Point-of-care testing (POCT or POC testing) describes diagnostic tests which are performed at or physically close to a patient. This distinguishes POCT from traditional testing, which involves extracting specimens from a patient and transporting them to a laboratory for analysis. Settings for POC tests range from in-hospital bed sites and primary care offices to patient homes.

Over a half century of use
The POCT era is considered to have begun in 1962, after development of a system that measured blood glucose levels during cardiovascular surgery. The year 1977 saw the US launch of the first POC test for application wholly outside a hospital – the so-called ‘epf’ rapid pregnancy test.

POCT is increasingly used to diagnose and manage a range of diseases, from chronic conditions such as diabetes to acute coronary syndrome. One of the latest additions is a genetic test – CYP2C 19*2 allele for anti-platelet therapy.
Common POC tests includes “blood glucose testing, blood gas and electrolytes analysis, rapid coagulation testing, rapid cardiac markers diagnostics, drugs of abuse screening, urine strips testing, pregnancy testing, fecal occult blood analysis, food pathogens screening, hemoglobin diagnostics, infectious disease testing and cholesterol screening.” Nevertheless, just three tests – urinalysis by dipstick, blood glucose and urine pregnancy – are believed to account for the majority of POCT.

Turnaround time key to POCT appeal
The principal objective of POC testing is to reduce turnaround time (TAT) – a reference to the duration between a test and the obtaining of results which aid in making clinical decisions. In the past, such a process was unavoidable because of the sophistication and size of equipment required for the vast majority of medical diagnostic tests. However, technology developments have since made it possible to perform a growing number of tests outside of the laboratory.

Product miniaturization
Since the late 1980s, one of the key drivers of POCT has been product miniaturization with dedicated onboard integrated circuits. As described in a recent book on biomedical engineering, increasingly sophisticated microdevices have made it feasible to diagnose disease at point-of-care. These include “microfilters, microchannels, microarrays, micropumps, microvalves and microelectronics”, with their mechanical and electrical components “integrated onto chips to analyse and control biological objects at the microscale.” The authors list the key advantages offered by miniaturizing diagnostic tests as compared to centralized laboratory testing:  portability, small size and low power consumption, simpler operation, smaller reagent volumes, faster analysis, parallel analysis, and functional integration of multiple devices.

Healthcare reforms drive POCT
Healthcare reforms have also driven POCT demand.
Spending controls and hospital mergers have led to shorter stays and faster patient turnaround. There have been growing demand for tests in outpatient clinics and patient homes. Test results have been needed quickly, not only for reasons of clinical urgency but also to ease patient waiting lists and reduce backlogs in emergency departments. Accompanying this has been the closure of several large central laboratories, which have further enhanced demand for POCT.

Making a case
The case for POCT has grown with time. In 2004, it was associated with a significant reduction in the time to treatment initiation and a shorter length of stay. More recently, a POCT cardiac marker screening stage at six UK hospitals led to a marked increase in the percentage of successful home discharges.
Such breakthroughs will increase as POCT use grows further, and as the tests become more sophisticated.

Early POC tests were based on the simple transfer of traditional methods from a central laboratory, accompanied by their downscaling to smaller platforms.
Subsequently, unique and innovative assays were designed specifically for POCT (such as the rapid streptococcal antigen test). Wide arrays of POCT-specific analytic methods have also been developed, ranging from simple (such as pH paper for assessing amniotic fluid) to the ultra-sophisticated (for example, thromboelastogram for intraoperative coagulation assessment).

Contemporary POCT systems are usually based on test kits and portable, often handheld, instruments. Many tests are realized as easy-to-use membrane-based trips, often enclosed by a plastic cassette. This requires only a single drop of whole blood, urine or saliva, and they can be performed and interpreted by any general physician within minutes.

Hospital emergency departments
Given its time-sensitive relevance, one of the fastest growing users of POCT have been hospital emergency departments (EDs).
In 2008, a study in  ‘Academic Emergency Medicine’ simulated the impact of reduced turnaround times and established grounds for a “compelling improvement in ED efficiency.” Though its authors concluded that specific outcomes such as the length of stay and throughput in the emergency department warranted further investigation, they categorically recommended POCTs as a means to improve turnaround time.
Over recent years, favourable perspectives on POC tests in the ED have strengthened. At the end of last year, a study in ‘Critical Care’ found POCT increased the number of patients discharged in a timely manner, expedited triage of urgent but non-emergency patients, and decrease delays to treatment initiation. The study quantitatively assessed several conditions such as acute coronary syndrome, venous thromboembolic disease, severe sepsis and stroke, and concluded that POCT, when used effectively, “may alleviate the negative impacts of overcrowding on the safety, effectiveness, and person-centeredness of care in the ED.”
Other POCT users include ICUs as well as endocrinology, cardiology, gastroenterology and hematology.

