, was diagnosed with PD at the age of 45 and died at 65. Approximately 10 years before the diagnosis, Joy realized that Les had developed a different, slightly muskier smell. After meeting other people with PD, Joy found that they all had the same unusual aroma. Joy’s ability to detect PD by smell was confirmed in tests conducted by scientists at the University of Edinburgh and she is now working with Dr Perdita Barran at the University of Manchester to isolate the specific compounds that create the distinctive PD aroma. So far, a handful of compounds have been identified. Currently, a definitive clinical test would allow a conclusive diagnosis for patients suffering from the varied and vague early symptoms of PD. In the future, however, given the lack of identifiable risk factors and the fact that the changes responsible for PD as well as the development of the unusual PD aroma happen up to a decade before external physical symptoms appear, for any medication that will cure or at least prevent disease progression to have any real chance of success, screening of the apparently healthy, asymptomatic population will have to be carried out.
Zika virus: current methods of detection and corresponding limitations
, /in Featured Articles /by 3wmediaZika virus (ZIKV) has recently become a global threat owing to the link between infection, Guillain–Barré syndrome and serious neurological defects in unborn fetus and infants. There are major challenges associated with the detection methods that are currently available for the virus, and there is no point-of-care test to accurately and quickly detect ZIKV. Herein, we describe the advantages and disadvantages of the methods that are used presently, and provide an insight into developing technologies that will yield improved detection in the future.
by Devon Pawley, Dr Emre Dikici, Dr Sapna Deo and Prof. Sylvia Daunert
Background
Infectious diseases are a serious public health concern and are the leading cause of death in low income countries [1]. The World Health Organization (WHO) declared the potential impact of the Zika virus (ZIKV) a global public health emergency in 2016, and considers the virus an ongoing threat [2]. Of particular concern is its association with Guillain–Barré syndrome and the link between ZIKV infection of pregnant women and microcephaly, neurological impairment and distress in their offspring [3, 4].
The ZIKV belongs to the genus Flavivirus, and is most commonly transmitted via different species of mosquitoes of the Aedes genus frequently found in tropical environments [5, 6]. The virus has also been shown to be transmitted from mother to fetus, as well as during sexual intercourse between individuals through bodily fluids [7]. The virus is closely related to other flaviviruses, such as the dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), which often complicates correct diagnosis of ZIKV [8]. Although the virus was discovered in Uganda in 1947, the potential for the virus to infect mammals was not described until 1971 [9, 10]. Interestingly, the first clinical reports of perinatal transmission and association with Guillain–Barré syndrome due to ZIKV occurred in 2013 in French Polynesia following a major change in the virus epidemiology [11–14]. This outbreak was complicated by concurrent outbreaks of patients of DENV and chikungunya virus (CHIKV) transmitted by the same Aedes mosquito vector [15]. Since then, other reports from Brazil have chronicled a rapidly spreading epidemic that, once more, co-exists with transmission of DENV and CHIKV, and is characterized by fever, conjunctivitis, and a maculopapular rash [16]. More ominously, there are reports of microcephaly and ocular damage in aborted fetuses and infants born to mothers infected with ZIKV. In these cases, evidence of ZIKV infection came from the recovery of the virus from amniotic fluid, placental, and brain tissue. Additionally, it is known that the virus can persist in body fluids such as urine, saliva, and semen beyond the short time (<7 days) that it is present in blood, which becomes an important consideration when developing methods of ZIKV detection [17, 18].
Developing rapid diagnostics is central to prevent and control ZIKV spread, while also providing women with the necessary information to make informed decisions regarding pregnancy. It is particularly important to distinguish ZIKV infection from that of the structurally related DENV in areas where DENV is endemic and ZIKV is increasing in prevalence. Regions with the highest incidence of ZIKV infection also tend to be resource-limited. There is, therefore, an urgent and unmet need for rapid, simple, on-site, and cost-effective diagnostics that can specifically identify ZIKV and ZIKV-specific antibody (Ab) responses in body fluids.
Current ZIKV detection methods, although rapid (<30 min), are not cost effective and require specialized equipment and trained personnel. These methods are not ideal in resource-limited settings where the virus is frequently found. Additionally, these methods are regularly used concurrently for detection of ZIKV in more than one bodily fluid, most commonly urine and serum, to accurately identify the presence of the virus. Because 20–25% of infected individuals do not demonstrate symptoms, the short window of time in which ZIKV is actively present in the body is often missed [7]. Thus, tests for previous exposure to ZIKV are also performed in conjunction with tests for active infection. It is important to note that test development, validation, and optimization have proven difficult thus far due to the low amount of samples available.
Current ZIKV tests and their limitations
RNA nucleic acid tests (NATs)
The presence of active ZIKV can usually be detected early in the infection in bodily fluids using RNA NATs, such as the Trioplex real-time polymerase chain reaction (RT-PCR) Assay, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), reverse-transcription isothermal recombinase polymerase amplification (RPA) and reverse-transcription strand-invasion based amplification (RT-SIBA) assay [19, 20]. The Trioplex RT-PCR is currently the test used by the Centers for Disease Control and Prevention (CDC) for evaluating symptomatic pregnant women in conjunction with IgM serology. Briefly, the viral RNA is first converted to cDNA via reverse transcription. If the sample contains the desired DNA sequence, a specially designed probe will bind to the target area and is detected via fluorescence. RNA nucleic acid testing is highly sensitive and can identify extremely low concentrations of viral RNA, 1.93×104 genome copy equivalents per millilitre of serum according to the CDC, present during the first 10 days of ZIKV infection (21). However, NATs require expensive machinery, technical expertise, and are associated with high costs. Additionally, because viral RNA degrades rapidly in the body, NATs cannot detect prior exposure to ZIKV. Under updated recommendations of the CDC, negative NATs should be repeated with new sample extractions because of the low levels of virus present during infection.
