Page 257 – Clinical Laboratory int.

Diagnostically challenging cases: distinguishing primary from secondary ovarian tumours

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

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].

References
1. Prat J. Ovarian carcinomas, including secondary tumors: diagnostically challenging areas. Mod Pathol. 2005; 18(Suppl 2): S99–111.
2. Young RH. From Krukenberg to today: the ever present problems posed by metastatic tumors in the ovary. Part II. Adv Anat Pathol. 2007; 14: 149–177.
3. Leen SL, Singh N. Pathology of primary and metastatic mucinous ovarian neoplasms. J Clin Pathol. 2012; 65: 591–595.
4. Moore RG, Chung M, Granai CO, Gajewski W, Steinhoff MM. Incidence of metastasis to the ovaries from nongenital tract primary tumors. Gynecol Oncol. 2004; 93: 87–91.
5. de Waal YR, Thomas CM, Oei AL, Sweep FC, Massuger LF. Secondary ovarian malignancies: frequency, origin, and characteristics. Int J Gynecol Cancer 2009; 19: 1160–1165.
6. Yemelyanova AV, Vang R, Judson K, Wu LS, Ronnett BM. Distinction of primary and metastatic mucinous tumors involving the ovary: analysis of size and laterality data by primary site with reevaluation of an algorithm for tumor classification. Am J Surg Pathol. 2008; 32: 128–138.
7. Ji H, Isacson C, Seidman JD, Kurman RJ, Ronnett BM. Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 2002; 21: 391–400.
8. Groisman GM, Meir A, Sabo E. The value of Cdx2 immunostaining in differentiating primary ovarian carcinomas from colonic carcinomas metastatic to the ovaries. Int J Gynecol Pathol. 2004; 23: 52–57.
9. Bamford S, Dawson E, Forbes S, Clements J, Pettett R, Dogan A, Flanagan A, Teague J, Futreal PA, Stratton MR, Wooster R. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004; 91: 355–358.
10. Natrajan R, Reis-Filho JS. Next-generation sequencing applied to molecular diagnostics. Expert Rev Mol Diagn. 2011; 11: 425–444.
11. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science 2013; 339: 1546–1558.
12. Crobach S, Ruano D, van Eijk R, Fleuren GJ, Minderhout I, Snowdowne R, Tops C, van Wezel T, Morreau H. Target-enriched next-generation sequencing reveals differences between primary and secondary ovarian tumors in formalin-fixed, paraffin-embedded tissue. J Mol Diagn 2015; 17: 193–200.

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

Rapid DNA mutational profiling for Familial Hypercholesterolemia

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

Since the mapping of the human genome was completed over a decade ago, our knowledge of genetic drivers of disease continues to evolve at an ever-quickening pace. Consequently, genetic testing and pharmacogenomics have become common within the healthcare system and have generated the knowledge that has empowered us to both understand and influence our lifelong health through pre-emptive intervention.
Progress in medical genomics and its impact on healthcare cannot be understated; from genotyping patients to predict drug response, to stratifying patients according to the risk of a disease, molecular testing is having a very positive impact on many patient treatment pathways.
Undoubtedly, we are now more aware and in control of our health than ever before. It is no surprise then that the molecular diagnostic market has become the fastest growing segment of the IVD industry with assays serving the gamut of disease areas and breaking new boundaries in personalized healthcare. Despite the public appetite and availability of powerful molecular diagnostic assays that can unequivocally diagnose genetic disorders, their use has not gained universal acceptance. Many traditional diagnostic tests continue to under-diagnose, or diagnostic testing is not attempted, leading to missed opportunities for early and appropriate therapy intervention of potentially life-threatening diseases. One prime example where a molecular diagnostic approach can improve health is mutation profiling for Familial Hypercholesterolemia (FH).

Familial Hypercholesterolemia
Familial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism. It is a common autosomal dominant, or inherited, disease which affects the plasma clearance of LDL-cholesterol (LDL-C), resulting in premature onset of cardiovascular disease (CVD) and a higher mortality risk.

