Two novel biochemical tests, the ESBL NDP and the Carba NP tests, have been recently developed for the early detection of ESBL- or carbapenemase resistance traits in Enterobacteriaceae. Those tests are rapid, sensitive, specific and cost-effective. Implementation of those tests in clinical microbiology laboratories may significantly improve the management and outcome of patients.

by Dr L. Dortet, Dr L. Poirel and Prof. P. Nordmann

Multidrug resistance is now emerging worldwide at an alarming rate among Gram negatives bacteria, causing both community-acquired and nosocomial infections [1–3]. One of the most important emerging resistance traits in Enterobacteriaceae corresponds to the acquisition of resistance to broad-spectrum β-lactams, which is mainly associated with production of clavulanic acid inhibited extended-spectrum β-lactamases (ESBLs) [4, 5]. An ESBL is a β-lactamase that confers reduced susceptibility, i.e. resistance, to the oxyimino-cephalosporins (e.g. cefotaxime, ceftriaxone, ceftazidime) and monobactams (e.g. aztreonam). The hydrolytic activity of ESBLs can be inhibited by several β-lactamase inhibitors such as clavulanic acid and tazobactam. Noteworthy, ESBLs usually do not hydrolyse cephamycins (e.g. cefoxitin and cefotetan) and carbapenems (imipenem, meropenem). In the context of worldwide spread of multidrug resistance, ESBL producers that are mostly Escherichia coli and Klebsiella pneumoniae are not only found as source of hospital-acquired but also of community-acquired infections [4-6]. Consequently, the last line of therapy, carbapenems, is now frequently needed to treat severe infections. However, carbapenem-non-susceptible Enterobacteriaceae due to the production of a carbapenem-hydrolysing enzymes termed carbapenemases, have been reported increasingly [1, 7, 8], leaving us with almost no effective molecules.

Thus, the early detection of ESBL and carbapenemase producers in clinical microbiology is now of utmost importance for determination of appropriate therapeutic schemes and the implementation of infection control measures.

Recently, we have developed two novel tests for rapid identification of (i) ESBL-producing Enterobacteriaceae (ESBL NDP test) [9] and (ii) carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. (Carba NP test) [10–12]. We discuss here the clinical value of those tests.

Detection of ESBLs: Place of the ESBL NDP test in the diagnostic armamentarium
Current techniques for detecting ESBL producers are based on the determination of susceptibility to expanded-spectrum cephalosporins followed by the inhibition of the ESBL activity, mostly by clavulanic acid or tazobactam [13]. The double-disk synergy test, the “E-test” ESBL and the combined disk method have been proposed for that purpose. All those techniques consist of the identification of a synergy between an extended-spectrum generation cephalosporin (ESC) and an inhibitor of β-lactamase (i.e. clavulanic acid or tazobactam) after 18–24h of growth on Mueller-Hinton agar.

This synergy is visualized by (i) a “bouchon de champagne”-shaped image between the extended-spectrum generation cephalosporin and the clavulanate-containing disks for the double-disk synergy test, by (ii) a difference of minimal inhibitory concentration of more than three dilutions between ESC alone and association clavulanate-ESC for the “E-test” ESBL and by (iii) a difference of inhibition diameter of more than 5 mm between an ESC-containing disk and a combined disk containing the same ESC plus clavulanate.

Sensitivities and specificities of the double-disk synergy test and of the E-test are good, ranging from 80 to 95% [13]. However, due to the large diversity of ESBLs [6] that do not hydrolyse ESC similarly, several combinations of those molecules (cefotaxime, ceftazidime and cefepime) together with clavulanate should be tested. Based on the same principle, automated methods for bacterial identification and susceptibility testing are also used in the detection of ESBL-producing organisms. The performance of those systems varies and differs depending on the species investigated with a much higher sensitivity (80–99%) than specificity (50–80%) [13]. However, those tests require mostly overnight growths after isolation of the bacteria, meaning that up to 24–72 h can elapse before ESBL production is detected once the isolate has grown.

Molecular methods (PCR, hybridization, sequencing) based on the detection of ESBL genes have been developed as an alternative. Although classical PCR and DNA arrays necessitate isolation of the bacteria from the clinical sample, real-time PCR based techniques may be performed directly on clinical samples, leading to a decrease of the detection delay. However, these molecular techniques remain costly and require a certain degree of expertise, which is not accessible to non-specialized laboratories. Additionally, those detection methods are able to detect only known genes. They are usually not performed in a routine laboratory but restricted to epidemiological purposes.

Recently, a rapid and cost-effective biochemical test was developed for the detection of ESBL producers, namely the ESBL NDP test [9]. This test is based on a technique designed to identify the hydrolysis of the β-lactam ring of a cephalosporin (cefotaxime), which generates a carboxyl group, consequently acidifying a medium [Figure 1A]. It can either be performed in a 96-well microtiter plate or into a single tube [Figure 1B]. The acidity resulting from this hydrolysis is identified by the colour change using a pH indicator. Inhibition of ESBL activity is evidenced by adding tazobactam in a complementary well [Figure 1]. The ESBL NDP test may be performed on isolate colonies or directly from clinical samples. When performed on bacterial colonies, the overall sensitivity and specificity of the ESBL NDP test are 92.6% and 100% respectively. The ESBL NDP test can easily differentiate ESBL producers from strains that are resistant to expanded-spectrum cephalosporins by other mechanisms, and from those that are likely to be susceptible to expanded-spectrum cephalosporins. Sensitivity of the test is 100% when the ESBL is of the CTX-M-type. Of note, those CTX-M ESBLs have spread worldwide and have become the most predominant type of ESBL [14]. The ESBL NDP test possesses excellent sensitivity (100%) and specificity (100%) when performed directly from blood cultures. In that case, the gain of time for detection of ESBL producers is ~48h compared to the previously mentioned techniques. Additionally, the ESBL NDP test may also be performed directly on colonies grown on selective media used for the screening of colonized patients, leading to a gain of time of at least 24h for the identification of carriers of ESBL producers and consequently faster implementation of adequate hygiene measures that will further prevent the development of nosocomial outbreaks [2, 5].