Plaque-reduction neutralization test (PRNT)
PRNTs involve an intensely laborious process that is performed by the CDC or at a laboratory designated by CDC to detect neutralizing antibodies of a virus. If a sample has a negative ZIKV NAT and a non-negative or inconclusive serology result, a PRNT is required. PRNTs take several days to deliver a result as the process involves mixing the sample with live virus, growing this treated sample in a dish over a monolayer of host cells, and leaving the plate to incubate until plaques grow. Plaques grow when the sample added contains neutralizing antibodies, indicating previous exposure to the virus. Besides the inherent downfall of the time it takes from sample collection to plaque identification, PRNTs require specific equipment, trained personnel and do not provide information on active
ZIKV infection.
Serologic test for ZIKV
The first antibodies produced in response to initial exposure to ZIKV, IgMs, are manifested towards the end of the first week of infection. These antibodies, as well as neutralizing antibodies, can be detected via the Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA). A plate is coated with the anti-IgM capture, the patient’s sample is added and detection is achieved by consequential addition of an enzyme-conjugated anti-viral antibody. The enzyme interacts with a chromogenic substrate producing a colorimetric change, which can then be detected using a spectrophotometer. Important limitations to address include (1) length of assay time (2.5 days to complete); (2) detection of previous exposure to ZIKV only rather than active infection; (3) occurrence of false-negative and false-positive results. False-negatives occur when the samples were collected before IgMs have been generated, usually 4 days post-onset of symptoms or when the samples were collected after IgMs levels have fallen below detectable levels, approximately 12 weeks post-onset of symptoms. Equally, false-positives occur due to cross-reactivity with structurally similar antigens, most commonly other flaviviruses, such as DENV. Follow-up testing is necessary to rule out a false-positive result.
Active infection ELISA
Active ZIKV can be detected using a sandwich-format ELISA. Specific anti-ZIKV antibodies sandwich the virus, if it is present in the sample, and can be detected via an enzyme-conjugated secondary antibody in the same manner as the MAC-ELISA. Until recently, developing an accurate active infection ELISA proved difficult owing to the lack of specific antibodies towards ZIKV, which caused high instances of cross-reactivity with other structurally similar flaviviruses.
The previously described methods are conducted under an ‘Emergency Use Authorization’ issued by the FDA except for the active infection ELISA. In collaboration with Dr David Watkins and Dr Esper Kallas, our lab is working on developing a highly specific active infection ELISA using monoclonal antibodies isolated from ZIKV-infected patients in Sao Paulo, Brazil, that bind only to ZIKV and no other flaviviruses. Currently, our assay is under optimization to detect levels of ZIKV in urine and serum samples.
The advantages and limitations of the methods of ZIKV detection discussed above are summarized in Table 1.
Ongoing and future developments: point-of-care testing for active infection for ZIKV
Recently, paper-based detection methods have gained considerable interest because of the low cost, portability, stability at various storage conditions, and ease of use associated with their handling. These testing platforms do not require external equipment, allowing them to be carried out in remote and resource-limited areas, such as those where ZIKV flourishes. Thus, there is an emphasis on the translation of common assay principles to more portable and affordable platforms.
Lateral flow assays employ ELISA principles, and, as such, antibodies that are selective towards the desired antigen are immobilized onto a membrane. Briefly, the primary and secondary antibodies are dispensed onto the membrane via inkjet technologies and function as the test and control lines, respectively. The top portion of the membrane is laminated with an adsorbent pad to facilitate capillary action. A separate set of selective primary antibodies are conjugated to detection molecules such as gold nanoparticles, latex particles or coloured cellulose nanobeads and are immobilized onto the conjugate pad. The sample is added to the sample pad and then migrates, via capillary action, through the membrane to the conjugate pad. If the sample contains the antigen, the dried primary Ab conjugated to the coloured particles will be remobilized and the antigen will bind to these conjugated primary antibodies. The formed complexes will flow through the reaction matrix, which is usually a porous matrix such as nitrocellulose. The labelled antigen will then be captured by the immobilized primary antibodies forming a coloured band (Fig. 1). The control line will bind the coloured labelled primary antibodies regardless of the presence of antigen. This verifies that the test is working properly and the labelled conjugate can flow and bind to its respective antibody pair. When the antigen is present, the antibody/bead complex will bind to the antigen, and this Ab/antigen complex is captured by the antibody that is immobilized as the test line. One line at the control region indicates a functional but negative test and two lines indicates a functional positive test. Using our highly specific anti-ZIKV antibodies, we have additionally developed a sandwich-format lateral flow assay for the detection of ZIKV in urine that is currently under optimization.
DNA/RNA detection methods on paper are also of particular interest because of the high selectivity of hybridization. In 2015, the Whitesides group described a novel “paper machine” device that uses LAMP to detect a signal using a hand-held UV source and camera phone [22]. The paper-based device costs $1.83, an extreme improvement when compared with traditional nucleic acid testing. The only drawback of this device is that it requires incubation steps at 65 °C throughout the assay to dry the reagents present on the paper strip, which sometimes can be challenging in a point-of-care situation. Furthering the research on paper-based methods of viral RNA detection, our group described a different paper-based platform that has only one step involving incubation in a boiling water bath [23]. We have continued our pursuit to develop a point-of-care paper-based viral detection system and have constructed another test that utilizes RPA and requires incubation at much lower temperature, namely at 37 °C.
The threat of ZIKV creating serious health issues has not lessened and continues to afflict women who are pregnant and wish to become pregnant. Without proper methods of detection, the virus is difficult to characterize, document, and study. While many of the progressive paper-based platforms described herein are promising, none are currently FDA approved and on the market for use for the detection of ZIKV. It is, therefore, imperative that researchers continue to investigate and design innovative detection methods that can detect ZIKV in an easy, accurate, and affordable manner.
