Call for more physician awareness as prevalence of celiac disease leaps

, /in /by

Although known to the ancient Greeks, celiac disease was definitively demonstrated only in the late 1950s after development of the endoscope. In 1969, the new European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) codified the first diagnostic criteria for the disease.

Fast-growing challenge
The prevalence of celiac disease has exploded in recent years, above all in North America, Europe and Australia. The Association of European Coeliac Societies (AOECS) notes that those with the condition, but unaware of it, are compelled to live a life “filled with chronic pain and discomfort.”
If unchecked, inflammation caused by celiac disease seriously damages the lining of the small intestine, which produces enzymes for the digestion and absorption of food and essential nutrients. The malabsorption leads to diarrhea, weight loss and fatigue. Celiac disease eventually impacts on the bones, liver, brain and nervous system, in some cases seriously. AOECS includes “infertility, osteoporosis and small bowel cancer” in its list of long-term risk factors.

The role of prolamins
The principal protein responsible for celiac disease consists of prolamins, which are resistant to proteases and peptidases of the gut. They stimulate intestinal membrane cells in susceptible people to become permeable (or ‘leak’), by allowing larger peptides to bypass the sealant between cells, and thereby enter circulation.
The best understood prolamin is gliadin (in wheat). Other prolamins believed to play a role include hordein in barley, scelain in rye and zein in corn. The role of avenin in oats as a causative factor for celiac disease remains unclear. In Europe, however, the EU Commission requires that gluten-free oats are specially produced or processed to avoid contamination by wheat, rye and barley.

Prevalence growth ‘mystifying’, increase uneven
Estimates on the prevalence of celiac disease have leaped dramatically in recent years. It was previously believed that it affected about 1 in 1,500 people. However, new studies suggest a 15-fold higher rate, about 1 in 100 (1%) in both Europe, and the US. As the ‘New York Times’ observed last year, the spike in US prevalence of celiac disease is  “mystifying.”
In Europe, prevalence varies widely. In the 30–64 year age group, the rate in Finland is eight times higher than in Germany (2.4% versus 0.3%). In addition, Finland has also shown a doubling in prevalence over 20 years – a fact which “cannot be explained by better detection rates.”
There is a higher prevalence of celiac disease in people with other conditions, such as Type 1 diabetes, Down Syndrome as well as both hypo- and hyper-thyroidism.

Genetics and environment
The challenges of celiac disease are manifold.
Its etiology is unclear. The disease is caused by “a combination of immunological responses to an environmental factor (gluten) and genetic factors.” The latter consist of the cellular receptors for two versions of HLA (human leukocyte antigen), DQ2 or DQ8. The absence of either results in “a negative predictive value … close to 100%.” This explains why people of Chinese, Japanese and African descent – who lack the HLA allele – are rarely diagnosed with celiac disease, unlike Caucasians.
Nevertheless, in a confirmation of the role of environmental triggers, the presence of HLA-DQ2 or -DQ8 is “necessary but not sufficient to predispose people to celiac disease.” In addition, the genes may be transmitted to some family members, but not others. First- and second-degree relatives of people with celiac disease show prevalence rates of about four-and-a-half and two-and-a-half times that of the general population.

The no-man’s land of gluten sensitivity
The symptoms of celiac disease are also varied, since it affects people differently. One of the best illustrations of the scale of the diagnostic challenge is the University of Chicago’s Celiac Disease Center, which lists as many as 300 symptoms that may accompany the disease.
Celiac disease is also routinely confused with irritable bowel syndrome. Indeed, a paper in 2009 published in the ‘American Journal of Gastroenterology’ remarks about the ‘no-man’s land of gluten sensitivity’ lying between celiac disease and irritable bowel syndrome.
As the US-based Celiac Disease Foundation observes, such factors make the disease difficult to diagnose. In addition, some patients have no symptoms at all.
Europe’s AOECS notes that only about 12%-15% of celiac disease patients obtain a diagnosis. In many cases, moreover, the time between experiencing first symptoms and diagnosis is over 10 years.
Confounding matters further is a lack of physician awareness about the onset of symptoms. Surveys in the US have shown that only 35% of primary care physicians had ever diagnosed celiac disease.
Peaks in diagnosis occur in childhood and between the fifth and seventh decades of life. The female-to-male ratio in celiac disease is about 2:1.

Strict diet only answer
There is no cure for celiac disease.
A gluten-free diet is used to manage symptoms and promote intestinal healing. The diet is strict and demanding. Patients can relapse if gluten is reintroduced, for some even in trace quantities, and people preparing gluten-free meals are urged to do so separately from other foods.
The only recommended preventative action against celiac disease is to avoid wheat-containing foods in an infant’s diet for six months after birth. Gluten increases the risk of developing celiac disease by five times, “within the first 3 months or after 7 months” of age.
In Europe, a 2006 EU Commission Directive bans the use of gluten containing foods in infant formula. The US, however, has no similar rule and the Celiac Disease Center at the University of Chicago simply notes that “most baby formulas are gluten-free.”

Guidelines for celiac disease
The original 1969 diagnostic criteria for CD by the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) were revised in 1990, and most recently in 2011. Along with clinical guidelines from the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition, these reflect the current consensus for celiac disease in pediatric practice.
In adults, testing for celiac disease is recommended only for symptomatic individuals and those in high risk groups. Screening is explicitly ruled out in asymptomatic patients, for example by the American Association of Family Physicians (AAFP).
Health authorities in many countries follow guidelines from the World Gastroenterology Organization (WGO).
For the WGO, celiac disease is based on patients having “characteristic histopathologic changes in an intestinal biopsy,” along with clinical improvement after a gluten-free diet.
The WGO’s latest guidelines specify serological tests for identifying patients in whom biopsy might be warranted, and investigating high-risk patients (including first- and second-degree relatives). The tests include immunoglobulin A (IgA) endomysial antibody (EMA), IgA anti-tissue transglutaminase antibody (tTG) and IgA and immunoglobulin G (IgG) deamidated gliadin peptide (DGP) antibodies.  Small-bowel biopsy, however, is considered a ‘gold standard’ by the WGO.

The UK was one of the first countries to consider diagnosis and management of celiac disease in general practice. In 2008, the National Institute for Health and Clinical Evidence (NICE) published guidelines for testing both adults and children presenting a variety of symptoms. These are mainly gastrointestinal, which the WGO classifies as ‘classical’, but also include anemia and weight loss, which are grouped by the WGO as ‘atypical’. The list also extends to about 25 specific conditions, which extend well beyond Type 1 diabetes, dermatitis herpetiformis, thyroid disorders and Down Syndrome – long associated with an increased prevalence of celiac disease – to areas such as chronic fatigue syndrome, epilepsy, mouth ulcers, low-trauma fractures and sub-fertility.

In the US, the Agency for Healthcare Research and Quality (AHRQ) recommends WGO guidelines. However, new initiatives are expected after the recent formation of the North American Society for the Study of Celiac Disease (NASSCD). The Society was set up at the International Celiac Disease Symposium in Oslo, Norway last June.