Detection of carbapenemases: Place of the Carba NP test in the diagnostic armamentarium
In Enterobacteriaceae, carbapenem resistance may be related either to association of a decrease in bacterial outer-membrane permeability with overexpression of β-lactamases possessing no carbapenemase activity, or to the expression of carbapenemases [7]. The spread of carbapenemase producers is an important clinical issue since carbapenemases confer resistance to most β-lactams. A variety of carbapenemases have been reported, such as Ambler class A carbapenemases of KPC-type, metallo-β-lactamase (Ambler class B) of VIM-, IMP- and NDM-types, and Ambler class D carbapenemase of OXA-48-type [7]. In addition, the detection of carbapenemase producers is a major issue since they are usually associated with many other non-β-lactam resistance determinants, giving rise to multi- or even pandrug-resistant isolates [1, 3].

Potential carbapenemase producers are currently screened first by susceptibility testing based on breakpoint values for carbapenems. Additional non-molecular techniques have been proposed for in vitro identification of carbapenemase production. One of the commonly used techniques corresponds to the modified Hodge test (MHT), which has been used for years. Although the addition of zinc to the culture medium was recently shown to increase the sensitivity of this test [in particular for metallo-β-lactamase (MBL) producers], the MHT remains time-consuming (at least 24h) and may lack of specificity (frequent false-positives with Enterobacter spp. overexpressing their chromosomal cephalosporinase, and false-negatives results with many NDM producers). Other detection methods based on the inhibitory properties of several molecules do exist, either for KPC (e.g. boronic acid, clavulanic acid) or MBL (e.g. EDTA, dipicolinic acid) producers, therefore allowing discrimination between the diverse types of carbapenemases. All those methods are time-consuming since they do require isolation of the bacteria from the infected samples followed by at least an additional 24h period of time for performing the inhibitor-based technique. Several molecular methods such as simplex and multiplex PCRs, DNA hybridization and sequencing are also commonly used for the identification of carbapenemase genes in research laboratories and reference centres. Recently a real-time PCR (RT-PCR) technique has been used for detecting KPC producers directly from blood cultures. Although interesting, this molecular-based technique is costly and requires expertise in molecular techniques.

A rapid and cost-effective biochemical test, the Carba NP test, was recently developed to detect carbapenemase production from isolated colonies [12]. The principle of this test is the same as that of the ESBL NDP test, but uses  imipenem as substrate instead (Figure 2A). The Carba NP test differentiates carbapenemase producers (100% sensitivity and 100% specificity) from strains being carbapenem resistant due to non-carbapenemase-mediated mechanisms (Figure 2B) such as combined mechanisms of resistance (outer-membrane permeability defect associated with overproduction of cephalosporinase and/or ESBLs) or from strains that are carbapenem susceptible but express a broad-spectrum β-lactamase without carbapenemase activity (ESBL, plasmid and chromosome-encoded cephalosporinases). Interpretable positive results are always obtained in less than 1h total time, which is unique, making it possible to implement rapid containment measures to limit the spread of carbapenemase producers. The Carba NP test might be performed from colonies recovered from antibiogram (gain of time at least 24h) or from selective media used for screening of carriers (gain of time at least 48h). It was shown to detect carbapenemase producers not only in Enterobacteriaceae [11, 12] but also in Pseudomonas spp. [10]. Additionally, the Carba NP test has also been evaluated for detection of carbapenemase-producing Enterobacteriaceae directly from positive blood cultures [15]. In that case, the Carba NP test has 97.9% sensitivity and 100% specificity. This technique, once applied routinely in clinical laboratories, may guide the first line therapy for treating patients with sepsis, and therefore significantly change the patient outcomes, particularly in areas where carbapenemase producers are highly prevalent (such as Greece, Italy, Turkey, Israel, India). Additionally, when compared to molecular techniques, the Carba NP test may detect any carbapenemase production regardless of the corresponding gene being either known or unknown. Consequently, the Carba NP test is a useful tool for the detection of new carbapenemases that might eventually further disseminate, as recently shown with NDM-1 carbapenemase [8].

Conclusion
The ESBL NDP and the Carba NP tests are rapid, sensitive, specific and cost-effective biochemical tests for the early detection of the most important emerging resistance traits corresponding either to ESBL- or carbapenemase-producing Enterobacteriaceae. Implementation of such tests in the strategies of detection of multidrug-resistant bacteria may significantly improve the management and outcome of colonized and infected patients. Subsequently, the antibiotic stewardship would be improved leading to the decrease of the selective pressure that plays a crucial role in the emergence and spreading of multidrug-resistant bacteria.