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The authors
Devon Pawley, Emre Dikici PhD, Sapna Deo PhD, Sylvia Daunert PhD
Department of Biochemistry and Molecular Biology,
Miller School of Medicine, University of Miami,
Miami, FL 33136, USA
*Corresponding author
E-mail: sdaunert@med.miami.edu
Scientific Literature Review: Infectious diseases
, /in Featured Articles /by 3wmediaIdentifying volatile metabolite signatures for the diagnosis of bacterial respiratory tract infection using electronic nose technology: a pilot study
Lewis JM, Savage RS, Beeching NJ, Beadsworth MBJ, Feasey N, Covington JA. PLoS One 2017; 12(12): e0188879
OBJECTIVES: New point of care diagnostics are urgently needed to reduce the over-prescription of antimicrobials for bacterial respiratory tract infection (RTI). A pilot cross-sectional study was performed to assess the feasibility of gas-capillary column ion mobility spectrometer (GC-IMS), for the analysis of volatile organic compounds (VOC) in exhaled breath to diagnose bacterial RTI in hospital inpatients.
METHODS: 71 patients were prospectively recruited from the Acute Medical Unit of the Royal Liverpool University Hospital between March and May 2016 and classified as confirmed or probable bacterial or viral RTI on the basis of microbiologic, biochemical and radiologic testing. Breath samples were collected at the patient’s bedside directly into the electronic nose device, which recorded a VOC spectrum for each sample. Sparse principal component analysis and sparse logistic regression were used to develop a diagnostic model to classify VOC spectra as being caused by bacterial or non-bacterial RTI.
RESULTS: Summary area under the receiver operator characteristic curve was 0.73 (95% CI 0.61–0.86), summary sensitivity and specificity were 62% (95% CI 41–80%) and 80% (95% CI 64–91%) respectively (p=0.00147).
CONCLUSIONS: GC-IMS analysis of exhaled VOC for the diagnosis of bacterial RTI shows promise in this pilot study and further trials are warranted to assess this technique.
Cerebrospinal fluid B-lymphocyte chemoattractant CXCL13 in the diagnosis of acute Lyme neuroborreliosis in children
Barstad B, Tveitnes D, Noraas S, Selvik Ask I, Saeed M, Bosse F, et al. Pediatr Infect Dis J 2017; 36(12): e286–e292
BACKGROUND: Current markers of Lyme neuroborreliosis (LNB) in children have insufficient sensitivity in the early stage of disease. The B-lymphocyte chemoattractant CXCL13 in the cerebrospinal fluid (CSF) may be useful in diagnosing LNB, but its specificity has not been evaluated in studies including children with clinically relevant differential diagnoses. The aim of this study was to elucidate the diagnostic value of CSF CXCL13 in children with symptoms suggestive of LNB.
METHODS: Children with symptoms suggestive of LNB were included prospectively into predefined groups with a high or low likelihood of LNB based on CSF pleocytosis and the detection of Borrelia antibodies or other causative agents. CSF CXCL13 levels were compared between the groups, and receiver-operating characteristic analyses were performed to indicate optimal cutoff levels to discriminate LNB from non-LNB conditions.
RESULTS: Two hundred and ten children were included. Children with confirmed LNB (n=59) and probable LNB (n=18) had higher CSF CXCL13 levels than children with possible LNB (n=7), possible peripheral LNB (n=7), non-Lyme aseptic meningitis (n=12), non-meningitis (n=91) and negative controls (n=16). Using 18 pg/mL as a cutoff level, both the sensitivity and specificity of CSF CXCL13 for LNB (confirmed and probable) were 97%. Comparing only children with LNB and non-Lyme aseptic meningitis, the sensitivity and specificity with the same cutoff level were 97% and 83%, respectively.
CONCLUSION: CSF CXCL13 is a sensitive marker of LNB in children. The specificity to discriminate LNB from non-Lyme aseptic meningitis may be more moderate, suggesting that CSF CXCL13 should be used together with other variables in diagnosing LNB in children.
Neutrophil CD64 – A potential biomarker in patients with complicated intra-abdominal infections? A literature review
Dimitrov E, Enchev E, Halacheva K, Minkov G, Yovtchev Y. Acta Microbiol Immunol Hung 2018; doi: 10.1556/030.65.2018.011
Complicated intra-abdominal infections (cIaIs) represent a serious cause of morbidity and mortality. Early diagnosis and well-timed treatment can improve patients’ outcome, whereas the delay in management often result in rapid progression to circulatory collapse, multiple organ failure, and death. Neutrophil CD64 antigen expression has been studied for several years as infectious and sepsis biomarker and has several characteristics that make it good for clinical employment. It has been suggested to be predictive of positive bacterial cultures and a useful test for management of sepsis and other significant bacterial infections. Our review concluded that the neutrophil CD64 expression could be a promising and meaningful biomarker in patients with cIaIs. It shows good potential for evaluating the severity of the disease and could give information about the outcome. However, more large studies should be performed before using it in clinical practice.
Mycoplasma genitalium: accurate diagnosis is necessary for adequate treatment
Gaydos CA. J Infect Dis 2017; 216(suppl_2): S406–S411
BACKGROUND: Mycoplasma genitalium is very difficult to grow in culture but has been more able to be studied for disease associations since the advent of research molecular amplification assays. Polymerase chain reaction (PCR) and other molecular assays have demonstrated an association with adverse disease outcomes, such as urethritis or nongonococcal urethritis in men and adverse reproductive sequelae in women-for example, cervicitis, endometritis, and pelvic inflammatory disease, including an association with risk for human immunodeficiency virus. The lack of commercially available diagnostic assays has limited widespread routine testing. Increasing reports of high rates of resistance to azithromycin detected in research studies have heightened the need available commercial diagnostic assays as well as standardized methods for detecting resistance markers. This review covers available molecular methods for the diagnosis of M. genitalium and assays to predict the antibiotic susceptibility to azithromycin.