Screening: challenges, ethical issues
At the moment, the broader political response to celiac disease has been largely focused on regulating gluten-free foods.
On the horizon, however, are efforts by celiac disease patient groups to increase the scope of screening. The outlook for this, however, remains unclear – in spite of the experience of an exception such as Italy, where everyone is screened by the age of six.
In June 2005, ‘Best Practice & Research – Clinical Gastroenterology’ published a paper headlined ‘Coeliac disease: is it time for mass screening?’. The authors argued that since antibody screening “may have to be repeated during each individual’s lifetime,” HLA typing of people with DQ2 or DQ8 would allow for “one-off exclusion of a large percentage of the population”. However, they agreed that gene-based screening would be confounded by ethical issues. They also noted that the costs of screening versus prevented morbidity were unknown.

Raising awareness in healthcare professionals
In 2010, a Markov model study provided answers to both the above questions. The study, by an Israeli medical team found that even IgA anti-tTG antibody mass screening – accompanied by confirmatory intestinal biopsy –  was “associated with improved QALYs” (quality adjusted life years) as well as cost effectiveness. Nevertheless, the authors of the study also noted that shortening the delay to diagnosis “by heightened awareness of healthcare professionals” could be a valid alternative to screening.
In the years to come, it is clear that physicians at least are going to become far more aware of celiac disease.

p18 06

Blood-based tests for colorectal cancer screening

, /in /by

Worldwide, screening has been shown to reduce mortality and incidence of colorectal cancer. Despite its documented success, people still fail to participate and screening rates remain low in most countries. Given that patient-reported barriers include resistance to recommended fecal-based methods or endoscopy, blood-based tests have the potential to increase participation in colorectal cancer screening programmes.

by Dr Theo deVos

Background
Globally, colorectal cancer (CRC) is the third most common cancer in men and the second in women, with an estimated 1.36 million cases and causing an estimated 694,000 deaths in 2012 [1]. These rates are unnecessarily high since CRC is an excellent candidate for screening as evidenced by large randomized trials demonstrating reductions in mortality and incidence [reviewed in 2, 3]. Biologically, CRC usually develops slowly, going through a progression from non-cancerous polyp to cancer over a period of a decade or more. This biology readily lends itself to screening and early detection which has a significant positive impact on the effectiveness of intervention. For example, in the United States, 5-year survival is ~90 % if the tumour is confined locally when detected, ~70% if it has spread regionally, but only ~10% if distant metastases are present [4].

Colonoscopy is the predominantly recommended method for routine screening in some countries including the United States, as it enables detection and intervention in the same procedure. It is also the diagnostic follow-up for positive results of other screening tests. However, challenges with capacity and quality, financial concerns, and patient resistance have led to its lack of use as the primary screening modality in most settings. In some countries, flexible sigmoidoscopy is showing a resurgence, with reports demonstrating mortality and incidence benefits [2]. Table 1 displays a list of common CRC screening methods along with new methods coming on-line, today.

The first non-invasive tests for CRC were based on the detection of fecal occult blood (FOBT), and these have been further developed into immunological tests (FIT) using specific antibodies to detect hemoglobin. These tests are typically designed to allow patients to collect stool samples at home and ship the sample by mail to a central laboratory for testing. A newer alternative to fecal blood testing is the analysis of genetic/epigenetic markers in fecal material. This is the basis for the Cologuard test (Exact Sciences, WI, USA), a fecal DNA test recently approved by the US FDA [5]. Blood-based screening tests that measure tumour biomarkers in plasma or serum have been developed as a minimally-invasive alternative to fecal testing. DNA methylation tests based on SEPT9 have become available in Europe and are undergoing regulatory review in China. In addition, methylated SEPT9 testing is available as laboratory-developed tests (LDTs) in the USA, and a kitted version (Epi proColon®; Epigenomics AG, Germany) is currently undergoing US FDA premarket (PMA) review [6]. Another blood-based test, the ColonSentry risk test based on an expression panel is available as an LDT in the USA and in Japan.

Given the clear benefit of screening and the long standing availability of tests, the lack of participation is disappointing, and improving screening rates is a broadly accepted goal. As an example, the ‘80 by 2018’ campaign in the USA has set a goal of 80% adherence to screening guidelines by 2018 [7]. In order to meet this goal, barriers that prevent screening must be understood and overcome. There are numerous reports focused on understanding patient barriers to CRC screening. Although this is a complex issue involving costs, time, physician recommendation and several other factors, one consistent message from these studies is that the test methods themselves present barriers. Many patients are uncomfortable with all or part of the colonoscopy process and many are also uncomfortable with collecting and shipping fecal samples [8]. As a consequence, CRCs are diagnosed symptomatically in more instances than necessary, when the disease has spread beyond the primary site, resulting in greatly reduced survival rates. The availability of a screening test using a simple and common blood draw, which can be included as part of a regular check-up, has the potential to overcome some barriers and improve screening rates.

Blood-based screening
There are a number of approaches to the measurement of cancer biomarkers in the blood. The detection and quantification of circulating tumour cells represents an early approach, which was developed into a commercial system (e.g. CellSearch; Janssen Diagnostics, NJ, USA) though this analysis has not generally been used for cancer screening. Another alternative derives from the isolation and fractionation of circulating immune cells and the quantification of gene expression panels correlated with the disease by reverse-transcriptase PCR. This ‘sentinel concept’ is the basis for the ColonSentry test (GeneNews, Canada) in Table 1. A third alternative is the measurement of metabolic products by mass-spectrometry that are correlated with the presence of cancer. As an example, a commercial test (Cologic; Phenomenome, Canada) was developed based on the measurement of serum levels of GTA-446, an anti-inflammatory fatty acid. The most developed and perhaps simplest approach in this field is the measurement of cell-free genetic or epigenetic markers in plasma or serum that are highly correlated with the presence of cancer. As shown in Table 1, the methylated Septin9 biomarker and the Epi proColon® test were developed based on this approach.

Screening biomarkers in plasma and serum
The recognition that tumour DNA contains genetic and epigenetic changes that can serve as biomarkers dates back a number of decades. As reviewed recently, the list of biomarker reports for colorectal cancer grows ever longer [9]. Although numerous studies report on marker performance, the majority of studies include only a limited number of cases and controls, and only a small subset of markers have been rigorously tested in the clinical setting. Furthermore, a review of marker studies in ClinicalTrials.gov indicated very few ongoing CRC marker screening trials. Well validated markers include methylated SEPT9 described above, and the methylation of BCAT1 and IKZF1 sequences in plasma which have shown to be correlated with CRC [10] and are currently being tested in a clinical trial in Australia. There are many interesting genetic and epigenetic markers, but most await additional validation data that will support clinical utility.