Abbreviations
IMP, imipenemase; KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; VIM, Verona imipenemase; OXA, Oxacillinase

References
1. Schwaber MJ, Carmeli Y. JAMA 2008; 300: 2911–2913.
2. Spellberg B, Blaser M, et al. Clin Infect Dis 2011; 52(Suppl 5): S397–428.
3. Walsh TR, Toleman MA. J Antimicrob Chemother 2011; 67: 1–3.
4. Coque TM, Baquero F, Canton R. Euro Surveill 2008; 13.
5. Pitout JD, Laupland KB. Lancet Infect Dis 2008; 8: 159–166.
6. Poirel L, Bonnin RA, Nordmann P. Infect Genet Evol 2012; 12: 883–893.
7. Nordmann P, Dortet L, Poirel L. Trends Mol Med 2012; 18: 263–272.
8. Nordmann P, Poirel L, et al. Trends Microbiol 2011; 19: 588–595.
9. Nordmann P, Dortet L, Poirel L. J Clin Microbiol 2012; 50: 3016–3022.
10. Dortet L, Poirel L, Nordmann P. J Clin Microbiol 2012; 50: 3773–3776.
11. Dortet L, et al. Antimicrob Agents Chemother 2012; 56: 6437–6440.
12. Nordmann P, Poirel L, Dortet L. Emerg Infect Dis 2012; 18: 1503–1507.
13. Drieux L, Brossier F, et al. Clin Microbiol Infect 2008; 14(Suppl 1): 90–103.
14. Livermore DM, Canton R, et al. J Antimicrob Chemother. 2007; 59: 165–174.
15. Dortet L, Bréchard L, et al. J Antimicrob Chemother (submitted 2012).

The authors
Laurent Dortet, PhD, PharmaD, Laurent Poirel, PhD and
Patrice Nordmann, PhD, MD

Service de Bactériologie-Virologie, INSERM U914 “Emerging Resistance to Antibiotics”, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris Sud, K.-Bicêtre, France

E-mail: patrice.nordmann@bct.aphp.fr

Melanoma is the most malignant type of all skin neoplasms. Although current clinical, morphologic, pathologic, and biochemical methods provide insights into disease behaviour and outcome, melanoma is still an unpredictable disease. Once in an advanced stage, it remains a fatal neoplasm with few therapeutic options. Therefore, significant efforts still need to be made in finding suitable biomarkers that could aid or improve its early diagnosis, its correct staging, the discrimination of other pathological conditions as well as indicate patients’ prognosis or the most appropriate therapeutic regimes. On the other hand, well-defined diagnostic markers are necessary to avoid the apparent overdiagnosis of melanoma.

by Prof. J. Pietzsch, N. Tandler and Dr B. Mosch

Malignant melanoma: the need for biomarkers
Melanoma incidence and mortality have been steadily increasing in almost all countries and in fair-skinned populations in particular. For example, in Germany in 2009 incidence rates (mortality rates) of cutaneous melanoma were 17.4 (2.6) per 100 000 males and 16.0 (1.7) per 100 000 females, with cutaneous melanoma responsible for about 1.3% of all cancer deaths (Association of Population-based Cancer Registries in Germany, GEKID; http://www.gekid.de).

Considering variations between countries, 5-year survival for people of all races diagnosed with primary cutaneous melanoma <1.5 mm in depth is about 90%, amounting to 99% for local disease. The 5-year survival for people diagnosed with mucosal and intraocular melanoma is about 70%. However, 5-year survival is only 60–65% if the disease has spread within the region of the primary melanoma, dramatically dropping to below 10% if widespread. Although screening campaigns and intensive public health programmes resulted in decreasing incidence rates in, particularly, younger age groups, the incidence and burden of melanoma continue to rise. This is mainly due to the aging population, continued high recreational sun exposure habits, changing climate patterns, and increasing environmental contamination with carcinogenic agents [1, 2]. Thus, sensitive screening and early detection of high risk groups, and, on the other hand, personalization of therapy are the major principles of melanoma control. In this regard, biomarkers represent molecular attributes of the individual patient that will not only allow for detection and diagnosis, but also answer questions about the biologic behaviour of the tumour and metastases, mechanisms of resistance and/or sensitivity to therapy. Prospectively, melanoma therapy will substantially be improved by the use of biomarkers that (i) offer the potential to identify and treat melanoma before it is clearly visible or symptomatic, (ii) will facilitate easy detection without even minimal surgical procedure, and (iii) will also be candidates for population-based screenings. In this regard, this article briefly summarizes the current trends and perspectives in malignant melanoma biomarker research as recently reviewed and discussed in more detail by us [1, cf. references therein]. The characteristics of a good biomarker
Melanoma biomarkers can be divided into different categories. Most of them show higher expression in melanoma cells than in normal tissue and, therefore, are used as diagnostic markers. Other biomarkers may serve as prognostic or predictive markers because of their increased expression in advanced stages of disease, as indicators of treatment response and/or of disease recurrence during follow-up. Moreover, melanoma progenitor/stem cell markers are of potential use for identification of cell subpopulations that exhibit specifically critical properties like high carcinogenicity, metastatic potency, and treatment resistance.
The ideal serological biomarker should be a metabolically and analytically stable molecule detectable and/or quantifiable in the blood or other body fluid compartments, which are accessible by minimally invasive procedures. The biomarker should allow for the diagnosis of a growing tumour in a patient or for prediction of the likely response of a patient to a certain treatment, even earlier or better than by applying clinical imaging modalities. Hence, the biomarker must exhibit sufficient sensitivity and specificity in order to minimize false-negative as well as false-positive results [1, 3].

Importantly, at the moment, no ideal biomarker exists in the melanoma field. Pathologic characteristics of the primary melanoma, e.g., tumour thickness (Breslow index), mitotic rate, and ulceration are important prognostic factors. However, these characteristics can only be determined after localization and biopsy or surgical resection of the tumour. Regarding the points above, either circulating melanoma cells or melanoma-associated extracellular molecules provide suitable non-invasive analytical access.