Primary care remains principal user
The bulk of POC tests are however conducted by primary care physicians. 
In 2014, the ‘British Medical Journal’ published the findings of the first-ever survey of POCT use by primary care physicians in five countries (Australia, Belgium, the Netherlands, the UK and the USA). The study found that blood glucose, urine pregnancy and urine leukocytes or nitrite were the most frequently used POC tests. Overall, more respondents in the UK and the USA reported using POC tests than respondents in the other countries. The widest gap in use of POC test was for fecal occult blood, used by 83% of US doctors against only 2–18% of primary care clinicians in the other countries.
One of the key findings of the ‘British Medical Journal’ study, however, was that there was an unmet need for new POC tests. Included here were tests for D-dimer, troponin, chlamydia, gonorrhea, B-type natriuretic peptide, CRP, glycated hemoglobin, white cell count and hemoglobin, which were desired by more than half of respondents across all the five countries.

Fast growing market
Over the past two-and-a-half decades, the availability and use of POCT has steadily increased. By 2012, nearly 100 companies worldwide were developing, manufacturing or marketing POC tests. One study, cited by the National Institutes of Health in the US, places POCT sales in 2011 at about $15 billion (€13.5 billiion). Of this figure, the US accounted for a share of 55%, Europe for 30% and Asia for 12%. The market is projected to show compound annual growth of 4% to reach $18 billion (€16.2 billion) by 2016.
Further growth in the use of POCT is expected to be driven by increases in accuracy, reliability and convenience. Alongside, one of the biggest catalysts for increased POCT use may consist of quality standards.

The quality challenge
Issues about POCT quality continue to vex experts. Variability in the interpretation of POC test results is a widespread concern, given differences in the education and experience of staff who conduct the tests. In addition, POCT results may also not be comparable across sites (e.g. when patients travel) and differences in specimen types (serum, plasma or whole blood) can impact on results – as compared to those from a traditional central laboratory.
In a laboratory setting, analytical quality is usually assessed by QC (quality control) and QA (quality assurance) procedures. Their aim is “to monitor the stability of the analytical measurement system and to alert the operator to a change in stability”… “that may lead to a medically important error.” While these processes serve a laboratory well, it is unclear whether these processes are relevant, transferable and practical for monitoring quality on POCT devices.

Regulators and POCT in the US and the EU
Future developments are expected to be driven by regulatory bodies.
In the US, CLIA88 (Clinical Laboratory Improvement Amendments of 1988) provided a major impetus for growth in POCT. The rules, published in 1992, expanded the definition of ‘laboratory’ to include any site where a clinical laboratory test occurred (including a patient’s bedside or clinic) and specified quality standards for personnel, patient test management and quality.
One of CLIA88’s biggest contributions to POCT growth was to define tests by complexity (waived, moderate complexity and high complexity control), with minimal quality assurance for the waived category.
CLIA88 has been followed by US federal and state regulations, along with accreditation standards developed by the College of American Pathologists and The Joint Commission. These have established POCT performance guidelines and provided strong incentives to ensure the quality of testing.

In Europe, POCT devices are regulated under the 1998 European Directive 98/79/EC on in vitro diagnostic medical devices, although the term itself is not specifically mentioned. There have since been several amendments, most recently in 2011 (2011/100/EU), as well as standards based on the Directive’s framework.
However, at the European level, specific coverage of POCT is referred by international standard ISO 22870:2006, used in conjunction with ISO 15189 which covers competence and quality in medical laboratories. It is important to note that patient self-testing in a home or community setting is not covered by ISO standards.

The role of ICT
The role of ICT in driving the growth of POCT is also likely to become crucial. In the late 1990s, there were concerns that POCT implementation, especially in the real-time critical care context, was accompanied by little understanding of its information technology requirements.
However, the situation has since changed dramatically, especially as ICT is seen as the only appropriate interface between POC test results and computerized patient records – seen as the means to restructure clinical care pathways.
ICT is also accepted as the best means to standardize care protocols. In 2012, a study found that the impact of point-of-care panel assessment on successful discharge and costs varied markedly from one hospital to another and that outcomes depended on local protocols, staff practices and available facilities. In effect, the study highlighted the importance of optimizing clinical pathways to derive maximum benefit from the reduced turnaround times provided by POCT.