Laboratory considerations for a plasma-based screening test
The basic concept outlined in Figure 1 illustrates key points associated with development of a genetic/epigenetic screening test. CRC screening from blood samples imposes rigorous demands that impact the reduction to practice for a test including: (a) high volume (millions of tests); (b) low target copy number (~1 copy per mL); (c) fragmented DNA; (d) large sample size (e.g. 3.5 mL); and (e) kitted reagents. These are discussed using the methylated Septin9 test as a case study.

Blood draw and processing
Given that screening is a high volume activity, an inexpensive and standard sample collection method is beneficial. In this case, a simple blood draw using a standard collection tube (e.g. K2EDTA plasma collection tube) is performed at the clinic or draw station. Plasma or serum is separated and if necessary they can be re-centrifuged to ensure cell-free status. The emphasis is on preparing cell-free material to limit background contamination due to lysis of nucleated cells in the blood. While this has led to the use of specialized collections tubes (Streck, NE, USA) in the field of prenatal diagnostics, these have not been widely tested for colorectal cancer screening. Cleared plasma can be tested immediately, or stored frozen for a period of time.

Nucleic acid extraction
In this step, cell-free nucleic acids are extracted from the plasma sample. While a number of commercial methods have been developed for this purpose, it remains the Achilles heel of the process. Given the wide range in target concentration, and particularly the exceptionally low copy number expected for early cancers (in the single copy per mL range) [6], as well as the fragmented nature of cell-free DNA, the extraction methods must be designed to handle large samples (e.g. 3–4 mL of plasma), and be able to isolate fragmented DNA. The use of magnetic particles for purification coupled with modified binding and wash buffers designed to capture the full range of DNA fragments has simplified the extraction, and with the development of liquid handling platforms that can process larger volumes, this step is becoming automatable. While the reduction from 3.5 mL plasma to 100 µL of DNA eluate would raise concerns for PCR inhibition, for DNA methylation tests, it is possible to reduce the wash steps because the DNA is extensively purified in the bisulfite treatment process.

Bisulfite treatment
The bisulfite treatment process is required if the target is DNA methylation-based. Recent improvements in bisulfite conversion technology have simplified the treatment. The change to ammonium bisulfite allows for liquid reagents – a key attribute for kit development. In combination with elevated temperatures, bisulfite incubation time is reduced to less than 1 hour, enabling single shift turn-around times for tests. Furthermore, the reaction can be purified using a magnetic particle extraction that takes advantage of the same particles used for the initial DNA extraction. This process can also be automated on a standard liquid handling platform to improve throughput and quality.

Real-time PCR
For genetic (mutation)-based tests, the test can be performed immediately following initial DNA extraction, though it is important to increase the stringency of DNA washes to limit the potential for PCR inhibition. In the final steps, either genetic or epigenetic markers are measured by real-time PCR. For screening applications, the target concentration dictates the conditions and interpretation of the PCR reaction. For example, in the methylated Septin9 test, the final recovered bisulfite converted template DNA is split into three wells and run in three PCR reactions. Although the PCR reaction is run as a real-time assay, the test is essentially a qualitative end point test, since a well is called positive if a PCR curve occurs at any cycle during the course of the reaction. In addition, the results of the three reactions are combined to produce a final interpretation for a patient sample. For the CE-marked Epi proColon 2.0 product, the sample is called positive if two of three wells are positive. For the Ep proColon product undergoing US FDA PMA review, the sample is called positive if any of three wells are positive. This allows for a greater emphasis on a specific test parameter – for sensitivity (any well-positive) or test specificity (two out of three wells positive).

Summary
The use of genetic and epigenetic biomarkers for cancer screening is a field still in its infancy that has great opportunities for growth. Because these biomarkers can be used as indicators of disease, they also have diagnostic and prognostic potential that will be incorporated into the clinical-decision making process. For CRC screening, test kits are already available in Europe and other countries, and are currently under review by both the US and Chinese FDA organizations. In the US, LDTs are currently marketed, and together, all progress represents significant opportunities to generate positive momentum. The introduction of simple, blood-based screening would provide a viable alternative to patients refusing or avoiding current well established methods. The convenience factors of sample collection and processing by health professionals also avoids the challenges of faulty sampling, handling, and mailing associated with at-home self-collected tests. Finally, given the extensive collection of promising biomarkers on the horizon, mechanisms are needed now to expedite clinical utilization and validation to drive further improvements in test performance.

References
1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from: http://globocan.iarc.fr, accessed on 12/09/2014.
2. Kuipers EJ, Rösch T, Bretthauer M. Colorectal cancer screening – optimizing current strategies and new directions. Nat Rev Clin Oncol. 2013; 10: 130–142.
3. Brenner H, Stock C, Hoffmeister M. Effect of screening sigmoidoscopy and screening colonoscopy on colorectal cancer incidence and mortality: systematic review and meta-analysis of randomised controlled trials and observational studies. BMJ 2014; 348: g2467.
4. American Cancer Society. Colorectal Cancer Facts & Figures 2014-2016. Atlanta: American Cancer Society, 2014.
5. Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014; 370(14): 1287–1297.
6. Potter NT, Hurban P, White MN, Whitlock KD, Lofton-Day CE, Tetzner R, Koenig T, Quigley NB, Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma. Clin Chem. 2014; 60(9): 1183–1191.
7. National Colorectal Cancer Round Table. Tools & Resources – 80% by 2018. http://nccrt.org/about/80-percent-by-2018/
8. Gimeno García AZ. Factors influencing colorectal cancer screening participation. Gastroenterol Res Pract. 2012; 2012: 483417.
9. Toiyama Y, Okugawa Y, Goel A. DNA methylation and microRNA biomarkers for noninvasive detection of gastric and colorectal cancer. Biochem Biophys Res Commun. 2014; doi: 10.1016/j.bbrc.2014.08.001.
10. Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, Duesing KR, Buckley MJ, Dunne R, Beetson I, Rand KN, McEvoy A, Thomas ML, Baker RT, Wattchow DA, Young GP, Lockett TJ, Pedersen SK, Lapointe LC, Molloy PL. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer 2014; 14: 54.

