Page 217 – Clinical Laboratory int.

Recent trends in malignant melanoma biomarker research

, 26 August 2020/in Featured Articles /by 3wmedia

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

A novel approach in the diagnostics of renal cell cancer: Image guided optical biopsy

, 26 August 2020/in Featured Articles /by 3wmedia

Optical coherence tomography (OCT) has long been routinely used in ophthalmology, but recent studies in the field of renal cell carcinoma have demonstrated the ability of OCT to distinguish between renal malignancies and normal renal tissue. This suggests the possibility that, eventually, diagnosis by invasive biopsy could be replaced by non-invasive techniques.

by D. M. de Bruin, Dr P. Wagstaf, Dr K. Barwari, Prof. T. G. van leeuwen, Dr D. J. Faber, Prof. J. J. de la Rosette and Dr M. P. Laguna

The diagnosis of small renal masses
The diagnosis of small renal masses (SRMs) has seen a dramatic increase in presentation in recent decades. This change is mainly attributed to an increased use of abdominal imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI). However, the large imaging depth of such modalities is accompanied by a relatively low resolution of the obtained images, hindering conclusions at the level of histological composition. Recent studies have shown an inverse correlation between tumour size and malignancy, and up to 10 % of all extirpated (and thus deemed malignant) tumours appear to be benign on histopathological examination. This inverse relationship increases to 25% when small renal masses (SRM) (≤4 cm) are considered [1]. Therefore, pre-operative diagnosis of (small) renal tumours would be desirable. However, due to the high number of non-diagnostic biopsy results (up to 30 % in SRM), systematic use of pre-operative renal mass biopsies is still not recommended in the major guidelines [2–5].

Renal mass biopsy
Most renal biopsies are performed percutaneously and are supported by image guidance using computed tomography (CT) or ultrasound. The biopsies are normally performed under local anesthesia in an outpatient setting. When a renal tumour is evaluated, a biopsy can deliver one of two results: diagnostic (benign or malignant) or non-diagnostic, the later including the presence of necrosis, fibrosis and normal renal parenchyma with absence of tumour cells [Figure 1]. When the biopsy is diagnostic, other characteristics such as histopathologic subtype and grade can also be assessed [4, 6, 7].

Conceptually a failed biopsy means that there is no tumour tissue available for assessment in the biopsy specimen, although other types of tissue might be present in the sample. The reason for a failed biopsy could be a technical failure of the puncture method (e.g. misfire or malfunctioning of the biopsy gun) or incorrect sampling caused by imperfect image guidance. Incorrect sampling is sometimes unavoidable due to the nature of renal tumours, which may contain necrotic and fibrotic tissue, or be mixed in nature with solid and cystic components. Also, the presence of normal renal tissue implies that the sampling is incorrect, as very few renal masses are composed of normal renal tissue. The presence of fibrotic, inflammatory, fatty or necrotic tissue in the specimen means that a differential diagnosis between malignant and benign tumour cannot be made. Besides the fact that histopathological analysis requires time, it is also subject to a certain degree of discordance among different pathologists [8].

A diagnostic imaging tool that allowed real-time visualization of micro-scale tissue architecture and subsequent differentiation of tissue type during the procedure would accelerate and simplify the overall diagnostic procedure.

Optical imaging
Optical diagnostic imaging comprises a novel group of imaging modalities that provide information by assessing differences between incident and detected light caused by the interaction of light with tissue. Scattering and absorption are tissue-specific optical properties and, by assessing these interactions,
diffeent tissue types can be distinguished.

Optical imaging has shown potential in several medical fields where they are employed routinely in various forms, ranging from pulse oximeters to fundus cameras, and experimental reports show promising results in the field of oncology [9].

Optical coherence tomography (OCT) is a technology developed in the early 1990s for ophthalmological applications [10] and is routinely used in that setting in current clinical practice. OCT is the optical equivalent of ultrasound, using light instead of sound to produce micrometer-scale resolution, cross-sectional images up to a depth of about 2 mm in renal tissue [Figure 2]. Resolutions up to 5 µm can be achieved, being 100–250 times higher than high-resolution ultrasound [11] and approaching that of microscopy. An image produced by OCT resembles the tissue structures observed in histology and can, therefore, be considered as an ‘optical biopsy’ [12] [Figure 2]. Moreover, data extracted from the original OCT images can be used for functional quantitative analysis after careful calibration of the OCT system. This finally results in a ‘functional optical biopsy’. The imaging depth is primarily limited by the scattering of light by cellular structures, hindering the return of reflections to the receiver. This scattering causes the light intensity to attenuate as it penetrates deeper into the tissue and this attenuation of OCT signal can be quantified by measuring the decay of signal intensity per unit depth. Using Lambert–Beer’s law and after careful calibration of the OCT system, a tissue specific attenuation coefficient (μOCT mm-1) can be derived [13–15]. Because malignant tissue displays an increased number, larger and more irregularly shaped nuclei with a higher refractive index and more active mitochondria, the μOCT is expected to be higher compared to normal and benign tissue [Figure 3].