Current and potential biomarkers for malignant melanoma
Melanoma cells release many proteins and other molecules into the extracellular fluid. Some of these molecules can end up in the bloodstream and hence serve as potential serum biomarkers. From a pathobiochemical point of view these biomarkers comprise molecules released by (i) necrotic cell content release, (ii) active secretion by melanoma cells, and (iii) ectodomain membrane shedding, including enzymes, soluble proteins/antigens, melanin-related metabolites, and circulating cell-free nucleic acids [1] [Table 1]. These molecules exhibit different prognostic and predictive values in melanoma diagnosis, staging, and treatment monitoring [1, 3–5].

Serum lactate dehydrogenase
In the American Joint Committee on Cancer (AJCC) staging system, serum lactate dehydrogenase (LDH) is the only serum biomarker that was accepted as a strong prognostic parameter in clinical routine for melanoma classifying those patients with elevated serum levels in stage IV M1C [3, 6].

Despite many promising results, there are also some limitations in measuring LDH as melanoma biomarker. First of all, LDH is not an actively secreted enzyme. Thus, LDH is only released through cell damage and cell death, which occur more frequently in malignant neoplasms. However, there are also false-positive values through hemolysis, hepatocellular injuries like hepatitis, myocardial infarction, muscle diseases, and other infectious diseases with high amounts of necrotic cells [3]. Moreover, LDH is non-specific for melanoma and elevated levels are also found in many other benign and malignant diseases.

Tyrosinase mRNA
An indicator for the presence of circulating melanoma cells and increased probability of the occurrence of metastases is the detection of tyrosinase mRNA in peripheral blood. Although the serological analyte is actually a nucleic acid isolated from circulating melanoma cells tyrosinase often is considered as an enzyme biomarker in melanoma [1, 3].

Due to the fact that tyrosinase mRNA is detected through nested RT-PCR the analytical sensitivity is very high. It is possible to detect one melanoma cell among 106 of normal blood cells. In recent decades, however, tyrosinase mRNA expression was determined in many different studies resulting in a wide range of variability (30–100%). One reason might be the transient presence of tumour cells in the bloodstream. On the other hand, non-standardized protocols for PCR-based techniques contribute to the observed variability, lower sensitivity, and different thresholds for melanoma cell detection.

Matrix metalloproteinases and cyclooxygenase-2
Further enzyme markers comprise matrix metalloproteinases and cyclooxygenase-2, with the latter detected via certain circulating eicosanoid products of the enzyme reaction [1, 7].

S100 calcium binding proteins
In addition, the S100 family of calcium binding proteins gained importance as both potential molecular key players and biomarkers in the etiology, progression, manifestation, and therapy of neoplastic disorders, including malignant melanoma. Moreover, S100 proteins receive attention as possible targets of therapeutic intervention moving closer to clinical impact.

In this regard, to-date, the best-studied S100 protein in melanoma is S100B [8, 9]. Increased S100B serum levels in melanoma patients chiefly have been attributed to the loss of cell integrity and proteolytic degradation as a result of apoptosis and necrosis of tumour cells. S100B seems to be the most promising serum marker for advanced melanoma, even more specific and sensitive than LDH, but is  not yet applied in the clinical routine [1, 10].

Another member of the S100 family, the metastasis-associated protein S100A4 influences cell motility, angiogenesis, and apoptosis. The mechanism by which S100A4 stimulates metastasis is still under investigation; however, extracellular S100A4 seems to be of major importance in this context and, therefore, possibly might serve as a blood marker. Despite some early promising results on the use of S100A4 serum levels as a prognostic marker in melanoma, the greatest problem might be the low protein concentration in the blood which impedes clinical relevance [1]. This seems to be also true for other S100 proteins that are suggested to be biomarker candidates of melanoma. As more specific reagents for individual S100 proteins are being generated, their potential diagnostic and prognostic usage will increase substantially [1, 9].

Other candidate biomarkers
Other soluble proteins considered as melanoma biomarker candidates are given in Table 1. Furthermore, various non-protein biomarkers are potential targets for melanoma biomarker research. Those comprise metabolites of the melanin synthesis pathways, originating from the amino acid L-tyrosine, and cell-free nucleic acids [1].

Future directions in melanoma biomarker discovery
As well as the markers discussed above, other proteins, some of them possibly representing melanoma progenitor/stem cell-like markers, can be detected in circulating melanoma cells, at least as demonstrated in animal models. This includes ATP-binding cassette (ABC) multidrug transporters and neuroepithelial intermediate filament nestin [1, 5]. These markers offer the potential to predict the risk of progression to metastatic disease states, treatment resistance, and disease relapse. Lack of sufficient sensitivity, specificity, and accuracy are the most relevant limitations of a single blood-based melanoma biomarker for clinical use.

By contrast, a cluster of biomarkers for one disease would be a better diagnostic tool with much higher sensitivity, specificity, and clinical accuracy. Therefore, new investigations, called ´proteomic profiling´, focus on the identification of multiple co-expressed biomarkers or signature biomarker patterns, which allow early detection, staging, therapeutic monitoring and prognostic predictions [4, 11, 12].

