Identifying 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.

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.

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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