Early diagnosis of FH is very advantageous as by the time heterozygous FH sufferers enter early adulthood they will have accumulated years of continuous build-up of fatty or lipid masses in arterial walls and are at one hundred-fold greater risk of a heart attack than their non-FH peers. If left untreated, men and women with heterozygous FH with total cholesterol levels of 8–15 mmol/L typically develop coronary heart disease (CHD) before age 55 and 60, while homozygotes with total cholesterol levels of 12–30 mmol/L typically develop CHD very early in life and if untreated die before age 20.
Clinical diagnosis of FH relies on five criteria: family history, clinical history of premature CHD, physical examination for xanthomas and corneal arcus, very high LDL cholesterol on repeated measurements, and / or a causative mutation detected by molecular genetics. To formally quantify this, a number of sets of statistically and genetically validated criteria have been devised; namely the Dutch Lipid Clinic Network Criteria and the Simon Broome Criteria. These classify suspected cases into definite, possible and probable diagnoses of FH. In the absence of definitive diagnosis through detection of a causative mutation using molecular genetics, clinical diagnosis could miss a considerable proportion of FH patients, particularly those with a mild phenotype and the pediatric population in whom the phenotype has not appeared yet.

The UK, US and international guidelines now recommend that probable or possible FH patients undergo a DNA test to confirm the diagnosis of FH. Recommendations also advocate that once an activating mutation has been found in one family member (the index case), cascade screening of that mutation in first degree relatives of the index case should proceed. Cascade screening using a molecular assay can thus identify index family members who may otherwise be asymptomatic.

The good news is that if detected early, FH can be treated successfully with lipid lowering therapy and lifestyle changes. In comparison to other hyperlipidemias, FH therapy tends to be more aggressive, so definitive diagnosis has additional benefits in determining care packages. Statin drug therapy significantly reduces the morbidity and mortality from premature coronary disease in FH, particularly if affected individuals are identified and treated in childhood or early adulthood. Accurate and early diagnosis of specific mutations can result in a better overall outcome for patients through the prescribing of tailored treatments to reduce morbidity and mortality from premature cardiovascular disease. Different mutations can dictate different directions of management, such as the poorer response to lipid-lowering therapy with certain LDLR mutations. The identity of the gene involved can potentially aid the clinician to decide on how aggressive the treatment strategy will be.

Mutation diagnosis also provides clarity, and can help with an individual’s understanding and acceptance of their condition. Also a greater compliance with cholesterol lowering medication is observed with those who have been genetically diagnosed with FH.

Mutational profiling of FH
Currently, ~1200 mutations have been documented worldwide in LDLR; these affect all functional domains of the LDL receptor protein and include single-nucleotide mutations, copy number variations, and splicing mutations throughout the LDLR gene. A single mutation, Arg3500Gln, is the only common FH-related mutation in APOB, while c.1120G>T mutation is predominately detected in PCSK9. Heterozygous LDLR, APOB, and PCSK9 mutations are found in >90%, ~5%, and ~1%, respectively, of heterozygous FH subjects with a causative mutation. Prevalence varies geographically.

The abundance of different FH mutations can make genetic testing labour-intensive and costly, with many laboratories defaulting to performing expensive and lengthy Next Generation Sequencing (NGS) tests in an effort to ensure a comprehensive mutational screen. However, as our understanding of the genetic drivers of FH, as well as common population-specific mutations, increases, novel assays and techniques are being developed to meet the needs facing clinical genetics laboratories, including cost, throughput and time to result.

Randox Laboratories have developed The Familial Hypercholesterolaemia (FH) Arrays I and II that are rapid, simple and accurate diagnostic tests which enable simultaneous detection of 40 FH-causing mutations (20 mutations per array) within the LDLR, ApoB and PCSK9 genes. The assay is based on multiplex PCR followed by biochip array hybridization. Using mutation rate data from a study of 500 UK and Ireland families with genetically-confirmed FH, the Randox FH Arrays are capable of detecting approximately 71% of activating mutations in this population. The mutations will also be detected in other geographical regions.