The author
Theo deVos PhD
Epigenomics Inc.,
Seattle, WA 98107, USA
E-mail: theo.devos@epigenomics.com

p22 1

Tumour markers for the diagnosis of mucinous ovarian cancer

, /in /by

The aim of this study was to determine the accuracy of CEA, CA 15.3, CA 19.9 and CA 125 for diagnosis of mucinous ovarian cancer (MOC). We studied 94 women with mucinous ovarian tumour, 82 were NOT MOC (68 mucinous ovarian cystadenomas and 14 mucinous borderline ovarian tumour) and 12 were MOC. All MOC patients were in FIGO stage I or II. No statistically significant differences were found between MOC and NOT MOC patients according to CEA and CA 15.3 (P>0.05). AUC values were 0.862 (P=0.0002) and 0.829 (P=0.0021) for CA 19.9 and CA 125 respectively. In conclusion, preoperative CA 19.9 and CA 125 levels showed high diagnosis efficacy to predict whether a mucinous ovarian tumour is benign or malignant.

by Dr J. D. Santotoribio, A. Garcia-de la Torre, C. Cañavate-Solano, F. Arce-Matute, M. J. Sanchez-del Pino and S. Perez-Ramos

Introduction
Ovarian cancer is the fifth leading cause of cancer-related death in women in developed countries and has one of the highest ratios of incidence to death [1]. Epithelial ovarian cancer is a heterogeneous disease with a heterogeneous distribution pattern [2]. Epithelial ovarian cancer set by the World Health Organization recognizes eight histological tumour subtypes: serous, mucinous, endometrioid, clear cell, transitional cell, squamous cell, mixed epithelial and undifferentiated [3]. Mucinous ovarian cancer (MOC) is an epithelial ovarian cancer that contains cysts and glands lined by mucin-rich cells and historically accounted for approximately 11.6% of all primary epithelial ovarian carcinomas [4]. MOC should be considered separate from the other epithelial ovarian cancers as metastatic primary disease and recurrent mucinous cancers have a substantially worse prognosis than other epithelial ovarian cancers [5]. Tumour markers are biochemical substances found in the blood which may be measured for the diagnosis of cancer. The major challenge of developing a screening test using serum tumour markers, is that it must be highly specific (because of the low prevalence of ovarian cancer) in order to avoid detection of numerous false positives [6]. The most common tumour markers in clinical chemistry are carcinoembryonic antigen (CEA), cancer antigen 15.3 (CA 15.3), cancer antigen 19.9 (CA 19.9) and cancer antigen 125 (CA 125). CEA and CA 15.3 have been found at elevated levels in patients with epithelial ovarian cancer [7–9]. Preoperative elevated CA 19.9 levels are related to a higher probability of MOC [8, 10]. A diagnostic approach based on the use of CA 125 has been suggested for the early diagnosis of ovarian cancer, although premenopausal women may have higher serum CA 125 levels than in postmenopausal women [11–13]. Also, in mucinous borderline ovarian tumours have found a significant relation with elevated CA 125 [14, 15]. Another tumour marker for diagnosis of ovarian cancer, serum human epididymis protein 4 (HE4), has lowest concentrations in mucinous tumours and displays no difference in serum concentration between benign or malignant mucinous ovarian tumours [12, 13].

The aim of this study was to determine the accuracy of CEA, CA 15.3, CA 19.9 and CA 125 for diagnosis of MOC in patients with mucinous ovarian tumors.

Materials and methods
Women with mucinous ovarian tumours diagnosed between 2004 and 2012 were included in the study. We excluded patients with other tumours that could elevate the tumour markers. Before biopsy and after obtaining an informed consent, blood specimens were drawn by venipuncture in gel separator serum tubes and centrifuged at 4000 rpm for 4 min. The following variables were analysed: CEA, CA 15.3, CA 19.9 and CA 125. We measured the serum concentrations of the tumour markers by electrochemiluminescence immunoassay (ECLIA) in MODULAR E-170 (ROCHE DIAGNOSTIC®). The reference range values provided by our laboratory are: CEA (0–3.4 ng/mL), CA 15.3 (0–30 U/mL), CA 19.9 (0–37 U/mL) and CA 125 (0–35 U/mL). After surgery, histology and stage were determined according to the International Federation of Gynecologists and Obstetricians (FIGO) classification. Patients were classified into two groups according to the diagnosis of ovarian biopsy: NOT MOC (mucinous ovarian cystadenomas and mucinous ovarian borderline tumour) and MOC. For all statistical comparisons a value of P<0.05 was considered significant. The accuracy of serum tumour markers was determined using receiver operating characteristic (ROC) techniques by analysing the area under the ROC curve (AUC). The optimal cut-off value was considered with higher than 95% specificity. Statistical analysis was performed using the software MEDCALC®. Results
We enrolled 94 women aged between 15 and 80 years old (median age was 43). Eighty-two patients (87.2 %) were NOT MOC (68 mucinous ovarian cystadenomas and 14 mucinous ovarian borderline tumours) and 12 patients (12.8 %) were MOC. Thirty-two patients were postmenopausal and 62 patients were premenopausal. All MOC patients were in FIGO I or II stages.

Descriptive statistics of serum tumour markers in MOC and NOT MOC patients are shown in Table 1. No statistically significant differences were found between MOC and NOT MOC patients according to CEA and CA 15.3 (P>0.05). The frequency of abnormal serum levels CA 19.9 and CA 125 in MOC and NOT MOC patients are shown in Table 2. AUC, optimal cut-off value, sensitivity and specificity of ROC curves for diagnosis of MOC using CA 19.9 and CA 125 are displayed in Table 3.

No statistically significant differences were found between premenopausal and postmenopausal women for CEA, CA 15.3, CA 19.9 and CA 125. Also, these tumour markers were not statistically significant for the diagnosis of mucinous borderline ovarian tumours (P>0.05).

Discussion
In the literature, CEA has been noted to be elevated in almost one third of all ovarian carcinomas. CEA is much more likely to be elevated in mucinous ovarian carcinomas than in non-mucinous ovarian carcinomas [5, 7, 8]. CA 15.3 has been found to be elevated levels in patients with advanced epithelial ovarian cancer [8, 9]. However, in this study, CEA and CA 15.3 were not useful to differentiate benign from malignant mucinous ovarian tumours.

In the recent paper of the guidelines on the recognition and initial management of ovarian cancer from the National Institute for Health and Clinical Excellence (NICE) stated that general practitioners should measure serum CA 125 in primary care in women with symptoms that suggest ovarian cancer [11]. Also, a diagnostic approach based on the use of CA 125 in association with ultrasonography has been suggested for the early diagnosis of ovarian cancer [11, 12]. The major drawback of using CA 125 as a screening strategy is that up to 20% of ovarian cancers do not express the antigen, and also that abnormal serum levels CA 125 may be found in patients with benign ovarian tumours [12, 13]. Recently, another tumour marker for ovarian cancer has been proposed, serum human epididymis protein 4 (HE4), frequently overexpressed in ovarian cancers, especially in serous and endometrioid histology [6, 12, 13]. However, HE4 has lowest concentrations in mucinous tumours and shows no difference in serum concentrations between benign or malignant mucinous ovarian tumours [12, 13]. Serum CA 19.9 presents low efficiency for the diagnosis of serous ovarian cancer, but preoperative elevated CA 19.9 levels could be related to a higher probability of MOC [8, 10]. In this paper, CA 125 false positive results (abnormal serum levels) were found in 31.7 % of NOT MOC patients and false negative (normal serum levels) in 33.3 % of MOC patients. CA 19.9 false positive results were found in 19.5 % of NOT MOC group and false negative in 16.6 % of MOC group. All MOC patients had abnormal serum CA 19.9 and/or CA 125 levels, and 60.98 % NOT MOC patients presented normal CA 19.9 and CA 125 (Table 2). Both tumour markers showed similar sensitivity (50%) in MOC diagnosis and slightly higher specificity with CA 19.9 (97.6%) than with CA 125 (95.1%) (Table 3).