Head and neck cancers (HNC) are a globally prevalent malignancy. Despite the efforts of reducing several known etiological factors such as smoking and drinking to lower the incidence of HNC at the population level, the incidence of oropharyngeal cancers (OPC) is on the rise. OPC is caused by human papillomavirus (HPV) and most prevalent in a younger age group. This review critically examines the epidemiology, biology and laboratory detection of OPC and provides future insights into combating this debilitating disease.

by X. C. Sun, P. Tran and Dr C. Punyadeera

Introduction
Head and neck cancers (HNC) are the sixth most prevalent neoplasm in the world with approximately 650 000 cases diagnosed each year [1–5]. Oral and oropharyngeal squamous cell carcinomas (OSCC & OPSCC) together constitute 90% of malignancies in the head and neck region. Several known traditional etiological factors such as tobacco and alcohol use are recognized in the development of these cancers. More recently, human papillomavirus (HPV) infection is recognized as an additional risk factor for the development of a subset of HNCs, mainly OPSCC [6].

In recent decades, the overall incidence of HNC caused by smoking and alcohol is on the decline. In contrast, HPV^+ve OPSCC is on the rise. In developed countries such as the United States of America, the incidence of HPV+ve OPSCC is escalating, with predictions that more than 50% of patients will be HPV^+ve by 2030 [7]. Interestingly, patients who are HPV^+ve OPSCC are relatively younger than HPV^-ve HNC patients and are therefore less likely to have any history of chronic or excessive alcohol or tobacco use but are more likely to engage in social habits that increase the likelihood of HPV transmission (oral sex). The clear distinction between HPV^+ve OPSCC and HPV^-ve cases provides multiple downstream inputs that can be applied into clinical treatment modalities. Conversely, it provides an exciting opportunity for the development of early diagnostic and screening methods to combat HNC at a population level through prevention strategies.

HPV^+ve OPSCC are both clinically and biologically distinct tumour entities compared with HPV^-ve counterparts. Classically, HPV^+ve OPSCC patients present with a molecular profile that includes retinoblastoma (pRB) pathway inactivation, p53 degradation and p16 upregulation. Clinically, HPV^+ve OPSCC patients often present with smaller primary tumours but more advanced nodal disease, similar rates of metastasis and differing patterns of metastasis [8, 9]. In addition, patients with HPV^+ve tumours have better prognosis with 5-year survival at 75% (c.f. 25% for HPV^-ve patients). There are a number of techniques for the diagnosis and detection of HPV^+ve OPSCC, including histopathology, polymerase chain reaction (PCR) and immunohistochemistry (IHC).

Biology
Upon integration of HPV DNA into the host genome, E6 and E7 viral oncoproteins activate a number of pathways within the host cell. The primary molecular target of E7 is the Rb protein and the E7 viral oncoprotein reprogrammes terminally differentiated epithelial cells to re-enter the cell cycle. E7 disrupts the Rb–E2F complex leading to the release of E2F, subsequently resulting in cyclin A and E activation and entry of the cell into S phase. As a consequence p16 is overexpressed [10, 11]. The E6-associated protein (E6-AP) is a specific ubiquitin-ligase that binds to the viral E6 oncoprotein, resulting in p53 degradation. E6 and E7 have also been shown to interfere with growth inhibitory cytokines [such as tumour necrosis factor-α (TNFα)] and to disrupt the mitochondrial apoptotic pathway by interfering with pro-apoptotic BAK and BAX [10]. E6 and E7 alone are insufficient to cause malignant cell transformation; however, due to their interference with proliferation checkpoints and apoptotic pathways, it is certain that the accumulation and damaged DNA, mitotic defects and integration of foreign DNA substantially increase the risk of malignant progression [10].