METHODS: A PubMed (US National Library of Medicine and National Institutes of Health) search was conducted for literature published between 2000 and 2016, using the search terms ‘Mycoplasma genitalium’, ‘M. genitalium’, ‘diagnosis’, and ‘detection’.
RESULTS: Early PCR diagnostic tests focused on the MPa adhesion gene and the 16S ribosomal RNA gene. Subsequently, a transcription-mediated amplification assay targeting ribosomes was developed and widely used to study the epidemiology of M. genitalium. Newer methods have proliferated and include quantitative PCR for organism load, AmpliSens PCR, PCR for the pdhD gene, a PCR-based microarray for multiple sexually transmitted infections, and multiplex PCRs. None yet are cleared by the Food and Drug Administration in the United States, although several assays are CE marked in Europe. As well, many research assays, including PCR, gene sequencing, and melt curve analysis, have been developed to detect the 23S ribosomal RNA gene mutations that confer resistance to azithromycin. One recently developed assay can test for both M. genitalium and azithromycin resistance mutations at the same time.
CONCLUSIONS: It is recommended that more commercial assays to both diagnose this organism and guide treatment choices should be developed and made available through regulatory approval. Research is needed to establish the cost-effectiveness of routine M. genitalium testing in symptomatic patients and screening in all individuals at high risk of acquiring and transmitting sexually transmitted infections.
Prognostic value of secretoneurin in patients with severe sepsis and septic shock: data from the Albumin Italian Outcome Sepsis Study
Røsjø H, Masson S, Caironi P3,4, Stridsberg M, Magnoli M, et al. Crit Care Med 2018; doi: 10.1097/CCM.0000000000003050
OBJECTIVES: Secretoneurin directly influences cardiomyocyte calcium handling, and circulating secretoneurin levels seem to improve risk prediction in patients with myocardial dysfunction by integrating information on systemic stress, myocardial function, and renal function. Accordingly, in this study, we hypothesized that secretoneurin would improve risk prediction in patients with sepsis and especially in patients with septic shock as these patients are more hemodynamically unstable.
DESIGN: Multicentre, interventional randomized clinical trial.
SETTING: Multicentre, pragmatic, open-label, randomized, prospective clinical trial testing fluid administration with either 20% human albumin and crystalloids or crystalloid solutions alone in patients with severe sepsis or septic shock (The Albumin Italian Outcome Sepsis).
PATIENTS OR SUBJECTS: In total, 540 patients with septic shock and 418 patients with severe sepsis.
INTERVENTIONS: Either 20% human albumin and crystalloids or crystalloid solutions alone.
MEASUREMENTS AND MAIN RESULTS: We measured secretoneurin on days 1, 2, and 7 after randomization and compared the prognostic value of secretoneurin for ICU and 90-day mortality with established risk indices and cardiac biomarkers in septic shock and severe sepsis. High secretoneurin levels on day 1 were associated with age and serum concentrations of lactate, bilirubin, creatinine, and N-terminal pro-B-type natriuretic peptide. Adjusting for established risk factors and cardiovascular biomarkers, secretoneurin levels on day 1 were associated with ICU (odds ratio, 2.27 [95% CI, 1.05–4.93]; p=0.04) and 90-day mortality (2.04 [1.02–4.10]; p=0.04) in patients with septic shock, but not severe sepsis without shock. Secretoneurin levels on day 2 were also associated with ICU (3.11 [1.34–7.20]; p=0.008) and 90-day mortality (2.69 [1.26–5.78]; p=0.01) in multivariate regression analyses and improved reclassification in patients with septic shock, as assessed by the net reclassification index. Randomized albumin administration did not influence the associations between secretoneurin and outcomes.
CONCLUSIONS: Secretoneurin provides early and potent prognostic information in septic patients with cardiovascular instability.
Adaptation of the Amoebae Plate Test to recover Legionella strains from clinical samples
Descours G, Hannetel H, Reynaud JV, Ranc AG, Beraud L, Kolenda C, et al. J Clin Microbiol 2018; doi: 10.1128/JCM.01361-17
The isolation of Legionella from respiratory samples is the gold standard for Legionnaires’ disease (LD) diagnosis and enables epidemiological studies and outbreak investigations. The purpose of this work was to adapt and evaluate the performance of an amoebic co-culture procedure (the amoebae plate test, APT) to the recovery of Legionella strains from respiratory samples, in comparison with axenic culture and a liquid-based amoebic co-culture (LAC). Axenic culture, LAC, and APT were prospectively performed on 133 respiratory samples from patients with LD. The sensitivities and times-to-result of the three techniques were compared. Using the three techniques, Legionella strains were isolated in 46.6% (n=62) of the 133 respiratory samples. The sensitivity of axenic culture was 42.9% (n=57), that of LAC was 30.1% (n=40), and that of APT 36.1% (n=48). Seven samples were positive by axenic culture only; for these there were less than 10 colonies in total. Five samples, all sputa, were positive by an amoebic procedure only (5/5 by APT, 2/5 by LAC); all had overgrowth by oropharyngeal flora with axenic culture. The combination of axenic culture with APT yielded a maximal isolation rate (i.e. 46.6%). Overall, the APT significantly reduced the median time for Legionella identification to 4 days, versus 7 days for LAC (p<0.0001). The results of this study promote the substitution of LAC by APT, which could be implemented as a second-line technique on culture-negative and microbial overgrown samples, especially sputa. They provide a logical basis for further studies in both clinical and environmental settings.