In some studies [12, 13], significantly higher serum CA 125 levels were found in premenopausal women than in postmenopausal women; in our case this is not significant (P>0.05). In other study, up to 61% of women with borderline ovarian tumours had elevated CA 125 [14]. In mucinous borderline ovarian tumours with papilla formation, others authors found a significant relation between elevated CA 125 [15]. In our patients, CA 125 and CA 19.9 were not statistically significantly different (P>0.05) for the diagnosis of mucinous borderline ovarian tumours.

In conclusion, preoperative CA 19.9 and CA 125 levels showed high diagnosis efficacy to predict whether a mucinous ovarian tumour is benign or malignant.

References
1. emal A, Siegel R, Xu J, Ward E. Cancer statistics. CA Cancer J Clin. 2010; 60: 277–300.
2. Sung PL, Chang YH, Chao KC, Chuang CM. Task Force on Systematic Review and Meta-analysis of Ovarian Cancer. Global distribution pattern of histological subtypes of epithelial ovarian cancer: a database analysis and systematic review. Gynecol Oncol. 2014; 133: 147–54.
3. Lee KR, Tavassoli FA, Prat J,  et al. WHO histological classification of tumours of the ovary. In: Pathology and genetics of tumours of the breast and female genital organs. Edited by Tavassoli FA, Devilee P. IARC Press 2003; 113–161.
4. Nolen B, Marrangoni A, Velikokhatnaya L,  et al. A serum based analysis of ovarian epithelial tumourigenesis. Gynecol Oncol. 2009; 112: 47–54.
5. Frumovitz M, Schmeler KM, Malpica A, et al. Unmasking the complexities of mucinous ovarian carcinoma. Gynecol Oncol. 2010; 117: 491–496.
6. Husseinzadeh N. Status of tumour markers in epithelial ovarian cancer has there been any progress? A review. Gynecol Oncol. 2011; 120: 152–157.
7. Tholander B, Taube A, Lindgren A, et al. Pretreatment serum levels of CA-125, carcinoembryonic antigen, tissue polypeptide antigen, and placental alkaline phosphatase in patients with ovarian carcinoma: influence of histological type, grade of differentiation, and clinical stage of disease. Gynecol Oncol. 1990; 39: 26–33.
8. Terzic M, Dotlic J, Likic I, et al. Diagnostic value of serum tumour markers evaluation for adnexal masses. Cent Eur J Med. 2014; 9: 210–216.
9. Gemer O, Oustinov N, Gdalevich M, et al. Pretreatment CA 15-3 levels do not predict disease-free survival in patients with advanced epithelial ovarian cancer. Tumori. 2013; 99: 257–260.
10. Kelly PJ, Archbold P, Price JH, et al. Serum CA 19.9 levels are commonly elevated in primary ovarian mucinous tumours but cannot be used to predict the histological subtype. J Clin Pathol. 2010; 63: 169–173.
11. Sturgeon CM, Duffy MJ, Walker G. The National Institute for Health and Clinical Excellence (NICE) guidelines for early detection of ovarian cancer: the pivotal role of the clinical laboratory. Ann Clin Biochem. 2011; 48: 295–299.
12. Molina R, Escudero JM, Augé JM, et al. HE4 a novel tumour marker for ovarian cancer: comparison with CA 125 and ROMA algorithm in patients with gynaecological diseases. Tumour Biol. 2011; 32: 1087–1095.
13. Escudero JM, Auge JM, Filella X, et al. Comparison of serum human epididymis protein 4 with cancer antigen 125 as a tumour marker in patients with malignant and nonmalignant diseases. Clin Chem. 2011; 57: 1534–1544.
14. Morotti M, Menada MV, Gillott DJ, et al. The preoperative diagnosis of borderline ovarian tumours: a review of current literature. Arch Gynecol Obstet. 2012; 285: 1103–1112.
15. Alanbay I, Aktürk E, Coksuer H, et al. Comparison of tumour markers and clinicopathological features in serous and mucinous borderline ovarian tumours. Eur J Gynaecol Oncol. 2012; 33: 25–30.

The authors
J. D. Santotoribio1,2,*, A. Garcia-de la Torre1,2, C. Cañavate-Solano1,2, F. Arce-Matute1, M. J. Sanchez-del Pino2, S. Perez-Ramos1,2
1Clinical Biochemistry Laboratory, Puerto Real University Hospital, Cadiz, Spain
2Dept. of Biomedicine, Biotechnology and Public Health, University of Cadiz, Cadiz, Spain

*Corresponding author
E-mail: jdsantotoribioc@gmail.com

26541 CLI 1410 DIRUI

FUS & Microscopy Comparison

, /in /by
26645 CLI Analyticon Urilyzer Sed

Automating the Gold Standard Urine Sediment

, /in /by
26587 Vision Sperm Rev 1

Vision Sperm – Sperm analysis

, /in /by
26740 ARK newSeptember2014 voriconazole 92x132

ARK Voriconazole Assay

, /in /by
p25 02

Ovarian reserve and beyond: AMH’s role in women’s reproductive health

, /in /by

The human gene for anti-Müllerian hormone (AMH) was isolated and sequenced 20 years ago [1], with the first immunoassays developed in 1990 [2,3].  Since then, our understanding of this hormone has significantly increased, with most clinical use today focusing on women’s reproductive health. AMH’s ability to reflect the number of small antral and pre-antral follicles present in the ovaries, and therefore the ovarian reserve, has led to AMH measurement being used in a wide array of clinical applications.

One of the first was as a tumour marker in the diagnosis and follow up of women with ovarian granulosa cell tumours (GCT) [4, 5]. More recently, with the dramatic improvements in the treatment of childhood cancers, attention is focused on AMH to assess the likelihood of gonadal damage and infertility after treatment.  It is also being used to investigate the toxicity of different therapeutic regimens, in the choice of those treatments, and the prediction (and potential preservation) of fertility in young women and children following cancer therapy.

Sensitive diagnostic marker for GCT
GCT accounts for 2-3% of all ovarian tumours, with two distinct types: the juvenile and the adult form. The more common adult form generally presents in women at around 50 years. A majority have endocrine manifestations as a direct consequence of hormone secretion by the tumour [6].

GCTs have the potential to secrete estradiol, Inhibin (A and B) and AMH. Inhibin and AMH are the more useful biomarkers since estradiol is only produced in 50-60% of GCT patients and is dependent on stimulation by testosterone from adjacent theca cells. While serum total Inhibin is secreted in almost all GCT and has been shown to successfully detect recurrence following surgery, it is also increased in some epithelial ovarian tumours and fluctuates significantly within the menstrual cycle. AMH is more specific to GCT as expression is limited to ovarian granulosa cells and it does not change substantially over the menstrual cycle.