Detection methods
A number of diagnostic methods are currently available to evaluate whether a tumour is HPV^+ve. These methods include both indirect as well as the direct methods; i.e. the latter includes the detection of HPV genomic DNA (gDNA). Besides clinical examination, current methods for the diagnosis of HPV status include tissue biopsy staining for p16 (indirect method). Biopsies may fail when tumours are too small to access or when they are located in hidden anatomical sites [10]. Other methods include the detection of HPV gDNA using PCR and in situ hybridization (ISH) as well as the detection of HPV viral transcripts E6 and E7 by PCR.

p16 detection by IHC is widely used in cervical cancer cases for the detection of HPV and it is being studied extensively in the field of HNC [12]. During immortalization of host cells, the E7 protein binds to Rb, resulting in the compensatory overexpression of the tumour suppressor gene p16 in HPV-infected tumour cells. Therefore, IHC detection of p16 is considered as an indirect surrogate marker to determine the presence of HPV [11]. However, there are pitfalls associated with p16 IHC detection. A number of studies have shown suboptimal specificity of IHC [10, 11]. As a consequence of the extreme anatomical and biological heterogeneity in HNC, elevation of p16 by non-viral materials may contribute to a considerable false positive rate [11]. Although it has been reported that p16-positive patients have a better prognosis and increased radiosensitivity, it has been advised that p16 detection by IHC alone cannot accurately identify HPV infection in HNC [12].

Detection of HPV gDNA is a widely used method because of its high sensitivity and cost-effectiveness. Common primers (MY09/MY11 and GP5/GP6) that target the L1 open reading frame are used to detect wide-spectrum HPV genotypes [11]. However, standard PCR primers do not allow detection of specific HPV genotypes [10]. In addition, the target L1 region could also be deleted upon viral integration, which may affect sensitivity of the test [10, 11]. Although, specific E6 and E7 primers have been designed and used to overcome L1 deletion, this method still lacks the ability to distinguish stromal/tumour and episomal/integrated DNA materials and is prone to contamination interference, which undermines the clinical usefulness [11].

The HPV DNA ISH method is unique because of its high specificity and the ability to be evaluated microscopically. The visible hybridization signals that precipitate within the nuclei help distinguish integrated and episomal DNA [11]. It is noteworthy that the presence of HPV DNA detected by ISH significantly correlates with p16 detection by IHC. [10]. However, ISH methods carry lower sensitivity compared to its excellent specificity [11]. The detection of HPV-16 viral transcripts E6 and E7 can highlight whether a patient is suffering from persistent infection – information that is clinically more valuable [12]. However, because of the fragile nature of mRNA, formalin-fixed paraffin-embedded (FFPE) specimens are often not ideal for RNA analysis and frozen fresh specimen are required [12].

The detection of HPV-specific IgG in serum is a useful biomarker to determine previous and current HPV infection status [13]. Serological biomarkers are not site-specific, and can arise due to HPV infections at sites other than the oral cavity, hence potentially affecting the specificity of the assay.

The effect of Gardasil™
From treatment and management of HPV-related diseases, the paradigm of HPV care has shifted to a preventative approach since the breakthrough introduction of the HPV vaccine, Gardasil™ (Merck & Co.). The biologic basis of HPV vaccines relies on the mechanism of neutralizing antibodies generated against virus-like particles (VLP), which consist of the major capsid protein HPV L1 [10]. The quadrivalent HPV 6/11/16/18 vaccine Gardasil™ was licensed by the FDA to prevent cervical, vaginal and vulvar infections in women in 2006 and genital warts in men in 2009, followed by the licensing of bivalent HPV 16/18 vaccine Cervarix™ (GlaxoSmithKline), in women in 2009 [14].

The benefits of HPV vaccination for the oral cavity include not only the biologically-plausible direct effect on oral infections, but also the sequential oral infection reduction following genital infection reduction, due to the sexually transmitted nature of HPV. To date, there are only a few studies examining the effect of Gardasil on oral infection; however, the results showed a promising outlook with high vaccine efficacy (as high as 93% in 4 years time, as recorded by randomized controlled trial in Costa Rica) [15] and reduced viral prevalence (oral prevalence dropped to 1.4% from 9.3% in the 15–23 age group in youth clinics in Sweden) [16].

Future outlook
As previously mentioned, HPV-related OPSCC involve a new segment of the population, which is distinctively different to the traditional HNC patient cohort caused by excessive smoking and drinking. This requires clinicians to conduct thorough cancer screening of at-risk groups. Such screening programmes should pay particular attention to cervical lymph nodes as some subtypes of HNC, especially OPSCC, involve hard-to-examine areas for clinical visual examination.