Design, implementation, and interpretation of amplification studies for prion detection
Haley NJ, Richt JA, Davenport KA, Henderson DM, Hoover EA, Manca M, et al. Prion 2018; doi: 10.1080/19336896.2018.1443000
Amplification assays for transmissible spongiform encephalopathies (TSEs) have been in development for close to 15 years, with critical implications for the post-mortem and ante-mortem diagnosis of human and animal prion diseases. Little has been published regarding the structured development, implementation and interpretation of experiments making use of protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC), and the goal with this Perspectives manuscript is to offer a framework which might allow for more efficient expansion of pilot studies into diagnostic trials in both human and animal subjects. This framework is made up of approaches common to diagnostic medicine, including a thorough understanding of analytical and diagnostic sensitivity and specificity, an a priori development of amplification strategy, and an effective experimental design. It is our hope that a structured framework for prion amplification assays will benefit not only experiments seeking to sensitively detect naturally-occurring cases of prion diseases and describe the pathogenesis of TSEs, but ultimately assist with future endeavours seeking to use these methods more broadly for other protein misfolding disorders, including Alzheimer’s and Parkinson’s disease.
A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis
Dao TNT, Lee EY, Koo B, Jin CE, Lee TY, Shin Y. Anal Biochem 2017; 544: 87–92
Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.
Long-term follow-up and quantitative hepatitis B surface antigen monitoring in North American chronic HBV carriers
O’Neil CR, Congly SE, Rose MS, Lee SS, Borman MA, Charlton CL, et al. Ann Hepatol 2018; 17(2): 232–241
INTRODUCTION: Quantitative hepatitis B surface antigen (qHBsAg) combined with HBV DNA may be useful for predicting chronic hepatitis B (CHB) activity and nucleoside analogue (NA) response.
MATERIAL AND METHODS: In this retrospective cohort study qHBsAg levels were evaluated according to CHB disease phase and among patients on treatment. Random effect logistic regression analysis was used to analyse qHBsAg change with time in the NA-treated cohort.
RESULTS: 545 CHB carriers [56% M, median age 48 y (IQR 38–59), 73% Asian] had qHBsAg testing. In the untreated group (44%), 8% were classified as immune tolerant, 10% immune clearance, 40% inactive, and 43% had HBeAg-CHB and the median HBsAg levels were 4.6 (IQR 3.4–4.9), 4.0 (IQR 3.4–4.5), 2.9 (IQR 1.4–3.8), and 3.2 log IU/mL (IQR 2.6–4.0), respectively; p<0.001. In the NA-treated group (28% entecavir, 68% tenofovir, 4% lamivudine), no significant change in qHBsAg levels occurred with time, 19% of patients on long-term NA had sustained qHBsAg <2 log10 IU/mL.
CONCLUSION: qHBsAg titres were associated with CHB phase and remained stable in those on long-term NA. A significant number of treated patients had low-level qHBsAg, of which some may be eligible for treatment discontinuation without risk
of flare.
Plasmonic nanowire interstice sensor for the diagnosis of prostate cancer
, /in Featured Articles /by 3wmediaExtracellular microRNAs recently provided valuable information including the site and the status of cancers. miR141 and miR375 are the most pronounced biomarkers for the diagnosis of high-risk prostate cancer. Here, we describe attomolar detection of miR141 and miR375 released from living prostate cancer cells through the use of a plasmonic nanowire interstice (PNI) sensor.
by Dr Taejoon Kang and Professor Bongsoo Kim
Background
Prostate-specific antigen
Prostate cancer (PC) represents 27% of all cancers in men and the second leading cause of cancer death for men worldwide [1]. In 2017 for the USA alone, there were approximately 161 360 cases of PC. PC has been diagnosed by digital rectal examination and the prostate-specific antigen (PSA) test. PSA is the only tissue-specific biomarker that can aid the early diagnosis of PC. The PSA blood test, however, has limited diagnostic accuracy for PC because PSA can be increased owing to other factors including benign prostatic hyperplasia or prostatitis as well as PC. The US Preventive Services Task Force even recommended that physicians should not routinely perform PC screening based on serum PSA levels [2]. Clearly, new biomarkers are needed to overcome this problem.
Recently, it has been reported that the level of free PSA (f-PSA) is decreased in men who have PC compared with those with benign conditions [3]. Therefore, various immunoassay technologies including enzyme-linked immunosorbent assay, fluorescence immunoassay, surface plasmon resonance (SPR), electrochemical immunosensor, dark-field microscopy, chemiluminescence, surface-enhanced Raman scattering (SERS), and dynamic light scattering have been employed for the quantitative analysis of f-PSA [3].
RNAs as prostate cancer biomarkers
Long noncoding RNAs (lncRNAs, ≥200 nucleotides) are often expressed in a disease-, tissue- or developmental-specific manner. Since lncRNAs are highly dysregulated in several cancer types and exhibit a high degree of tissue- and disease-specificity, lncRNAs are regarded as candidates for cancer diagnostic biomarkers [4]. Prostate Cancer Antigen 3 (PCA3) is a prostate-specific lncRNA that is overexpressed by 60- to 100-fold in >90% of prostate tumours compared to benign prostatic tissue. Urinary PCA3 has been used as a diagnostic biomarker for PC with a sensitivity of 58–82% and a specificity of 56–76%. The sensitivity and accuracy of PCA3 are increased when used in combination with α-methylacyl-CoA racemase. Urinary PCA3 is now widely used for PC diagnosis and has been approved by the US Food and Drug Administration (FDA). MicroRNAs (miRNAs) are single-stranded, small, and noncoding RNAs. The expression patterns of miRNAs in tissue and blood samples of patients are often closely associated with disease types and also disease stages, hinting that certain miRNAs can be compelling diagnostic markers [5]. In 2008, it was first reported that the level of miR141 is upregulated in the serum of metastatic PC compared with healthy controls and benign prostatic hyperplasia patients. Since then, miR141 and miR375 have been the most pronounced biomarkers for high-risk PC, including castrate-resistant PC and metastatic PC, which account for approximately 15% of PC diagnoses and have the potential to progress to a lethal phenotype [6].