In urology, the early research on OCT has been focused on tissue diagnosis predominantly in bladder and prostate cancer [12, 16] and, more recently, attention has turned to the field of renal cell carcinoma (RCC) and research is currently ongoing [17–20]. We were the first authors to publish data on the ability of OCT to differentiate renal malignancies from normal renal tissue using quantitative analysis. Subsequently, we performed an in vivo pilot study assessing the difference of the attenuation-coefficient of malignant renal tumours from normal renal parenchyma and benign tumours [18]. OCT-imaging took place using an in vivo OCT-probe during surgery, and a significant difference was found between the attenuation-coefficient of normal renal tissue and that of malignant tumours. Attenuation-coefficients of malignant and benign tumours did differ, although it is likely that the small sample size (3 benign tumours vs 11 malignant) is hindering a statistical significance, suggesting that a clear difference might be found in larger samples. Linehan et al. assessed qualitative differences of OCT images of different types of renal tumours showing that certain tumour subtypes do have different appearances on OCT-imaging; however, intriguingly, clinical distinction of tumours such as RCC from oncocytomas could not be demonstrated [19].

Future developments
Finally, anticipating the validation of results showing optical diagnostics being able to differentiate renal tissues, there is a potential role for the techniques in several clinical scenarios. Before going as far as replacing pathological examination as discussed earlier, the two techniques might be complementary with the real-time- and non-invasive nature of the optical techniques serving as guidance for correct needle placement in order to reduce the number of non-diagnostic biopsy results, as is already done in other malignancies, and the small in vivo probes necessary for such interventions are becoming commercially available. The technological configuration behind OCT allows for easy integration with diffuse reflectance spectroscopy (DRS) and Raman spectroscopy (RS). Moreover, the structural-imaging- and light-scattering based quantitative possibilities of OCT together with the quantitative light absorption sensitivity of DRS and the inelastic light scattering (and therefore biochemical) sensitivity of RS yields the full potential of a functional optical biopsy.

We would like to thank the Cure for Cancer Foundation (CFC) and the Technology Foundation (STW) for project funding. This work is part of the innovative Medical Imaging Technologies program (iMIT) of STW and the Novel Biopsy Methods program of CFC.

References
1. Tan H-J et al. Understanding the role of percutaneous biopsy in the management of patients with a small renal mass. Urology 2012; 79(2): 372–377.
2. Volpe A, Jewett MA. Current role, techniques and outcomes of percutaneous biopsy of renal tumors. Expert Rev Anticancer Ther 2009; 9(6): 773–783.
3. Motzer RJ et al. NCCN clinical practice guidelines in oncology: kidney cancer. J Natl Compr Canc Netw 2009; 7(6): 618–630.
4. Leveridge MJ et al. Outcomes of small renal mass needle core biopsy, nondiagnostic percutaneous biopsy, and the role of repeat biopsy. Eur Urol 2011; 60(3): 578–584.
5. Ljungberg B et al. EAU guidelines on renal cell carcinoma: the 2010 update. Eur Urol 2010; 58(3): 398–406.
6. Menogue SR et al. Percutaneous core biopsy of small renal mass lesions: a diagnostic tool to better stratify patients for surgical intervention. BJU Int 2012; doi: 10.1111/j.1464-410X.2012.11384.x.
7. Laguna MP et al. Biopsy of a renal mass: where are we now? Curr Opin Urol 2009; 19(5): 447–453.
8. Kümmerlin IP et al. Cytological punctures in the diagnosis of renal tumours: a study on accuracy and reproducibility. Eur Urol 2009; 55(1): 187–198.
9. Pierce MC, Javier DJ, Richards‐Kortum R. Optical contrast agents and imaging systems for detection and diagnosis of cancer. Int J Cancer 2008; 123(9): 1979–1990.
10. Huang D et al. Optical coherence tomography. Diss. Massachusetts Institute of Technology, Whitaker College of Health Sciences and Technology, 1993.
11. Fujimoto, JG et al. Optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy. Neoplasia 2000; 2(1–2): 9–25.
12. Crow P et al. Optical diagnostics in urology: current applications and future prospects. BJU Int 2003; 92(4): 400–407.
13. Faber DJ et al. Quantitative measurement of attenuation coefficients of weakly scattering media using optical coherence tomography. Optics Express 2004; 12(19): 4353–4365.
14. van Leeuwen TG, Faber DJ, Aalders MC. Measurement of the axial point spread function in scattering media using single-mode fiber-based optical coherence tomography. IEEE Journal of Selected Topics in Quantum Electronics 2003; 9(2): 227–233.
15. de Bruin DM et al. Optical phantoms of varying geometry based on thin building blocks with controlled optical properties. J Biomed Opt 2010; 15(2): 025001.
16. Cauberg EC et al. Quantitative measurement of attenuation coefficients of bladder biopsies using optical coherence tomography for grading urothelial carcinoma of the bladder. J Biomed Opt 2010; 15(6): 066013.
17. Barwari K et al. Advanced diagnostics in renal mass using optical coherence tomography: a preliminary report. J Endourol 2011; 25(2): 311–315.
18. Barwari K et al. Differentiation between normal renal tissue and renal tumours using functional optical coherence tomography: a phase I in vivo human study. BJU Int 2012; 110(8 Pt B):E415–20.
19. Linehan JA et al. Feasibility of optical coherence tomography imaging to characterize renal neoplasms: limitations in resolution and depth of penetration. BJU Int 2011; 108(11): 1820–1824.
20. Onozato ML et al. Optical coherence tomography of human kidney. J Urol 2010; 183(5): 2090–2094.