As a result of the sexually transmitted nature of HPV, some studies have advocated routine sexual behaviour education in clinical practice. However, this practice carries inherent controversies of sexual harassment and confidentiality [17]. Public awareness campaigns have been argued to be a more efficient preventative means in altering patients’ behaviour. Studies have shown that many oral health practitioners have limited knowledge with regards to HPV-related HNC and HPV vaccinations [17]. Professional bodies and health authorities are required to address this knowledge gap by establishing new clinical guidelines and using continuing educational methods, in order to effectively control the rising trend of HPV-related HNC.

Conclusion
All current diagnostic methods require excision of tumour tissue and this can be challenging when they are located in hidden sites. Efforts have been made globally to develop a less invasive, more cost-effective and clinically-relevant test. Serology tests that detect HPV-specific IgG have been shown to indicate viral presence and are linked with prognostication; however, this method inherently lacks site specificity [10]. Oral specimens, more specifically oral rinse, have shown promise in this field. Oral rinse samples not only are non-invasive and cost-effective, the proximity of collection to the area of interest ensures the localized sampling field. It is also important to note that shedding of normal cells into the oral cavity/oral pharynx may interfere with and/or decrease the HPV detection level [10]. OraRisk^® HPV test, uses oral rinses for HPV detection [10]. Translational collaborations between scientists and clinicians have resulted in an assortment of tumour markers and diagnostic techniques for OPSCC. However, these need to be tested in clinical trials to determine the cost-effectiveness.

Acknowledgments
This work is supported by Garnett Passé & Rodney Williams Memorial Foundation and the Queensland Centre for Head and Neck Cancer funded by Atlantic Philanthropies, the Queensland Government and the Princess Alexandra Hospital.

References
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2. Nagadia R, Pandit P, Coman WB, Cooper-White J, Punyadeera C. miRNAs in head and neck cancer revisited. Cell Oncol (Dordr). 2013; 36(1): 1–7.
3. Ovchinnikov DA, Cooper MA, Pandit P, Coman WB, Cooper-White JJ, Keith P, Wolvetang EJ, Slowey PD, Punyadeera C. Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients. Transl Oncol. 2012; 5(5): 321–326.
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The authors
Xiaohang Charles Sun¹, Peter Tran¹ and Chamindie Punyadeera*² MSc, PhD
¹School of Dentistry, The University of Queensland, Brisbane, Australia
²The Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.

*Corresponding author
E-mail: chamindie.punyadeera@qut.edu.au

Tumours found in the ovaries can be either from primary ovarian tumour processes or metastases (secondary tumours) foremost from colorectal cancer (CRC), appendiceal tumours or stomach cancer. Correctly distinguishing between these tumour subsets using hematoxylin-eosin staining in combination with immunohistochemistry can be problematic [1–3], but is crucial for correct treatment choice. Mutation profiles, generated in a fast and cost-effective way by (targeted) Next Generation Sequencing (NGS), can assist in correctly diagnosing ovarian tumours.

by Stijn Crobach and Prof. Hans Morreau

Background
The ovaries are a preferential location for metastases from, among others, colon, stomach, appendiceal, breast and endometrium carcinomas. The percentage of secondary ovarian tumours (metastases), varies in several reports ranging from 8–30% [4, 5]. Several reasons can be given to explain why the range of percentages is so broad. First, studies are different by design. Some studies are based on autopsy findings, others on prophylactic oophorectomies. Second, differences in incidence of primary tumours can cause a variance in patterns of metastases. For example, stomach cancer has a higher incidence in Japan than in many other countries; therefore, metastases of stomach cancer to the ovaries are expected to be more common in Japan. In general, however, the gastrointestinal tract (GIT) seems to be the main source of ovarian metastases [5].

Macroscopic and histologic approaches
A gross distinction between primary and secondary ovarian tumours can be made taking tumour size and unilaterality versus bilaterality into account [6]. Following the decision tree depicted in Figure 1, it is possible to estimate whether an ovarian tumour is a primary tumour or a metastasis. A unilateral ovarian tumour with a diameter larger than 10 cm is probably a primary tumour. All bilateral and unilateral tumours smaller than 10 cm are much more likely to be metastases.