The assay can be completed from extracted DNA to an easy-to-interpret result report in 3 hours, with the requirement for only 20ng of genomic DNA per array. The system can be used to detect small base changes, insertions and deletions within the same multiplex PCR, allowing addition of new FH mutational targets if required. The arrays are designed for use on the Evidence Investigator (Randox Laboratories Limited, Crumlin, UK). This instrument has been developed alongside Randox’s proprietary Biochip Array Technology (BAT), a multiplex testing platform founded on ELISA principles that currently has application within clinical immunoassays, drug development R&D, clinical research, forensic and clinical toxicology, veterinary drug residues and molecular diagnostics.

FH Array I and II workflow
Randox’s multiplex assays, such as FH Array I and II, have been specifically designed to detect the most common mutations, provide a cost-effective and clinically relevant alternative to NGS testing. Targeting the most commonly detected mutations in a given population enables diagnosis within hours rather than months. Where a mutation is identified in an index patient, cascade testing of family members only requires the mutation in question to be targeted; therefore negating the use of broad profiling approaches such as NGS in this setting.

Conclusion
FH is a common yet underdiagnosed condition that poses a significant risk to public health worldwide. In 2008, cardiovascular diseases were the leading cause of non-communicable deaths worldwide, with an estimated mortality rate of 17 million people. Raised cholesterol was attributed to 2.6 million deaths. Understanding a person’s genetic predisposition to cardiovascular disease through genetic testing will allow patients to receive appropriate therapeutic and interventional treatment to reduce morbidity and mortality associated with cardiovascular disease.
Pioneering multiplex diagnostic assays, tailored to incorporate the relevant FH-causing mutations, provide a promising future for both genetic laboratories, where a rapid, cost-effective approach to determine mutational status in cases of suspected FH is enabled, and the patient, whose treatment and care pathway is managed effectively.

The author
Martin Crockard, PhD
Randox Laboratories Ltd.
55 Diamond Road, Crumlin, Co. Antrim
U.K.

Complete HPV detection and typing in cervical cancer prevention

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

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

Introducing the DxN VERIS molecular diagnostics system from Beckman Coulter, Inc.

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

Delegates at ECCMID 2015, held in Copenhagen from 25th to 28th April 2015, attended a symposium introducing Beckman Coulter’s new DxN VERIS Molecular Diagnostics System.* DxN VERIS provides a fully automated sample to result platform with true single sample random access, integrating sample introduction, nucleic acid extraction, reaction setup, real-time PCR amplification, detection and results interpretation into a single system that is set to revolutionize laboratory workflows.
Speakers from four of the 10 DxN VERIS beta study sites shared their experiences and results from comparative evaluations of this new system.

Meeting molecular diagnostic needs
By way of introduction, Hervé Fleury described the molecular diagnostic needs in Europe, where laboratories are becoming fewer and larger, both in the public and private sectors. The number of molecular scientists available for routine tasks is also decreasing, he said, and there is a need for the level of automation, from preanalytics to analytical, that the DxN VERIS will bring.
He then described the DxN VERIS technology, which is able to provide results in approximately 75 minutes for DNA targets and in around 110 minutes for RNA targets, performing in excess of 150 and 100 results in 8 hours for DNA and RNA targets respectively. CE marked DxN VERIS assays for human cytomegalovirus (CMV) and hepatitis B virus (HBV) are already available, in addition to assays for hepatitis C virus (HCV) and human immunodeficiency virus (HIV). DxN VERIS products in the pipeline include assays for Chlamydia trachomatis and Neisseria gonorrhea (CT/NG), MRSA (screen), Clostridium difficile, respiratory virus multiplex and human papilloma virus (HPV).