Although GCT is extremely rare, it is noted for its late recurrence, usually within four-six years, but can be up to 10-20 years after removal of the primary tumour. AMH disappears within days of removal of the ovaries [7] and, following tumour resection, a rise in AMH precedes clinical detection, making it an extremely sensitive marker for the early detection of tumour recurrence.

Lane’s 1999 study followed 56 patients post operatively and showed that AMH was useful in evaluating the completeness of tumour removal [4]. In addition, serial AMH measurements were able to detect recurrence on average three months prior to clinical detection. A second study, which followed 31 patients for up to seven years, confirmed these observations [5]. This group used an AMH assay 20 times more sensitive than previously used and, when comparing both assays found discrepant values in six out of 31 patients. The more sensitive assay accurately reflected the clinical situation and was elevated up to 16 months earlier in patients with tumour recurrence.

However, there is still insufficient published information on which to assess the sensitivity and specificity of AMH for the diagnosis of GCT.  This is due to small patient numbers, the insensitivity of older assays and the lack of solid reference values in pre-menopausal women and children. The advent of more sensitive, fully automated assays will facilitate more robust studies.

Assessment of ovarian damage
The relationship between AMH and the number of small growing follicles (and therefore the number of primordial follicles or ovarian reserve) makes it useful for assessing the gonadal toxicity of cancer therapy and loss of ovarian reserve.  Levels fall rapidly with the onset of cancer treatment, with subsequent recovery dependant on degree of ovarian damage. AMH appears to identify which treatments may spare the ovaries, or are most toxic to them, and may give clinicians additional information to direct therapeutic choices in children and women of childbearing age with cancer.

Radiotherapy is a well-known cause of ovarian damage, even at low radiation levels. Women who have undergone pelvic or total body irradiation are likely to have low or undetectable AMH levels [9, 10]. The gonadal toxicity of alkylating agents is also well established. In a study involving young women with lymphoma, those receiving alkylating agents showed little or no recovery in AMH levels following treatment whereas those receiving alternative chemotherapy showed good recovery. 

Childhood cancer and fertility
Childhood cancer treatment has improved dramatically with survival rates of more than 90%.  However, the consequences of treatment may be permanent damage to the ovaries, affecting fertility. AMH is detectable in females of all ages rising steadily throughout childhood. Several studies have confirmed its role as a clinically useful marker to assess impairment of ovarian reserve in those receiving treatment for cancer  [11, 12, 13].

Brougham showed that AMH decreased during chemotherapy in both prepubertal and pubertal girls, becoming undetectable in 50% of patients; recovery occurred in the low to medium risk groups after completion of treatment, yet remained undetectable in the high risk group.  Inhibin B was undetectable in most patients before treatment and FSH showed no relationship with treatment. Thus AMH indicates a more useful  assessment of residual ovarian reserve, revealing partial loss or ovarian failure.

It is clear that a woman can suffer a significant loss of ovarian reserve without any lasting effects on her fertility, for example following removal of an ovary.  For survivors of childhood cancer this may mean that only a substantial loss of ovarian reserve would have a clinical impact. Indeed, recent work has shown that there is a high number of successful pregnancies in lymphoma survivors, despite low AMH levels [14].  In a study of 84 childhood cancer survivors they achieved pregnancy rates similar to controls despite impaired ovarian reserve [15]. However, a 10-year follow up study of childhood cancer survivors, now in their 30s, showed that the percentage of childless women in this group was greater than in the normal Danish population, particularly in the group of women who received the most gonadotoxic treatment burden. Their pregnancy rate and outcome was especially poor [16].  The truth is difficult to discern on current evidence and more work is required on long term follow up, with fertility and age at menopause as end points.

The real value of measuring AMH in young women surviving cancer would be to forecast long-term reproductive outcome and take steps to preserve their fertility.

Reproductive outcomes in adult women
The same fertility concerns exist for women of childbearing age.  Using AMH values to assess ovarian reserve and individualize risk, more invasive methods of fertility preservation may be appropriate for women with a low AMH, while those with high values for their age may decide  to start cancer treatment without delay.

Most evidence comes from breast cancer studies and is based on the assumption that a woman with a higher pre-treatment AMH before chemotherapy will be more likely to retain ovarian function. A prospective study in women with newly diagnosed breast cancer linked high levels of AMH detected before treatment with retaining long-term ovarian function five years after surgery [17]. Pretreatment serum AMH was seen to be markedly higher in women who continued to have menses. The predictive value of AMH for post-chemotherapy ovarian function has subsequently been confirmed [18] allowing the development of prediction tools combining age and AMH [18].

Individualizing breast cancer adjuvant chemotherapy
Adjuvant endocrine therapy has been shown to reduce the likelihood of reocurrence and improve overall survival rates in hormone receptor-positive (HR-positive) breast cancer. However, it appears that ovarian function after chemotherapy has direct implications on the choice of therapy.  Aromatase inhibitors (AIs) are more effective in postmenopausal women than tamoxifen [19]. However, in premenopausal women, AIs may cause a rise in estrogen levels due to reactivation of ovarian function. Consequently, even in women who have developed chemotherapy-induced ovarian failure, tamoxifen is the standard of care [20, 21].

It has been suggested that all women who are premenopausal prior to chemotherapy, even those in their late 40s and early 50s, should be treated with adjuvant tamoxifen therapy or, if they are going to receive an aromatase inhibitor, should have their ovaries removed or chemically suppressed [22]. For the latter group, these strategies are invasive and are associated with increased side effects. Consequently, being able to predict permanent ovarian failure using information other than the patient’s age is relevant.

Data from recent studies [8, 17] suggest that  pre-chemotherapy assessment of serum AMH concentrations, possibly in combination with inhibin B, may provide important information about the likelihood of developing permanent ovarian failure with chemotherapy. In addition, this could help identify a patient population in which it would be safe to treat with upfront AI monotherapy. The expanding number of studies available all add to our understanding of the role of AMH in ovarian function, its ability to predict a woman’s ovarian reserve for her fertility and the impact of cancer treatment on reproductive health.