Detection methods for nucleic acid biomarkers
For the detection of nucleic acid biomarkers, polymerase chain reaction (PCR) is the most extensively used analytical tool. Although PCR is considered the gold standard for the detection of gene biomarkers, it has drawbacks including a long amplification time and the risk of erroneously amplifying contaminants or unrelated gene sequences. To overcome these limitations, PCR-free assays have been developed by taking various sensing approaches such as fluorescence resonance energy transfer, colorimetry, SPR, electrochemistry, SERS, and so on. These methods have contributed to the advance of cancer diagnosis by reducing the drawbacks of PCR. SERS is a fascinating phenomenon that significantly increases the Raman signal of molecules located within nanoscale metallic interstices (hot spots). SERS has been employed for the sensitive detection of nucleic acid because of its single-molecule sensitivity, molecular specificity, and insensitivity to quenching. It is known that the SERS enhancement strongly depends on the detailed morphology of the metal nanostructure. Although a number of promising nanostructures that can be used as efficient SERS-active platforms have been proposed, it still remains a challenging task to develop a practical SERS sensor that can detect multiple nucleic acid biomarkers simultaneously while retaining high sensitivities. The use of single-crystalline noble metal nanowires (NWs) is highly advantageous for SERS-based detection because of their well-defined geometries, atomically smooth surfaces, and simple fabrication process [7]. Previously, we developed several noble metal NW-based SERS sensors including plasmonic nanowire interstice (PNI) sensor, particle-on-NW sensor, NW on a graphene sensor, and nanogap-rich Au NW sensor [8–15]. Among them, PNI nanostructures have been widely employed for the detection of several biochemical molecules. Particularly, by combining the PNI nanostructure with the bi-temperature hybridization process, we were able to detect miRNAs with near-perfect accuracy of single nucleotide polymorphism (SNPs) and at the extremely low detection limit of 100 aM. Here, we introduce a PNI sensor which can detect the extracellular miR141 and miR375 released from living PC cells into a culture medium. This sensor shows an extremely low detection limit of 100 aM for both miR141 and miR375, and a wide dynamic range from 100 aM to 100 pM, covering the typical concentration range of extracellular miRNAs in the bloodstreams of patients. Additionally, the PNI sensor can completely discriminate the single-base mismatches of miR141 and miR375. This excellent sensing capability of the PNI sensor enables the simultaneous detection of miR141 and miR375 released from the cells of PC cell lines (LNCaP and PC-3), showing the potential applicability to a novel PC diagnostic method.
Specific and sensitive detection of miRNA
To accurately determine the expression patterns of miRNAs in biological fluid samples, it is necessary to overcome the inconsistent measurement results caused by low specificities and complicated sensing procedures. For the ultra-specific and ultra-sensitive detection of miRNAs, we applied miRNA-specific bi-temperature hybridizations to Au NW surfaces, where short miRNAs can readily crawl into the narrow hot spots of the PNI sensor for effective SERS detection. The probe locked nucleic acid (LNA)-modified PNI sensors were incubated with miRNAs at 42 °C and subsequently incubated with Cy5-labeled reporter LNA at 64 °C (Fig. 1a). If the target miRNAs have perfectly complementary sequences to both probe and reporter LNAs, sandwiched complexes of probe LNA-miRNA-reporter LNA can be stably formed on a PNI sensor, providing strong SERS signals of Cy5. In contrast, when the sample only contains single-base mismatched miRNAs, little signal was observed. Figure 1(b) displays the intensity of the Cy5 1580 cm−1 band plotted as a function of the miR141 (magenta) and miR375 (blue) concentrations. Both intensities were quite linearly increased throughout the concentration range from 100 aM to 100 pM in spite of the different sequences of miR141 and miR375. To investigate the specificity of a PNI sensor, we prepared four kinds of single-base mismatched miRNAs (miR141 A, miR141 B, miR375 A, and miR375 B). The miR141 A and miR375 A had a mismatched single base on the probe LNA recognition site, respectively, and the miR141 B and miR375 B had a mismatched single base on the reporter LNA recognition site. Figure 1(c,d) shows the plot of Cy5 1580 cm−1 band intensity obtained from the PNI sensors for perfectly matched and single-base mismatched miRNAs. The concentration of all miRNAs was 100 pM. When the single-base mismatched miRNAs (miR141 A, B and miR375 A, B) were present, featureless SERS signals were obtained from the PNI sensors. In contrast, significantly strong SERS signals were measured from the PNI sensors in the presence of miR141 and miR375 with intact sequences. In the miRNA sensing procedure using the PNI sensor, the unstable single-base mismatched miRNA–LNA hybridized structures were destroyed at the temperature over Tm. Therefore, we near-perfectly excluded the possibility of detecting single-base mismatched miRNAs.
Detection of miRNAs released from cells in culture
The PNI sensors were also employed to detect miR141 and miR375 released from the living PC cells. We prepared four types of media in which different human cancer cell lines were cultured. The cultured cell lines were LNCaP (PC cells), PC-3 (PC cells), RWPE-1 (noncancerous prostate epithelial cells), and HeLa (cervical cancer cells). For the detection of miR141 and miR375 using PNI sensors, the total extracellular miRNA released from the cells into the media were isolated and purified. Figure 2(a,b) represent the extracellular miR141 and miR375 levels determined by the PNI sensor for LNCaP, PC-3, RWPE-1, and HeLa, respectively. The levels of miR141 and miR375 in LNCaP and PC-3 culture supernatants were higher than those in RWPE-1 and HeLa, indicating that the PNI sensor can detect extracellular miRNAs released from living PC cells accurately. The well-defined PNI nanostructure which provides a highly reproducible SERS hot spot line, straightforward probe LNA immobilization, and simple miRNA–LNA hybrid formation with equalized stabilities seems to collectively contribute to the observed equally enhanced and highly reproducible SERS signals for miR141 and miR375.