Excellent performance criteria
All four speakers at the ECCMID Symposium described excellent analytical and clinical performance criteria for the VERIS assays evaluated.
Jacques Izopet reported very good analytical performance results for all four VERIS assays that are currently available (table 1). In addition, these assays demonstrated good agreement with an alternative method (Cobas^® Ampliprep/ Cobas^® TaqMan™). Significantly, in a patient monitoring setting, the VERIS CMV assay demonstrated overlapping patterns compared to this alternative for plasma samples and compared to a whole blood reference method (figure 1).

Rafael Delgado then went on to present the results from his evaluation of the VERIS CMV and HBV assays. At his laboratory, both assays were extremely sensitive and specific, exhibited a high linearity and repeatability, and showed good correlation with an alternative method (Cobas Ampliprep/ Cobas TaqMan*) (figure 2). In addition, the system demonstrated no carry over when known high positive samples were interspersed among known negative samples.

Rafael Delgado concluded that the overall performance and easy to use design of the DxN VERIS platform facilitated the introduction of this technology in the laboratory and that the DxN VERIS CMV and HBV viral load assays are helpful new solutions for patient management.

In his evaluation of the DxN VERIS HBV assay, Duncan Whittaker observed excellent precision (within and between run), with a standard deviation of ≤ 0.12, and a limit of detection of 7.99 IU/mL, which is less than the manufacturer’s claim of 10 IU/mL. He described the existing method at the Sheffield laboratory as very manual (with separate extraction and amplification systems) which was adequate when they received just 10-12 HBV samples every two weeks but which struggles to cope now that they are receiving up to 80 samples per week.

Duncan Whittaker reported that the quantitative results from the VERIS assay were similar to their exisitng method (Qiagen) (figure 3) with improved precision at lower levels (table 2). He was also able to demonstrate excellent performance and reproducibility across HBV genotypes. In conclusion, he stated that the DxN VERIS Molecular Diagnostics System offers significant improvements in laboratory workflow and time.

Finally, Giovanni Gesu also shared his results from the evaluation of the DxN VERIS HBV assay. At the Niguarda ca’ Granda Hospital in Milan, DxN VERIS HBV demonstrated excellent within and between run precision (SD ≤ 0.156), linearity (1.63 – 8.82 log IU/mL) and sensitivity (limit of detection 6.82 IU/mL), and performed well compared to an alternative HBV real time method (Abbott m2000).

In order to demonstrate the potential workflow and throughput efficiences that the DxN VERIS platform could achieve, Giovanni Gesu applied the throughput capabilities of this new system to a typical day in his laboratory, in which 33 CMV, 17 HBV, 26 HCV and 21 HIV samples were received. With samples arriving at two hour intervals throughout the day between 10am and 4pm, the true single sample random access capability of the DxN VERIS platform combined with assay runtimes of around 70 minutes for DNA tests and around 110 minutes for RNA tests, would mean that samples would not need to be batched and that all results could be reported by 6pm on the same day (figure 4).

Conclusions
In conclusion, each of the speakers at the ECCMID Symposium agreed that the analytical performance of the DxN VERIS assays evaluated was excellent, and they compared well to other molecular diagnostic assays currently available. In addition, the sample-to- result automation and true single sample random access of the DxN VERIS Molecular Diagnostics System offer workflow improvements and laboratory efficiencies.

For further information about the DxN VERIS Molecular Diagnostics System and the DxN VERIS assays currently available, please contact: Tiffany Page, Senior Pan European Marketing Manager Molecular Diagnostics, Email: info@beckmanmolecular.com

^{*Not for sale or distribution in the U.S.; not available in all markets.
** TaqMan® is a registered trademark of Roche Molecular Systems, Inc. Used under permission and license.}

_{Beckman Coulter, the stylized logo, DxN and VERIS are trademarks of Beckman Coulter, Inc. Beckman Coulter and the stylized logo are registered in the USPTO.}