References
1. Cate RL, Mattaliano RJ, Hession C, et al.  Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells. Cell 1986; 45, 685-698.
2. Hudson PL, Dougas I, Donahoe PK, et al. An immunoassay to detect human Müllerian inhibiting substance in males and females during normal development. J Clin Endocrinol Metab. 1990; 70, 16-22.
3. Josso et al. An enzyme linked immunoassay for anti-müllerian hormone: a new tool for the evaluation of testicular function in infants and children. JCEM 1990; 70, 23-27.
4. Lane AH, Lee MM, Fuller AF Jr, et al. Diagnostic utility of Müllerian inhibiting substance determination in patients with primary and recurrent granulosa cell tumors. Gynecol Oncol 1999; 73, :51–55.
5. Long WQ, Ranchin V, Pautier P, et al. Detection of minimal levels of serum anti-Müllerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 2000; 85, 540–544.
6. Bjorkholm E, Silfversward C. Prognostic factors in granulosa-cell tumors. Gynecol Oncol. 1981;11, 261–274.
7. LaMarca A, De Leo V, Giulini S, et al. Anti-Müllerian hormone in premenopausal women and after spontaneous or surgically induced menopause. J Soc Gynecol Invest 2005; 12, 545–548.
8. Henry, NL, Xia R, Schott AF, McConnell D, et al. Prediction of Postchemotherapy Ovarian Function Using Markers of Ovarian Reserve. The Oncologist 2014; 19, 68–74.
9. Lie Fong S, Laven JS, Hakvoort-Cammel FG, et al. Assessment of ovarian reserve in adult childhood cancer survivors using anti-Mullerian hormone. Hum Reprod 2009;24, 982–990
10. Gracia CR, Sammel MD, Freeman E, et al. Impact of cancer therapies on ovarian reserve. Fertil Steril 2012; 97, 134–140 e131.
11. Bath LE, Wallace WH, Shaw MP, et al. Depletion of ovarian reserve in young women after treatment for cancer in childhood: detection by anti-Müllerian hormone, inhibin B and ovarian ultrasound. Hum Reprod 2003; 18, 2368–2374.
12. van Beek RD, van den Heuvel-Eibrink MM, Laven JS, et al. Anti-Müllerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin’s lymphoma during childhood. J Clin Endocrinol Metab 2007; 92, 3869–3874.
13. Brougham MF, Crofton PM, Johnson EJ, et al. Anti-Müllerian hormone is a marker of gonadotoxicity in pre- and postpubertal girls treated for cancer: a prospective study. J Clin Endocrinol Metab 2012; 97, 2059–2067.
14. Janse F, Donnez J, Anckaert E, et al. Limited value of ovarian function markers following orthotopic transplantation of ovarian tissue after gonadotoxic treatment. J Clin Endocrinol Metab 2011; 96, 1136–1144.
15. Dillon KE, Sammel MD, Ginsberg JP, et al. Pregnancy After Cancer: Results From a Prospective Cohort Study of Cancer Survivors. Pediatr Blood Cancer. 2013 Dec; 60(12), 2001-6.
16. Nielsen SN, Andersen AN, Schmidt KT, et al. A 10-year follow up of reproductive function in women treated for childhood cancer. Reprod Biomed Online 2013; 27, 192–200.
17. Anderson RA, Cameron DA. Pretreatment serum anti-müllerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer. J Clin Endocrinol Metab 2011; 96, 1336–1343.
18. Anderson RA, Rosendahl M, Kelsey TW, et al. Pretreatment anti-Müllerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer. Eur J Cancer 2013;49, 3404–3411.
19. Burstein HJ, Prestrud AA, Seidenfeld J et al. American Society of Clinical Oncology clinical practice guideline: Update on adjuvant endocrine therapy for women with hormone receptor-positive breast cancer. J Clin Oncol 2010; 28, 3784–3796.
20. Smith IE, Dowsett M, Yap Y-S et al. Adjuvant aromatase inhibitors for early breast cancer after chemotherapy-induced amenorrhoea: Caution and suggested guidelines. J Clin Oncol 2006; 24, 2444–2447.
21. Burstein HJ, Mayer E, Patridge AH et al. Inadvertent use of aromatase inhibitors in patients with breast cancer with residual ovarian function: Cases and lessons. Clin Breast Cancer 2006;7, 158–161.
22. Henry NL, Xia R, Banerjee M et al. Predictors of recovery of ovarian function during aromatase inhibitor therapy. Ann Oncol 2013; 24, 2011–2016.

The author
Sherry Faye, PhD
Director, Global Scientific Affairs,
Beckman Coulter Diagnostics
Brea, CA, USA

C167 Horizon fig2

Clinical application of NGS – ensuring quality

, /in /by

Advances in Next Generation Sequencing (NGS) are bringing much higher throughput and rapidly reducing costs, whilst facilitating new mechanisms for disease prediction. Consequently, the clinical applications of NGS technologies are continuing to develop, with the potential to change the face of genetic medicine [1].

by Hannah Murfet (BSc, PCQI), Product Quality Manager, Horizon Discovery

Applications of NGS in a clinical context are varied, and may include interrogation of known disease-related genes as part of targeted gene panels, exome sequencing, or genome sequencing of both coding and non-coding regions. However, as NGS moves further into the clinic, care must be taken to ensure high levels of quality assurance, rigorous validation, recording of data, quality control, and reporting are maintained. [1] [2]
Guidelines specific to NGS are beginning to emerge and to be adopted by clinical laboratories working with these technologies, in addition to those mandated by clinical accreditation and certification programmes. In this article we give an overview of the specific guidance set out by the American College of Medical Genetics and Genomics in its September 2013 report ‘ACMG clinical laboratory standards for next-generation sequencing’, and the New York State Department of Health’s January 2014 document ‘Next Generation Sequencing (NGS) guidelines for somatic genetic variant detection’.

Quality Assurance
Quality assurance (QA) in the clinical context comprises maintenance of a desired level of quality for laboratory services. Typically, quality management systems take a three tier hierarchy. At the highest level the policies define the organisation’s strategy and focus. Underneath this sit the procedures, which define and document instructions for performing business/quality management or technical activities. Underpinning both of these tiers are accurate records.
In the case of New York State Department of Health guidelines, there is clear focus on the requirement for SOPs, which can be broken down into two levels. The first level states the required flow of information, demonstrating the sequence of events, and associated responsibilities or authorities. The first level procedures are best kept at a relatively high level, and may reference more specific and detailed level two processes.
Testing sequences may be incorporated into one or more level one processes, depending on the complexity of the clinical laboratory’s operations. An overview of the typical testing sequence is shown in the figure below.
Level two processes are best documented as clear ‘how to’ guides, detailing all responsibilities, materials and procedures necessary to complete the activity. For laboratory-focused activities, validation study inputs and outputs can establish clear and consistent protocols, supporting training and laboratory operation.
Accurate record keeping should include which instruments were used in each test, as well as documentation of all reagent lot numbers. Any deviations from standard procedures should be recorded, including any corrective measures [1]. Templates may be generated to ensure consistency in output records for both testing and reporting.
In addition to documented processes, implementation of predetermined checkpoints or key performance indicators should be included to permit the monitoring of QA over time. Once established, these may act as a trigger for assay drift, operator variability, or equipment issues.
In the US, compliance to the HIPAA Act (Health Insurance Portability and Accountability Act) must be implemented to ensure traceability and protection of patient data, and many authorities mandate record retention periods, including CLIA who dictate that records and test reports must be stored for at least two years [1].
Clinical laboratories may look to further certification to ensure tight QA, such as the implementation of ISO 15189, especially in countries where no formal accreditation schemes are in place. [3]