Conclusion
We have developed a PNI sensor that can detect extracellular miR141 and miR375 released from the cultured cells of PC cell lines. The proposed PNI sensor exhibited a low detection limit of 100 aM, a wide dynamic range from 100 aM to 100 pM, and a perfect discrimination of single-base mismatches in target miRNAs. By using the PNI sensor, we were able to estimate the absolute amount of the released miR141 and miR375 from each PC cell line. The highly sensitive and exactly quantifiable PNI sensor could be useful for the precise diagnosis of PC patients and will be further valuable for detecting other disease-related extracellular miRNAs.
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The authors
Taejoon Kang*1 PhD, Bongsoo Kim*2 PhD
1Hazards Monitoring Bionano Research Center, KRIBB, Daejeon 34141, Republic of Korea
2Department of Chemistry, KAIST, Daejeon 34141, Republic of Korea
*Corresponding author
E-mail: kangtaejoon@kribb.re.kr;
bongsoo@kaist.ac.kr
Biomarkers for the prediction of clinically significant prostate cancer
, /in Featured Articles /by 3wmediaProstate cancer is the most common cancer in men and diagnosis involves a combination of assessments. Levels of the biomarker prostate-specific antigen (PSA) are commonly measured, but do not always equate to cancer status. Testing of PSA in combination with other biomarkers may help to improve diagnostic and prognostic accuracy, as well as minimizing overdiagnosis as well as unnecessary intervention. This article provides a summary of the information currently known about biomarkers showing promise for use in prostate cancer screening.
by Dr Alexandra Tabakin, Dr Sung Un Bang and Dr Isaac Y. Kim
Introduction
Prostate cancer is the most commonly diagnosed cancer type in the USA and second leading cause of cancer deaths in men. In 2018, there will be an estimated 164 690 new cases with an estimated 29 430 deaths [1]. Although many prostate cancers are indolent in nature and can be safely monitored with active surveillance, a significant proportion of patients will require intervention with surgery, radiation, or other therapies. One of the major challenges in treating prostate cancer is risk stratification and differentiating clinically significant prostate cancers in order to avoid overdiagnosis and overtreatment [2]. To do so, patients undergo risk stratification, which conventionally includes a combination of prostate-specific antigen (PSA) screening, digital rectal exam (DRE), trans-rectal ultrasonography (TRUS)-guided biopsy. Currently clinically insignificant prostate cancer, according to Epstein criteria, is defined as cancer with preoperative PSA <10ng/ml, clinical stage <T1c, Gleason score <6, PSA density <0.15, <2 positive biopsy cores, and <50% cancer involvement in any core [3–5]. However, as we learn more about the heterogeneity of prostate cancer tumours, various serum biomarkers have been investigated to improve diagnostic and prognostic accuracy and minimize treatment. In this review, we discuss various biomarkers and their utilization for the prediction of clinically significant prostate cancer.
Serum biomarkers
PSA
PSA, or prostate-specific antigen, is a serine protease released by the prostate. PSA gained notoriety in the 1980s when it was reported to have various uses including screening, monitoring disease progression, and detecting recurrence of the cancer. As PSA screening for prostate cancer increased, the incidence of prostate cancer also rose in the 1990s. However, PSA only has a 25–40% specificity rate for prostate cancer and can be elevated with infection, trauma, and benign prostatic hyperplasia (BPH) [6]. In fact, about 15% of men with a low level of PSA (<4.0 ng/ml) have prostate cancer [7].
Because the harms of biopsy and prostate cancer treatment may outweigh the benefits in some patients with clinically insignificant prostate cancer, efforts have been made to improve the validity of PSA in differentiating benign conditions and prostate cancer. One such effort is the use of free PSA; those with an elevated PSA and a lower serum percentage of free PSA (%f-PSA) are more likely to have BPH rather than prostate cancer [8]. The Prostate Health Index (PHI) formula incorporates serum total PSA, free PSA, and the [−2]proPSA to discriminate Gleason 3+4 and greater cancers with 90% sensitivity and 17% specificity [9]. 4K score is another novel test which is comprised of total PSA, free PSA, intact PSA, and human kallikrein-related peptidase 2 to detect clinically significant prostate cancer and discern those who would benefit from a prostate biopsy while preventing 30–58% of biopsies [10].
PAP
Prostatic acid phosphatase (PAP) was the first popularized prostate cancer serum biomarker, but was eventually replaced by PSA, as it is less sensitive in diagnosing prostate cancer and detecting recurrence. Elevated PAP level has been associated with a high risk of bone metastases [11], significantly shortened overall survival [12] as well as disease-free survival [13], and increased risk of biochemical recurrence [14]. Interestingly, recent studies have shown that PAP may be useful in detecting high-risk clinically significant prostate cancer patients; it is speculated that PAP may be associated with micro-metastatic disease prior to treatment, and therefore, predict response to treatment [15].
NLR, ANC, ALC
Inflammation and tissue microenvironment are important factors in cancer development. Although the temporal relationship is not well established, a lymphocyte-mediated immune response is thought to occur early in the development of prostate cancer. Neutrophil-to-lymphocyte ratio (NLR) compares the activity of both neutrophils and lymphocytes in the inflammatory response. NLR is a useful prognostic biomarker in mCRPC, where a higher score correlates less relative lymphocyte activity and a worse prognosis. When looking at localized low-risk prostate cancer, studies have shown that NLR is not associated with upstaging, upgrading, or biochemical recurrence. However, increased absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) were associated with upstaging and lower 5-year biochemical recurrence-free survival. More development on these markers may guide clinicians in re-stratifying patients who meet conventional criteria for low-risk prostate cancer [16].