Abbreviations
Biomarker abbreviations: 6H5MI2C, 6-hydroxy-5-methoxyindole-2-carboxylic acid; CEACAM, carcinoembryonic antigen-related cell adhesion molecule 1; CYT-MAA, cytoplasmic melanoma associated antigen; MAGE, melanoma associated antigen-1; MART-1, melanoma antigen recognized by T-cells 1; MIA, melanoma inhibitory activity; MMP, matrix metalloproteinase; sICAM, soluble intercellular adhesion molecule 1; sVCAM, soluble vascular cell adhesion molecule 1; TA90, tumour-associated antigen 90; VEGF, vascular endothelial growth factor; YKL-40, heparin- and chitin-binding lectin YKL-40 (syn. human cartilage glycoprotein-39)

Method abbreviations: ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography; IHC, immunohistochemistry; IP, immunoprecipitation; LIA, luminescence immunoassay; RT-PCR, reverse transcription polymerase chain reaction; TMA, tissue microarray

This publication summarizes a comprehensive review article on protein and non-protein biomarkers in melanoma recently published by the authors [1, cf. references therein].

References
1. Tandler N, Mosch B, Pietzsch J. Protein and non-protein biomarkers in melanoma: a critical update. Amino Acids 2012; 43: 2203–2230.
2. De Giorgi V, Gori A, Grazzini M, et al. Epidemiology of melanoma: is it still epidemic? What is the role of the sun, sunbeds, Vit D, betablocks, and others? Dermatol Ther 2012; 25: 392–396.
3. Vereecken P, Cornelis F, Van Baren N, et al. A synopsis of serum biomarkers in cutaneous melanoma patients. Dermatol Res Pract 2012; 2012: 260643.
4. Palmer SR, Erickson LA, Ichetovkin I, et al. Circulating serologic and molecular biomarkers in malignant melanoma. Mayo Clin Proc 2011; 86: 981–990.
5. Mimeault M, Batra SK. Novel biomarkers and therapeutic targets for optimizing the therapeutic management of melanomas. World J Clin Oncol 2012; 3: 32–42.
6. Balch CM, Gershenwald JE, Soong SJ, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009; 27: 6199–6206.
7. Kruijff S, Hoekstra HJ. The current status of S-100B as a biomarker in melanoma. Eur J Surg Oncol 2012; 38: 281–285.
8. Nicolaou A, Estdale SE, Tsatmali M, et al. Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone. FEBS Lett 2004; 570: 223–226.
9. Pietzsch J. S100 proteins in health and disease. Amino Acids 2011; 41: 755–760.
10. Weide B, Elsässer M, Büttner P, et al. Serum markers lactate dehydrogenase and S100B predict independently disease outcome in melanoma patients with distant metastasis. Br J Cancer 2012; 107: 422–428.
11. Solassol J, Du-Thanh A, Maudelonde T, et al. Serum proteomic profiling reveals potential biomarkers for cutaneous malignant melanoma. Int J Biol Markers 2011; 26: 82–87.
12. Pham TV, Piersma SR, Oudgenoeg G, Jimenez CR. Label-free mass spectrometry-based proteomics for biomarker discovery and validation. Expert Rev Mol Diagn 2012; 12: 343–359.

Acknowledgements
Nadine Tandler is the recipient of a fellowship from the Europäische Sozialfonds (ESF).

The authors
Jens Pietzsch*, PhD, MD, Nadine Tandler, MSc and Birgit Mosch, PhD
Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany

*Corresponding author
E-mail: j.pietzsch@hzdr.de

Primary hepatic tumours are one of the most aggressive and resistant forms of cancer. Early diagnosis of liver cancer and the development of more accurate markers for biological classification are crucial to improving the clinical management and survival of patients. This article discusses the emerging use of microRNAs for the diagnosis of liver cancer.

by Dr Luc Gailhouste and Dr Takahiro Ochiya

Liver cancer and diagnosis
Primary liver cancer is mainly represented by hepatocellular carcinoma (HCC) and accounts for almost 90% of primitive hepatic malignancies. Statistically, HCC is the third most common cause of death from cancer worldwide [1] and is generally encountered in patients exhibiting an underlying chronic liver disease such as hepatitis B virus (HBV) and/or C virus (HCV) infection, alcohol abuse, or liver steatosis. Chronic hepatitis leads to fibrosis and gradually evolves into cirrhosis. Global studies estimate that approximately 80–90% of all HCCs arise from cirrhotic livers. Despite great advances in the treatment of the disease, hepatic cancer exhibits one of the lowest remission rates (less than 10% after five years), mainly due to its late diagnosis and high resistance to the conventional agents of chemotherapy. Indeed, as such a disease tends to remain asymptomatic, approximately 50% of newly diagnosed patients already exhibit late advancement.

Common HCC diagnostic methods include liver imaging techniques such as triphasic computed tomography scanning, magnetic resonance imaging (MRI), and abdominal ultrasound [2]. A panel of serological biochemical markers, including aminotransferases ALAT and ASAT, has also been used for several decades to monitor liver pathologies in a non-invasive manner.

Until recently, imaging tests were frequently combined with the non-invasive measurement of serum alpha-fetoprotein (AFP). Normally produced by the fetal liver, AFP decreases soon after birth whereas its high level in adults can be correlated with the appearance of malignant hepatic disease. However, the American Association for the Study of Liver Diseases (AASLD), in its practice guidelines, discontinued the use of the blood tumour marker AFP for surveillance and diagnosis due to the limited sensitivity and specificity of the method. When uncertainty regarding the diagnosis persists, a percutaneous biopsy followed by histological examination of the nodule is indicated [3]. This technique remains the gold standard method for determining the degree of underlying fibrosis and shows appreciable sensitivity (more than 80%) for HCC diagnosis.