Validation
Validation involves the in-depth assessment of protocols, tests, materials and platforms, providing confidence that critical requirements are being met. Test development and platform optimization should include factors such as determination of sample pooling parameters, and use of synthetic variants to create a strong data set, to compare tools and optimize the workflow. Validation of each entire test should be undertaken, using set conditions for sensitivity, specificity, robustness and reproducibility.  It should be noted that the first test developed may naturally carry a higher validation burden than subsequent tests developed for the same platform. Platform validation and quality management are also vital. [1,2]
Specific validation requirements for NGS as set out by the New York State Department of Health are listed below.  These guidelines may be used as a basic checklist for coverage, or to supplement more general accreditation or certification requirements, e.g. those required by CLIA or ISO 15189. [1]

  • Each reportable variant does not require confirmation every time it is encountered, as long as the variant and target area (gene) containing it was rigorously validated
  • Accuracy and validity of the bioinformatics must be demonstrated
  • Anything that is not exclusively based on a FDA-approved assay is considered to be a laboratory developed test and will require full validation over verification
  • Commercially available materials must be validated by the laboratory for use as a diagnostic tool where there are no clinical indications for use
  • Validation of a single version of all analyses software
  • Performance characteristics for each sample type must be established (e.g. FFPE)
  • Performance characteristics for each type of variant in the assay must be established, and each type of detection should be validated separately (e.g. SNV or structural variants)

Data
NGS has the potential to create huge amounts of data, meaning that accurate and efficient systems for data storage and collection are more essential than ever. Data protocols are generally established through the validation stages, then monitored at predetermined checkpoints with key performance indicators to ensure consistency and accuracy of service provision.
The list below gives an overview of NGS specific data requirements from the New York State Department of Health. [1]

Accuracy

  • Validation, including minimum 50 patient samples with representation for material type (e.g. FFPE), and variants across target areas, confirmed by an independent reference method
  • Minimum 10 positive samples for each type of variant
  • Recommended approach – sequence a well characterised reference sample to determine specificity
  • If vigorous validation of reported variants has not been completed in the original studies, ongoing confirmation by independent reference methods must be performed until at least 10 reference points have been independently validated
  • A disclaimer must be used where incidental findings of unknown significance are included, where there is no established confirmatory assay. The disclaimer must clearly state that the variant has not been verified

Robustness

  • Robustness is the likelihood of assay success. Adequate quality control measures must be in place to determine success of techniques such as extraction, library preparation or sequencing

Precision

  • Precision is related to within-run control
  • For each type of variant a minimum of 3 positive samples containing variants near the stated sensitivity of the assay must be analysed in triplicate in the same run using different barcodes
  • Renewable reference samples can be used to determine the analytical validity of the test. These can establish baseline data to which future modifications can be compared

Repeatability and Reproducibility

  • Repeatability and reproducibility is related to between-run controls, to determine ability to return identical results under identical (repeatability) or changed (reproducibility) conditions
  • For each type of variant a minimum of 3 positive samples containing variants near the stated sensitivity of the assay must be analysed in three separate runs, using different barcodes on different days, by two different technologists where possible
  • If multiplexing samples with distinct barcodes, it must be verified that there is no cross talk and that all target areas and variants are reproducible, independent of which patient/barcode combination is used
  • It is useful to consider instrument-instrument variability as well as inter-operator variability. Parameters for expected reproducibility should be established, and would typically be around 95-98%

Analytical Sensitivity and Specificity

  • Sensitivity and specificity refer to positive and negative percentage variability respectively, when compared to gold standard
  • All types of variants in three target areas with consistently poor coverage should be interrogated, as well as three target areas with consistently good coverage. These can be established with defined mixtures of cell line DNA (not plasmids), but must be verified with 3 – 5 patient samples
  • The limit of detection should be established
  • Confidence intervals for variant types must be determined

A minimum data set is expected, to establish key performance characteristics, including: base calling; read alignment; variant calling; and variant annotation.

Quality Control
In contrast to quality assurance where the infrastructure for quality is established to maintain the right service, quality control addresses testing and sampling to confirm outputs against requirements. Quality control takes place across all aspects of a process from reagents used, to software and in-assay controls.
Quality control of reagent lots is best implemented at the point of goods inspection. A clear label should be placed on the reagent under inspection, and testing performed to validate/confirm analytical sensitivity. Quality control of software updates can be handled through a version control and impact assessment process. All re-validation must be clearly documented and demonstrate consistency in analytical sensitivity.
Sample identity confirmation is essential, especially if samples are pooled. Proficiency testing protocols must be established to allow for execution as required by clinical accreditation bodies (such as CLIA). Quality control stops may be added to laboratory process before the sequencing run, to the run itself and at the end before data analysis.
Use of control materials /reagents at all stages of the sequencing procedure supports quality control. No Template Controls (NTC) should be used at all amplification steps; a negative Control should be used upon initial validation, and periodically thereafter; and a Positive / Sensitivity Control should be used in each sequencing run. [1]
Several different QC protocols may need to be followed, and quality control measures applied can vary depending on chosen methods and instrumentation, but they should always include procedures to identify sample preparation failures and failed sequencing runs. Documentation for QC protocols is best detailed in the relevant SOP.

Reports
Specific requirements around the generation, approval, issue and re-issue of reports are included as part of accreditation programmes, such as CLIA, and standards certifications, such as ISO 15189. The most essential reporting requirements related to NGS are as follows [1,2]:

  • The laboratory director is responsible for designing advantages and limitations of test offerings, ensuring healthcare providers can make informed decisions
  • Turnaround times for reports should ensure there are clear requirements for NGS test prioritisation, and should be clinically appropriate
  • All detected somatic variants should be recorded in a report, identifying each variant’s significance
  • Incidental findings including clinical relevance should be recorded
  • Limitations of the assay should be identified and reported on, including for which target areas the assay lacked sufficient coverage to confidently determine mutational status
  • Information comparing the level of exome vs. genome sequencing to an available disease specific panel test should be included


Conclusions
While complete understanding of the clinical implications of some variants is still to be fully understood, there are clear prospects emerging for NGS to support further development and adoption of companion diagnostics. As the overall picture for NGS evolves, sell-defined guidelines are being developed for everything from quality assurance to reporting.  It is expected that guidance and certification will continue to develop as NGS becomes an ever more common technology within the clinical laboratory.

References
1. American College of Medical Genetics and Genomics. (2013, September). ACMG clinical laboratory standards for next-generation sequencing.
2. New York State Department of Health. (2014, January). “Next Generation” Sequencing (NGS) guidelines for somatic genetic variant detection.
3. Horizon Discovery. (n.d.). ISO 15189: A Standard of Yin and Yang.

www.horizondx.com

Latest issue of Clinical laboratory

March 2026

CLi Cover MRCH 2026
Digital edition
All articles Archived issues

Free subscription

View more product news

Get our e-alert

The leading international magazine for Clinical laboratory Equipment for everyone in the Vitro diagnostics

Sign up today
clinlab logo blackbg 1

Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com

PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.