The histologic characteristics of metastatic GIT ovarian tumours can resemble primary endometrioid and mucinous ovarian tumours, but not serous papillary or clear cell tumours. Thus, based on histology a subset of primary ovarian tumours does not cause diagnostic doubt about the origin of the malignancy. Furthermore, other histologic findings can assist in defining the malignancy. For example, on the one hand, surface involvement by malignant epithelial cells is much more often seen in metastases than in primary ovarian tumours. On the other hand, however, an expansile growth pattern is more often seen in primary ovarian tumours. So, with the help of histopathological findings the characterization of a primary origin or a metastatic process becomes more achievable.

Immunohistochemical approaches
The logical next step in differentiating primary ovarian tumours from metastases is with the use of immunohistochemistry. For example, primary ovarian tumours are classically positive for keratin 7 and negative for keratin 20, whereas colorectal tumours show the opposed staining pattern (keratin 7 negative, keratin 20 positive) [7]. Other markers can also be used, not only to rule out an ovarian origin of the tumour but also to get an idea about the location of the primary tumour. Positivity of intestinal markers [such as carcinoembryonic antigen (CEA) and caudal type homeobox 2 (CDX-2)] can be an argument for an intestinal origin of the tumour cells [8].

Furthermore, when a colon carcinoma is already diagnosed before the ovarian tumour is discovered, the staining profile of the metastasis can be compared with the primary tumour. However, in up to 38% of cases the detection of ovarian metastases precedes the detection of the primary tumours. Also, secondary primary ovarian tumours can occur in patients that anamnestically suffered from another malignancy, complicating the diagnostic procedures. In practice, immunohistochemistry is frequently not fully discriminating. As mentioned, primary ovarian tumours tend to have a Ker7+/Ker20− immunoprofile and colonic metastases a Ker7−/Ker20+ immunoprofile. Nevertheless, keratin 7 positivity can be seen in proximal located GIT tumours, and keratin 20 positivity can also be seen in primary ovarian malignancies. In Figure 2, a guided immunohistochemical decision scheme is shown for complex cases.

Molecular diagnostic approaches
With the combined use of clinical information, histologic features and immunohistochemical staining patterns, differentiating primary tumours from metastases is possible in a substantial subset of cases. With a history of a colorectal tumour and the presentation of a large ovarian mass a few years later showing a similar immunoprofile, it is not difficult to decide that this tumour is a metastasis. Nevertheless, there are cases that are not as clear-cut. In those cases tumour size, unilaterality versus bilaterality and the histologic findings are not discriminating enough to solve the challenge. New approaches using massive parallel DNA sequencing (Next Generation Sequencing; NGS) have emerged in recent years.

Cancer driver genes (oncogenes and tumour suppressor genes) can be screened for DNA mutations in different tumour types. In the Catalogue Of Somatic Mutations In Cancer (COSMIC; http://cancer.sanger.ac.uk/cosmic), literature on these profiles has been compiled [9]. It was hoped that comparing mutational profiles of primary ovarian tumours versus metastases from different organs would reveal specific mutation patterns and/or mutation types in different tumour types.

NGS enables the screening of a large number of genes in a fast and cost-effective way. Previously, Sanger DNA sequencing was used to detect mutations in clinically relevant genes. However, screening complete genes and multiple genes in this way is a time-consuming process. Now, with the introduction of the disruptive NGS technology, it is possible to sequence multiple genes at the same time. NGS will become a standard technique in diagnostics for identifying gene mutations, chromosomal rearrangements and RNA expression/mRNA patterns [10]. One would expect that large scale screening of molecular alterations will results in very specific profiles per tumour type. Each tumour type could be defined by subsets of mutated genes. However, recent studies show that the mutation profiles do not differ so much between tumour types [11]. A few well-known so-called cancer driver genes seem to be important in many malignancies. Other (passenger) mutations, which are also needed in tumorigenesis, seem to be interchangeable. Apparently, there is wide overlap in mutation profiles. Looking at mutations described in the COSMIC database or The Cancer Genome Atlas (TCGA) at the current time, similar mutations can be seen in both primary ovarian tumours and metastases, although with different frequencies. The latter would suggest that the applicability of such tests is limited. However, a more select approach shows that certain genes can be discriminatory.

For example, CTNNB1 mutations are found in primary endometrioid carcinoma of the ovary. CTNNB1 mutations are also found in colon tumours, but only in mismatch repair deficient colon tumours, that do not tend to metastasize to the ovary. This reasoning could also be followed for APC, which is frequently mutated in colon carcinomas but not typically in mucinous and endometrioid primary ovarian carcinomas. However, genes such as these, which show such a ‘black-and-white’ phenomenon, are sparse. Therefore, mutation profiles that are used to guide clinical decision taking will probably be based on combining information from multiple genes. Most of these genes will not provide significant differences on their own, but a combination of odds-ratios will make one diagnosis more probable than the other.