An important breakthrough in the clinical management of liver cancer would come from the accurate correlation of the alterations of cancer-related genes and the tumour phenotype. Although HCC lesions can be broadly distinguished by histological or immunological assessment, their prognosis and clinical evolution vary greatly from one individual to another. The discovery of innovative and effective biomarkers ensuring an early diagnosis of the disease correlated with the etiology, the pathogenic tendency, and the malignancy of the tumour could significantly enhance the molecular assessment of HCC and its classification in order to maximize the positive response of therapeutics.

MicroRNAs: biogenesis and mechanism of action
MicroRNAs (miRNAs) constitute a group of evolutionary conserved small non-coding RNAs of approximately 22 nucleotides that accurately regulate gene expression by complementary base pairing with the 3’-untranslated regions (3’-UTRs) of messenger RNAs (mRNAs) [4]. These post-transcriptional regulators were first evidenced in C. elegans by Ambros and co-workers who discovered that lin-4, a gene known to control the timing of nematode larval development, did not code for a protein but produced small RNAs that specifically bind to lin-14 mRNA and repress its translation.

miRNA biogenesis is a multistep process that has been reviewed extensively [Figure 1]. An essential feature of miRNAs is that a single miRNA can recognize numerous mRNAs, and, conversely, one mRNA can be recognized by several miRNAs. These pleiotropic properties enable miRNAs to exert wide control over a plethora of targets, attesting to the complexity of this mechanism of gene expression regulation. Several reports have described the key role of these post-transcriptional regulators in the control of diverse biological processes such as development, differentiation, cell proliferation, and apoptosis. The alterations of miRNA expression have also been reported in a wide range of human diseases, including cancer [5].

In HCC, the atypical expression of miRNAs frequently contributes to the deregulation of critical genes known to play an essential role in tumorigenesis and cancer progression. The current consensus is that cancer-related miRNAs function as oncogenes or tumour suppressors [6]. As for other malignancies, two situations can occur in HCC: (i) tumour suppressor miRNAs can be downregulated in liver cancer and cause the upregulation of oncogenic target genes repressed in normal hepatic tissues, increasing cell growth, invasion abilities, or drug resistance; (ii) oncogenic miRNAs, also called oncomirs, can be upregulated in HCC and can downregulate their target tumour suppressor genes, potentially leading to hepatocarcinogenesis.

miRNA as a diagnostic tool
As miRNA signatures are believed to serve as accurate molecular biomarkers for the clinical classification of HCC tumours, the availability of consistent technologies that enable the detection of miRNAs has become of interest for both fundamental and clinical purposes. The most current detection methods commonly used are microarray and real-time quantitative polymerase chain reaction (RT-qPCR).

Microarray analysis presents the advantage of offering a high speed of screening by employing various miRNA probes within a single microchip. However, the technique has lower sensitivity and specificity than RT-qPCR, which is the most widely used method.

miRNA RT-qPCR is based on the use of stem–loop primers, which can specifically bind to the mature miRNA during reverse transcription, granting a high degree of accuracy to the method [7]. Analysis of miRNAs by RT-qPCR is a cost-effective technique and, due to its efficiency, a valuable way to validate miRNA signatures. Moreover, the development of RT-qPCR protocols has improved the sensitivity of miRNA detection down to a few nanograms of total RNAs. This amount can be easily and routinely obtained by extracting total RNAs from a small fragment of a hepatic percutaneous biopsy.

A plethora of studies have already reported various miRNA profiles potentially reflecting HCC initiation and progression that could be employed as specific cancer biomarkers [8]. Comparative analysis of bibliographic data provides evidence of the persistent augmentation of miR-21 in cancer, regardless of the tumour origin. In the HCC, miR-21 is also frequently overexpressed where it acts as an oncogenic miRNA. The major overexpression of miR-21 is associated with the inhibition of the tumour suppressor PTEN and the poor differentiation of the tumour. The use of an miRNA-based classification correlated with the etiology and the aggressiveness of the tumour appears very promising, as it could significantly enhance the accuracy of the molecular diagnosis of HCC and its classification, leading to the consideration of more appropriate therapeutic strategies.

In this regard, Budhu and collaborators defined a combination of 20 miRNAs as an HCC metastasis signature and showed that this 20-miRNA-based profile was capable of predicting the survival and recurrence of HCC in patients with multinodular or single tumours, including those at an early stage of the disease [9]. Remarkably, the highlighted expression profile showed a similar accuracy regarding patient prognosis when compared to the conventional clinical parameters, suggesting the relevance of this miRNA signature. Consequently, the profiling of aberrantly expressed cancer-related miRNAs might establish the basis for the development of a rational system of classification in order to refine the diagnosis and the prediction of HCC evolution.

Tumour suppressor miRNA: the case of miR-122
The case of miR-122 is of prime interest, first, because it represents by itself more than half of the total amount of miRNAs expressed in the liver [10]. Remarkably, miR-122 is a key host factor required for HCV replication. A phase 2 clinical trial was recently initiated that reported the world’s first miRNA-based therapy targeting miR-122 in HCV-infected patients using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen [11]. Thus, a four-week miravirsen treatment by subcutaneous injection provided long-lasting antiviral activity and was well tolerated.

However, the experimental silencing of miR-122 resulted in increased expression of hundreds of genes normally repressed in normal hepatocytes. The miR-122 knockout mouse model displays hepatosteatosis, fibrosis, and a high incidence of HCC, suggesting the tumour suppressor role of miR-122 in the liver. In primary liver carcinoma, the existence of an inverse correlation was demonstrated between the expression of miR-122 and cyclin G1, which is highly implicated in cell cycle progression.