The authors
D. Martijn de Bruin^1,2,* Msc; Peter G. Wagstaff¹ MD; Kurdo Barwari¹ PhD, MD; Ton G. van Leeuwen² PhD; Dirk J. Faber² PhD; Jean J. de la Rosette¹ PhD, MD; M. Pilar Laguna¹ PhD, MD.

¹ Department of Urology, Academic Medical Center, Amsterdam, Meibergdreef 9, 1105 AZ, The Netherlands
² Department of Engineering & Physics, Academic Medical Center, Amsterdam, Meibergdreef 9, 1105 AZ, The Netherlands

*Corresponding author
E-mail: d.m.debruin@amc.uva.nl

MicroRNAs: New tools to tackle liver cancer progression

, 26 August 2020/in Featured Articles /by 3wmedia

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.
6. Gailhouste L, Ochiya T. Cancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma. Histol Histopathol 2012.
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

Clinical labs: in the frontline of new respiratory epidemics

, 26 August 2020/in Featured Articles /by 3wmedia

Simmering concerns about respiratory disease pandemics flared up again in mid-February after the death of a patient in Britain due to infection by a new coronavirus. The virus is part of a family which also includes the one that caused the deadly SARS (severe acute respiratory syndrome) crisis.

To recall, in the space of just seven months from November 2002, SARS spread from Hong Kong to over 37 countries, infecting over 8,000 people and killing 775. Its mortality rate was close to that of the 1918 Spanish flu outbreak – billed the ‘Mother of all Pandemics’, and 100 times more than typical influenza epidemics. SARS has since faded away, but the virus is probably lying dormant; it can also infect cats and dogs.

SARS, bird flu and swine flu
SARS outgunned the H5N1 influenza strain which also emerged out of Asia in 1997; this was largely due to the inability of the latter, best known as ‘bird flu’, to spread between people.

In 2009, another influenza strain, Type A/H1N1, involving a cocktail of genes from pigs, birds and humans, was identified in Mexico. By June, the World Health Organization (WHO) had declared the disease (dubbed ‘swine flu’) as a Level 6 pandemic , but this was due to the speed of its spread rather than mortality, which was less than the common flu.

The new coronavirus
The numbers infected by the new coronavirus are small, just 12, so far. However, the virus has some troubling characteristics. Unlike swine flu, mortality is high, and typically accompanied by pneumonia and renal failure. Of the 12 infected so far, six have died, according to the WHO. Of equal concern is the possibility of human-to-human transmission, as opposed to bird flu.

This time, the new virus has its origins in the Middle East, with Saudi Arabia and Jordan accounting for seven infections and five deaths. In November 2012, the WHO reported cases from within one Saudi Arabian family. However, it was impossible to determine if the patients were infected separately but simultaneously (during travel), or whether the disease had spread between them. Europe hosts the remaining cases – one in Germany and four in Britain, including the Birmingham fatality. While the German patient had been in Qatar, in Britain, rather than the victim, it was his father who had travelled to the Middle East. Since then, the father is reported to have infected yet another family member. Prof. John Watson, head of the Respiratory Disease Unit at the British Health Protection Agency (HPA), noted that this suggested “that person-to-person transmission has occurred.”

Nevertheless, British health officials have been quick to ward off panic. The Birmingham victim is reported to have had a weakened immune system placing him in a vulnerable risk group. The HPA’s Deputy Chief Executive Dr. Paul Cosford has underlined that the disease appears “very difficult to catch.” Prof. Wendy Barclay of Imperial College London adds: “We’re an incremental step closer to worrying, but it isn’t a worry where we need to say there is a pandemic coming.”