Along with solutions at a mutational level, characterizing the transcriptome, methylation patterns and copy numbers of a tumour could also provide useful information. This field of ‘omics’ has developed rapidly in recent years. In diagnostically challenging cases from unknown primary tumours (UPT) or alternatively named carcinoma of unknown primary (CUP), expression array based assays were developed in order to identify the primary tumours. Genomics will also probably become effective in determining the origin of the tumour. Furthermore, in depth comparison of molecular features of synchronously presenting tumours at different sites might reveal whether the tumours have arisen independently or are clonally related. The readout of these tests can be seen in the context of increased odds-ratios. The use of such tests is still in a premature phase, and not used routinely in clinical practice.

Summary
In conclusion, a combination of the various molecular features will hopefully reveal specific molecular profiles that can be used to correctly identify the origin of malignancies in problematic cases. These techniques are applicable on ovarian tumours, to determine whether tumours are primary ovarian in origin or metastases to the ovaries [12].

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The authors
Stijn Crobach BSc; Hans Morreau MD, PhD
Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands

*Corresponding author
E-mail: j.morreau@lumc.nl

Cervical cancer is a major burden worldwide with significant mortality, especially in developing countries. Human papillomavirus (HPV) analysis is gaining ground as the primary screening modality for the early diagnosis and prevention of cervical carcinoma. Direct pathogen detection allows an infection to be identified before cell changes have even taken place. Thus, interventional measures can be applied before the cancer even develops, helping to reduce the overall incidence and mortality rates. The EUROArray HPV molecular diagnostic microarray provides highly sensitive detection and typing of all known high- and low-risk anogenital HPV in one reaction. With fully automated data analysis it is particularly well suited to the high-throughput requirements of routine screening.

Human papillomaviruses
Human papillomaviruses are uncoated double-stranded DNA viruses which infect epithelial cells of the skin and mucous membranes. They are transmitted by sexual contact. Infection is assumed to occur via tiny lesions in the basal cells of the epithelium. Thus, the most frequent place of infection is the transformation zone of the cervix, where dividing basal cells lie near to the surface. The size of the cells, their histology and the duration of the lesion can influence the number of cells infected. The course and outcome of the infection depends on the HPV type, the anatomy of the infection site and the differentiation status of the host cells.
Infections with HPV are always local and are not accompanied by viremia. Following infection, the viral DNA is replicated in the host cell nuclei. Viral proteins produced in the infected cells can trigger uncontrolled tumour-like growth of the cells. This is, depending on the infecting HPV subtype, mostly benign, leading to warts at the site of infection. However, some HPV types can induce malignant changes, particularly cervical cancer. A significant proportion of vaginal, penile, anal and head and neck carcinomas are also assumed to be caused by HPV infection.
HPV are the most frequent sexually transmitted viruses. The worldwide prevalence of HPV infection is estimated to be 2 to 44% in women and 4 to 45% in men, with regional variations depending on culture and the corresponding sexual activity. Viral transmission from mother to newborn during birth can also occur, even with subclinical infections. HPV infection does not lead to life-long immunity and reinfection with the same virus is possible.

HPV subtypes
Around 130 types of HPV have so far been described of which 30 infect exclusively the skin and mucous membranes in the anogenital area. HPV are divided into two groups according to their oncogenic potential. High-risk HPV cause cervical carcinoma. Low-risk HPV alone do not induce tumours, but cause non-malignant tissue changes. Concurrent infections with multiple HPV subtypes are common and known to increase the risk of malignant cell transformations.
Of the high-risk anogenital types, HPV 16 and HPV 18 are responsible for around 70% of cervical carcinomas. HPV 16 is found in 50 to 60% of cases and HPV 18 in 10 to 20%. Other types classified as high-risk by the WHO are 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66. Types 26, 53, 68, 73 and 82 have also been detected in cervical carcinoma and should be considered as high-risk types.
Of the low-risk types, HPV 6 and 11 are the main causative agents of genital warts (condylomata acuminata, fig warts). Further low-risk types are 40, 42, 43, 44, 54, 61, 70, 72, 81 and 89.