Urine biomarkers
PCA3
Urinary prostate cancer antigen 3 (PCA3) is among the most promising non-invasive biomarker among non-PSA based tests, as it is overexpressed in over 95% of prostate cancers [6]. PCA3 is a long noncoding RNA collected from shed prostate cells during urination. PCA3 is a more specific biomarker for prostate cancer because, unlike PSA, PCA3 levels are not influenced by prostate size, BPH, prostatitis, or the use of 5α-reductase inhibitors [17]. At the genetic level, PCA3 may help distinguish between prostate cancer and high-grade prostatic intraepithelial neoplasia (HG-PIN), as PCA3 is seldom expressed in HG-PIN [18]. Several studies have demonstrated that a higher PCA3 score correlates with clinically significant prostate cancer and larger tumour volume, therefore potentially aiding in selecting patients for active surveillance [19]. In 2012, the FDA approved the PROGENSA PCA3 assay predict men who would benefit from a repeat biopsy in those with a previous negative prostate biopsy [6]. In a recent meta-analysis of 46 clinical trials, the sensitivity and specificity of PROGENSA PCA3 was 65% and 73%, respectively [20].
Genetic mutations
TMPRSS2::ERG
TMPRSS2::ERG, or T2:ERG, gene fusions constitute 90% of gene fusions implicated in prostate cancer, as well as half of all prostate cancers [21, 22]. When the androgen responsive regulatory element TMPRSS2 and the gene for the transcription factor ERG are fused together, androgen-driven genes are overexpressed and tumorigenesis occurs [21]. When used alone, urinary T2:ERG RNA has a reported 86% specificity and 45% sensitivity in prostate cancer detection. However, when used in conjunction with PCA3, specificity and sensitivity improve to 90% and 80%, respectively [6, 23]. Moreover, this test may prevent up to 42% of unnecessary biopsies, limiting healthcare costs [24].
PTEN
Loss of the tumour-suppressor gene, PTEN, or phosphatase and tensin homologue, leading to activation of the PI3K/AKT/mTOR signalling has been found in both early stage and castrate-resistant prostate cancers. Preclinical data show that PI3K pathway activation is related to resistance to androgen deprivation, leading to disease progression and poor response to treatment. In mouse models, conditional deletion of PTEN initially led to the development of prostate hyperplasia and later on invasive and metastatic prostate cancers likely by modulating the p110β catalytic subunit of PI3K. In addition, ablation of p110β hindered AKT signalling and reduced tumorigenesis [25]. In a cohort of 77 men, loss of PTEN at initial prostate biopsy was predictive of the development of castrate-resistant prostate cancer, response to androgen deprivation therapy, and prostate-cancer-specific mortality [26]. Additionally, decreased PTEN expression has been associated with increased risk of biochemical and clinical recurrence after prostatectomy [27]. Therefore, the association between castrate-resistant prostate cancer and PTEN loss as well as PI3K/AKT/mTOR pathway activation suggests that PTEN may have prognostic value in prostate cancer risk stratification.
CHD1
The CHD1 gene, encoding chromodomain helicase DNA-binding protein 1, is commonly deleted in 10–26% of all prostate cancers. The loss of CHD1 affects the ability to repair DNA double-strand breaks via homologous recombination. CHD1 deletion has generally been associated with a poor prognosis. However, studies have demonstrated tumours with CHD1 deletions to be sensitive to both PARP inhibitors and carboplatin, both in vitro and in vivo, suggesting that future research may be able to identify to response to treatment based off of tumour genotypes [28].
Circulating biologics
Circulating tumour cells
Circulating tumour cells (CTCs) are defined as cells that leave the site of a primary cancer, travel through the bloodstream, and settle at other sites in the body where they grow into new tumours, or metastases [29]. In 2004, CellSearch Circulating Tumor Cell System was approved by the FDA as an assay for enumerating CTCs. In recent studies, CTC count, deemed the ‘liquid biopsy’, has been shown to have a role in predicting overall survival and response to treatment, risk stratification, and detecting metastases. In addition, using CTCs to detect biomarkers, such as ERG, PTEN, and AR may allow for personalized treatments based on tumour genomes [9, 30].
Circulating exosomes
Circulating prostate cancer-related exosomes are double-membrane vesicles carrying RNA and pro-oncogenic molecules, which induce malignant transformation in normal cells. ExoDx prostate Intelliscore urine exosome assay (developed by Exosome Diagnostics Inc.) detects exosomal RNA expression of ERG, PCA3 and SPDEF (SAM pointed domain containing ETS transcription factor) in voided urine samples. A score is generated that is able to predict high-grade prostate cancer (Gleason >7) with a negative predictive value of 91%. This assay may be useful in discriminating clinically significant prostate cancer and reducing the number of unnecessary biopsies [31].
Conclusion
The emergence and widespread usage of PSA as a routine screening test for prostate cancer allows for early detection and risk stratification. Testing for PSA in combination with novel biomarkers such as PCA3, TMPRSS2::ERG, PTEN, and others may improve our diagnostic abilities, given the heterogeneity of prostate cancers and their treatments. There are many challenges in establishing useful biomarkers including creating affordable and accessible testing, determining cut-offs, and determining accuracy. As the clinical utility of these biomarkers is further defined, we hope to better risk stratify patients and select appropriate treatments early on in diagnosis. Further research efforts should focus on the synergism between the Epstein criteria, biomarker utility, and how to best bring these tools from bench to bedside.
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The authors
Alexandra Tabakin MD,
Sung Un Bang MD, Isaac Yi Kim MD PhD
Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey and Division of Urology, Rutgers Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA
*Corresponding author
E-mail: kimiy@cinj.rutgers.edu
Book review: Oral anticoagulants
, /in Featured Articles /by 3wmediaAfter the 3rd volume devoted to parental anticoagulants, it stands to reason that the Clinical Development Department had to turn its attention to the other side of anticoagulant treatment – the oral anticoagulants – in this series launched in 2014 with the objective of publishing a Practical Manual every year.
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