Regarding the potential of miR-122 as a diagnostic biomarker in liver cancer, numerous studies have already reported the significant and specific downregulation of miR-122 expression in both human and rodent HCC models. Obviously, miR-122 was shown as downregulated in more than 70% of the samples obtained from HCC patients with underlying cirrhosis as well as in 100% of the HCC-derived cell lines [12].

To illustrate this statement, we analyzed the expression levels of miR-122 in 20 patients who exhibited HCC using RT-qPCR. Following RNA extraction from biopsies with the miRNeasy Mini Kit (Qiagen), 100 ng of total RNA was reverse-transcribed using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems). The expression levels of mature miR-122 were determined in each sample by RT-qPCR with Taqman Universal PCR Master Mix in a 7300 Real-Time PCR System from Applied Biosystems. The expression levels of miRNAs were normalized with respect to the endogenous levels of RNU6B. RT-qPCR data were obtained easily and rapidly by a routinely conventional method used in our laboratory. As a result, miR-122 expression was reduced more than threefold in HCC biopsies relative to the normal liver group (median 0.935 and 3.495, respectively; P<0.0001, Mann–Whitney U test) [Figure 2]. These data suggest that cancer-related miRNAs, such as miR-122, which are deregulated in HCC tissues, could be relevant with regard to the development of new diagnostic tools and the clinical management of liver cancer patients. Conclusions and emerging approaches
The expression profile of specific miRNAs has been found to reflect the biological behaviour of HCC tumours, such as aggressiveness, invasiveness, or drug resistance. As a consequence, miRNA investigations may offer opportunities to determine miRNA signatures that would provide valuable information to stratify and refine HCC diagnosis in terms of prognosis, response to treatment, and disease relapse. Recently, tumour-derived miRNAs have been efficiently detected in the serum of patients and characterized as potential non-invasive biomarkers for HCC.

The concept that miRNAs could serve as potential plasma markers for liver diseases is, thus, gaining attention. Due to its frequent deregulation in viral hepatitis, cirrhosis, and cancer as well as its specific and massive expression in the liver, the assessment of serum miR-122 could represent one reliable strategy for the non-invasive diagnosis of chronic liver pathologies. Although the process of assessing serum miRNAs remains under improvement, cancer-related circulating miRNAs represent an exciting and promising field of investigation for the development of more accurate technologies for the early diagnosis of HCC.

References
1. Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006; 6: 674–687.
2. Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002; 122: 1609–1619.
3. Ryder SD. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 2003; 52(Suppl 3): iii1–8.
4. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
5. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857–866.
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7. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.
8. Gailhouste L, Gomez-Santos L, Ochiya T. Potential applications of miRNAs as diagnostic and prognostic markers in liver cancer. Front Biosci 2013; 18: 199–223.
9. Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47: 897–907.
10. Girard M, Jacquemin E, Munnich A, et al. miR-122, a paradigm for the role of microRNAs in the liver. J Hepatol 2008; 48: 648–656.
11. Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol 2012; 199: 407–412.
12. Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67: 6092–6099.

The authors
Luc Gailhouste PhD and
Takahiro Ochiya PhD

Division of Molecular and Cellular
Medicine, National Cancer Center Research Institute, Tokyo, Japan

Coronaviruses are a group of positive sense, single-stranded RNA viruses that infect humans and animals. In a short period of time the SARS-associated coronavirus was identified and initial laboratory protocols for diagnosis of SARS were disseminated. The need for the early diagnosis of SARS is vital due to the difficulty in clinically diagnosing this infection and its rapid nosocomial transmission.

by Dr Hoon H. Sunwoo and Dr Arivazhagan Palaniyappan

Clinical background
Severe acute respiratory syndrome (SARS) is a life-threatening viral respiratory illness caused by a coronavirus known as SARS-associated coronavirus (SARS-CoV, but usually shortened to SARS). The SARS-CoV is associated with a flu-like syndrome, which may progress into pneumonia, respiratory failure, and sometimes death. It is believed that SARS-CoV originated in the Guangdong Province in southern China and the virus has subsequently spread around the world. China and its surrounding countries have witnessed the greatest numbers of SARS-related cases and death.

SARS history is short. SARS-CoV was first reported in 2002 in Asia and cases were reported until mid-year 2003. According to the World Health Organization (WHO), as of July 2003, a total of 8437 people worldwide became ill and 813 died during the SARS outbreak or epidemic. Illness was reported in more than 30 countries and on 5 continents. This new emerging disease represented the most recent threat to human health as it has been reported to be highly contagious. Infection with the SARS-CoV causes acute respiratory distress (severe breathing difficulty) and sometimes death.

SARS-CoV Diagnosis
Three major diagnosis methods are currently developed (i) viral RNA detection using quantitative reverse transcription (RT)-PCR, (ii) antibody detection using indirect fluorescence assay (IFA), and (iii) using both recombinant nucleocapsid protein (NP) and culture extract of SARS-CoV–based enzyme-linked immunosorbent assay (ELISA). ELISA based antibody detection tests with recombinant antigens are well known to offer higher specificity and reproducibility. Such tests are easy to standardize and less labour intensive than antibody detection by indirect IFA and thus avoids the requirement of growing SARS-CoV.

RT-PCR has been widely used for the rapid diagnostic of the viral genome in different clinical specimens. Early diagnosis of SARS-CoV infection, which involves viral RNA detection by RT-PCR, first targeted the polymerase (pol) 1b region of the 5’ replicase gene using different formats including one-step or two-step RT-PCR or real-time PCR assays. A comprehensive monitoring of the time periods of RT-PCR diagnosis after disease onset in different types of specimens such as tracheal and nasopharyngeal aspirates, throat swabs, nasal swabs and rectal swabs has also been studies. This study demonstrates that the peak detection rate for SARS-CoV occurred at 2 weeks after the onset of stool or rectal swab specimens and at week 4 for urine specimens [1]. It is likely that the current RT-PCR is not quite sensitive enough to detect the early diagnosis of SARS, showing that the detection rate for probable SARS was only 37.5–50%.