Cervical carcinoma
HPV infection is a prerequisite for the development of cervical carcinoma. However, HPV infection does not necessarily lead to cancer. Most infected women eliminate the virus within two years. If the virus remains detectable for longer than 18 months, the infection is considered to be persistent. A persistent infection, in particular with a high-risk HPV subtype, increases the risk of developing cervical carcinoma by around 300-fold.
HPV infections are often asymptomatic and tend to remain unnoticed. The initial stages of cervical carcinoma also proceed without pain, and the only symptom may be light bleeding. With increased tumour size, the cancer manifests with a blood-tinged, sweet smelling discharge.

Around 528,000 new cases of cervical carcinoma occur annually worldwide, making it the fourth most frequent cancer in women after breast, colorectal and lung cancers. It is also the fourth most common cause of cancer mortality, causing approximately 266,000 deaths in 2012 (International Agency for Research on Cancer).
In the early stages, treatment involves removal of the altered tissue by conisation. In later stages of the disease, the uterus and surrounding tissue must be removed.

Role of HPV detection and typing
Along with the current diagnostic gold standard, the Papanicolaou (Pap) test, HPV direct detection plays an important role in the early diagnosis of cervical carcinoma. In contrast to the Pap test, which is used to investigate cervical cells for pathological changes, PCR-based methods detect viral nucleic acids directly, and can thus identify an HPV infection at a very early stage before morphological cell changes have even occurred. Moreover, while the Pap test is based on subjective evaluation, HPV detection represents an objective as well as extremely sensitive test method.
In HPV screening it is crucial to differentiate between high- and low-risk types and also to discriminate between different high-risk viruses. A positive result for high-risk HPV indicates an increased risk for cervical carcinoma, which can then be minimized by more frequent follow-up examinations to detect morphological cell changes at an early stage. A positive result for low-risk HPV can help to clarify uncomfortable and embarrassing symptoms for patients. Since low-risk HPV can also cause mild dysplasia, HPV subtyping is also useful for excluding a high-risk HPV infection and a corresponding risk of cervical cancer in these cases. Women who are HPV negative can forgo Pap smears for a longer time interval, based on the recommendations of the respective professional societies.
The PCR detection strategy is a critical aspect of direct HPV analysis. Tests with primer or probe systems based on conserved genes like L1 may yield false negative results in some cases due to loss of these genes during integration of the viral DNA into the host DNA. The highest possible detection sensitivity is achieved using the viral oncogenes E6/E7. Detection of variable sequences in these genes enables differentiation of the different HPV subtypes.

Microarray for complete HPV typing
A standardized microarray based on PCR detection of E6/E7 has been developed for complete HPV typing in routine diagnosis. Using an extensive panel of specific primers and probes, the EUROArray HPV detects all thirty genitally relevant HPV subtypes in one test, distinguishing eighteen high-risk subtypes that may trigger cancer (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and twelve low-risk subtypes that cause benign warts (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89). Multiple infections are reliably identified, and primary and persistent infections can be differentiated.

Simple procedure with automated evaluation
The EUROArray procedure (Figure 1) is extremely easy to perform and does not require any in-depth molecular biology knowledge. DNA prepared from patient cervical smear samples is first amplified by a single multiplex polymerase chain reaction (PCR). The fluorescently-labelled PCR products are then incubated with biochip microarray slides (Figure 2) containing immobilized complementary DNA probes. Specific binding (hybridization) of the PCR products to their corresponding microarray spots is detected using a specialized microarray scanner.
In contrast to manually evaluated tests, the results are evaluated (Figure 3) and interpreted fully automatically by user-friendly software (EUROArrayScan). A detailed result report (Figure 4) is produced for each patient and all data is documented and archived. Meticulously designed primers and probes, ready-to-use PCR components and integrated controls all contribute to the reliability of the analysis. The entire EUROArray process from sample arrival to report release is IVD validated and CE registered, supporting quality management in diagnostic laboratories.

Conclusion
As evidence mounts about the efficacy of HPV testing for primary cervical cancer screening, multiplex microarrays are poised to become a major tool in prevention programmes worldwide. The EUROArray HPV, in particular, is ideally positioned for high-throughput HPV screening, providing fast and sensitive detection of all high- and low-risk anogenital HPV types combined with fully automated data analysis.

The author
Jacqueline Gosink, PhD
EUROIMMUN AG
Seekamp 31
23560 Luebeck
Germany
E-mail: j.gosink@euroimmun.de