Getting it right
These are reassuring words for the public, but hardly so for clinical laboratories. If any of the above assumptions are (or turn out to be) wrong, the challenge for labs will be herculean – as demonstrated during the SARS crisis. Indeed, Prof. Barclay’s statements were reported four days before the new virus took its first casualty in Britain.

Though coronaviruses are fragile (they are easily destroyed by detergents and survive outside a host organism for only a day or so), the severity of illnesses like SARS compel authorities to err on the side of caution – including enforcing quarantine (with its disturbing legal implications). The nature of such a response, in turn, places inordinately heavy demands on labs to get their diagnoses right, and be ready to ramp up scale exponentially. Complicating matters further is the fact that coronaviruses are a large family. Other than SARS, they also include the virus which causes the common cold.

Though several diagnostic tests have emerged since the SARS crisis, each has its limitations. Enzyme-linked immunosorbent assays (ELISA) detect antibodies to SARS reliably, but only 21 days after the onset of symptoms. Immunofluorescence assays (IFA) take half the time but require an immunofluorescence microscope and highly skilled staff. Polymerase chain reaction (PCR) tests are extremely specific, but less sensitive: though positive results strongly indicate SARS infection, negative results do not necessarily mean its absence.

Guidelines for respiratory disease epidemics
The WHO’s laboratory guidelines for SARS hint at the magnitude of the challenge of any new respiratory disease epidemic. Above all, its recommendations on interpreting results are cumbersome. Positive PCR requires at least two different clinical specimens from a patient, or the same specimen collected on two or more days, or two different assays or repeat PCR using the original clinical sample on each occasion of testing.

For ELISA and IFA testing, the WHO specifies a negative antibody test on acute serum, followed by a positive antibody test on convalescent serum, or an over four-fold rise in antibody titre between the acute and convalescent phase
sera, which must be tested in parallel.

So far, evidence of the origin of the new Middle Eastern coronavirus is sketchy. Genetic sequencing at a Dutch laboratory has established that the virus is not the one which causes SARS. Since then, phylogenetic analysis has shown its closest relatives are bat coronaviruses from Hong Kong.

Labs: frontline defence and court of last resort
A Health Canada study titled ‘Learning from SARS’ is an excellent evaluation of the role of laboratories – above all, that of lab personnel, during the crisis. One conclusion was that though the country’s Winnipeg-based National Microbiology Laboratory (NML) was “not designed for an epidemic response”, its personnel (and those from labs across the country) managed to quickly and effectively move into crisis management mode.

The study highlighted the unique role of laboratories as both a ‘first-line’ defence against a new threat as well as a ‘court of last resort’ to improve testing – in terms of diagnosis, surveillance, and response to epidemics.

One priority, according to the Health Canada study, is to standardize testing protocols and share data, to “see the whole picture” of an evolving epidemic. This, it argued, required laboratory information systems (LIS) that are “agile, modular, and rapidly modifiable for special purposes“, a lesson which has relevance for LIS designers even today. On its part, the WHO has mentored an international network of laboratories to identify best practices from the SARS
experience. This will clearly have a bearing on preparations for any new epidemic.

The impact of air travel
The challenge of respiratory system viruses is emphasized by the huge numbers of air travellers. Though little research has been done on the role of airplanes in respiratory epidemics, circumstantial evidence is strong. When SARS struck, 16 of 120 people on a single flight from Hong Kong to Beijing developed the disease, from just one index case. Conversely, the fall in air travel after the September 2011 US terror attacks sharply reduced flu incidence during the year.

Today, of some 8 million air passengers aloft every day, over 1 million cross international borders, just like the victims of the new virus in Britain and Germany. This is an area clearly in need of official attention. Indeed, in March 2003, the WHO recommended screening airline passengers for SARS but its impact was minimal, and questionable. Given the massive number of air travellers, it is clear that any new respiratory epidemic will first grow by leaps and bounds before any meaningful steps can be devised to control it.

The promise of biosensors
Some experts believe that airports should be provided with the means (and the authority) to screen passengers in an impending epidemic, for alternative causes. During the SARS crisis, such eliminative tests – even in a sophisticated setting like the US – were “ordered at the discretion of local clinicians”, diagnosed on “the basis of local interpretations” and many “were never reported to CDC.”

Today, at least one handheld, biosensor-based kit for diagnosing influenza A and B and respiratory syncytial viruses (RSV) – without having to send samples to the lab – is close to market. Deploying such devices at airports ought to be the next step, given the potential threat from the new Middle Eastern coronavirus as well as others that may arise in the future.

This would free laboratories to concentrate on their main task – to identify and confirm genuine, high-risk cases and direct their expertise to what Health Canada billed as their role as a ‘court of last resort’: to quickly master new diagnostic techniques and ensure a quicker response to containing epidemics.

Diagnosis of SARS-associated coronavirus

, 26 August 2020/in Featured Articles /by 3wmedia

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