The presence of specific antibodies against various viral components has been a classical diagnostics method. It has been found that anti-NP antibodies in patients’ sera are detected early and with high specificity during the infection. Three different methods, Western blot, ELISA and IFA, used both native and bacterially produced SARS antigens to evaluate serum samples obtained from SARS patients, 40 patients with non-SARS pneumonia, and 38 health individuals. A report indicated that 89% of the SARS patients’ sera were found to be positive to SARS-CoV NP antigen by Western blot that had a strong ability to detect antibodies against SARS. The sensitivity and specificity was reported to be 98.5 and 100% respectively [2]. There was no cross reactivity between the N195 protein and antibodies against chicken, pig and canine coronaviruses. The Western blot assay could distinguish patients with fewer caused by other diseases from that of SARS patients, through reducing the possibility of false positives.

Our earlier study also showed that different combinations of monoclonal antibody (mAb), bispecific antibody (bsmAb), and IgY polyclonal antibody detected the SARS-CoV NP by Immunoswab assay [3] and sandwich ELISA [4] with a sensitivity of 18.5 pg/ml of recombinant SARS-CoV NP antigen in-vitro [Figure 1]. Antibodies against the NP have longer a shelf life and occur in greater abundance in SARS patients than antibodies against other viral components such as the spike protein (SP), membrane and envelope protein. This may be due to the presence of higher levels of NP, compared with other viral proteins, after SARS-CoV infection. A recombinant NP-based IgG ELISA was more sensitive than a recombinant S-protein-based IgG ELISA for diagnosis of SARS-CoV in serum [5–6], due to the highly immunogenic region of N2. It may help in explaining the present results that show less sensitivity of SP detection, compared to a previous NP detection study [4].

Recent studies demonstrate that mAbs and bsmAb could be useful reagents for the diagnosis of SARS-CoV, as well as for functional analysis of SP during infection. Further, the present study shows the development of a novel sandwich ELISA test with a potential use for the diagnosis of SARS-CoV infections based on bsmAb that recognize simultaneously the SP of SARS-CoV and the enzyme peroxidase [7] [Figure 2]. In addition to allowing the rapid diagnosis of SARS infection, the availability of diagnostic tests will help to address important questions such as the period of virus shedding during convalescence, the presence of virus in different body fluids and excreta, and the presence of virus shedding during the incubation period. Until a certain degree of standardization and quality assurance has been achieved for the SARS-CoV laboratory tests, test results must be used with utmost caution in clinical situations. It is strongly advisable to closely check on updated recommendations by the WHO and relevant national organizations regarding the availability and use of such tests.

Limitations
All tests for SARS-CoV available so far have limitations. Extreme caution is therefore necessary when management decisions are to be based on virological test results. In particular, false negative test results (due to low sensitivity, unsuitable sample type, or time of sampling, etc.) may give a false sense of security; in the worst case, they could allow persons carrying the SARS virus, and therefore capable of infecting others, to escape detection.

To aid in the better understanding of SARS, the WHO recommends that sequential samples be stored from patients with suspected or probable SARS – and also close contacts who are not ill themselves – for future use. This is particularly important for the first case(s) recognized in countries that have not previously reported SARS. Data on the clinical and contact history should also be collected in order to obtain a better understanding of the shedding pattern of the virus and the period of transmissibility. Such patient samples should be suitable for viral culture, PCR, antigen detection, immunostaining and/or serological antibody assays. The WHO also encourages each country to designate a reference laboratory for investigation and/or referral of specimens from possible SARS patients.

Future SARS outbreaks
Although the threat of SARS to public health seems to have passed, international health officials continue to remain vigilant. The WHO monitors countries throughout the world for any unusual disease activity (http://www.who.int/csr/sars/en/). Therefore, if another SARS outbreak is to occur, it should be possible to limit the spread of infection using the same measures implemented during the 2002/3 pandemic.

References
1. Chan PK, To WK, Ng KC, Lam RK, Ng TK, et al. Laboratory diagnosis of SARS. Emerg Infect Dis 2004; 10: 825–831.
2. He Q, Chong KH, Chng HH, Leung B, Ling AE, et al. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol 2004; 11: 417–422.
3. Kammila S, Das D, Bhatnagar PK, Sunwoo HH, et al. A rapid point of care immunoswab assay for SARS-CoV detection. J Virol Methods 2008; 152: 77–84.
4. Palaniyappan A, Das D, Kammila S, Suresh MR, Sunwoo HH. Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y. Poult Sci 2012; 91: 636–642.
5. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP. Characterization of novel coronavirus associated with severe acute respiratory syndrome. Science 2003; 300: 1394–1399.
6. Woo PC, Lau SK, Wong BH, Tsoi HW, Fung AM, et al. Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 2005; 43: 3054–3058.
7. Sunwoo HH, Palaniyappan A, Ganguly A, Bhatnagar PK, et al. Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked  immunoassay. J Virol Methods 2013; 187: 72–78.

The authors
Hoon H. Sunwoo* PhD and Arivazhagan Palaniyappan PhD
Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, Alberta, Canada T6G 2E

*Corresponding author
E-mail: hsunwoo@ualberta.ca