Published guidelines provide different approaches for laboratories to follow when investigating celiac disease. The aim is to minimize time to diagnosis and reduce unnecessary investigations. Variability between IgA tissue transglutaminase tests must be considered when implementing the local diagnostic strategy. Determining best practice depends on the assays used, expertise available, cost and local clinical audit of outcomes.
by K. Swallow, Dr G. Wild, Dr W. Egner and Dr R. Sargur
Background
Celiac disease is a common autoimmune condition affecting approximately 1 : 100 in the UK [1, 2]. Early diagnosis is key to appropriate management. Symptoms are eliminated by following a gluten-free diet after a confirmed diagnosis. Following best practice guidance can reduce repeated visits to GP practices and outpatient departments, and numerous requests for laboratory tests.
In recent years, guidelines for the diagnosis and management of celiac disease have been published by the National Institute of Health and Care Excellence (NICE) [1] and the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) [3]. The guidelines provide algorithms for laboratory testing strategies and recommendations about when a duodenal biopsy should be performed in both symptomatic and asymptomatic patients. There is a perceived need to avoid invasive procedures in children by using optimal in vitro testing.
Guidelines: recommendations for diagnostic testing in symptomatic patients
There are some similarities and several notable differences between the guidelines given for symptomatic patients. Both NICE and ESPGHAN guidance recommend IgA tissue transglutaminase antibodies (TTG Ab) as the first-line screening test. ESPGHAN also recommend testing all patients for IgA deficiency. NICE only recommend checking for IgA deficiency if the IgA TTG Ab result is negative. After this initial test the recommended pathways by the two groups differ; however, both base the strategies on the level of positivity of the IgA TTG Ab result.
NICE pathway after IgA TTG Ab testing
NICE recommend that if the TTG Ab test is positive the patient should be referred for confirmatory biopsy. If it is ‘equivocal’, IgA endomysial antibodies (EMA) should be tested in order to determine if the TTG Ab result is potentially false positive or if the patient should be referred to a gastroenterologist. A patient with negative IgA TTG Ab should be checked for IgA deficiency and IgG TTG/EMA performed if they are found to be deficient. The guidelines do not provide information regarding the definition of the ‘equivocal’ range for TTG Ab assays.
ESPGHAN pathway after IgA TTG Ab testing
For symptomatic patients the diagnosis can be made with or without the need for duodenal biopsy, dependent on the serological test results. As with NICE, all patients with positive IgA TTG Ab should be referred to a gastroenterologist. They also suggest using IgG TTG/EMA if the patient is IgA deficient.
ESPGHAN stratify the level of positivity based upon levels above the normal range or ‘upper limit of normal’ (ULN). If the TTG Ab result is >10× ULN the decision to go to biopsy is made after further testing EMA and HLA-DQ2/DQ8. If all tests (TTG Ab, EMA and HLA-DQ2/DQ8) are positive, a patient may be diagnosed without the need for duodenal biopsy. Biopsy is recommended if EMA and/or HLA typing is negative, or if TTG is positive but <10× ULN.
Guidance for testing in asymptomatic patients (screening)
Several conditions, including IgA deficiency, autoimmune disease (type I diabetes, hypothyroidism, pernicious anemia), Down syndrome, or a having a first degree relative with celiac disease, are associated with an increased risk of the condition. Both sets of guidance recommend that screening should be considered in these groups. NICE testing follows the same pathway as recommended for symptomatic patients. ESPGHAN guidance takes a different approach, recommending HLA-DQ2/DQ8 as the first line test since virtually all celiac cases have these haplotypes. TTG Ab titres are then used to determine if EMA and/or biopsy are required. In this algorithm all patients would need a biopsy to confirm a diagnosis. In contrast NICE state that HLA typing should not be used in initial diagnosis, but can be of use to gastroenterologists in certain cases. Cost of testing will be a factor, as will the availability of an adequate testing strategy for HLA typing.
Laboratory testing for celiac disease: things to consider
Serological tests
The IgA TTG Ab test is an integral part of both published guidelines. This test does not have an international reference preparation, therefore kits available from different manufacturers all perform in a slightly different manner. Monitoring performance of different TTG Ab assays via the UK National External Quality Assessment Service (NEQAS) external quality assurance scheme [4] provided evidence of the lack of consensus between assays from different manufacturers and between laboratories using the same assay. The same sample can generate a range of results when measured as ULNs, with the potential for different pathways being followed depending on the laboratory performing the test. This emphasizes that currently a generic statement about the level of positivity cannot easily be used across the board without local validation of outcomes.
Clinical audit plays an important role in determining whether published guidelines work when there is poor standardization of assays. Audit of your cohort using your assays should be performed. Data published about our experience of serological assay performance when following different testing strategies highlights that false positive IgA TTG Ab results, even at very high titres, can be problematic (Fig. 1) [5]. Other centres have also noted false positive IgA TTG Abs at high levels [6, 7]. Conversely, there are a number of reports that show following ESPGHAN guidance works well [2, 8, 9] and that avoiding biopsy could be reasonably justified in children with high IgA TTG Ab titres.
In all guidelines the EMA test by immunofluorescence is used as a secondary test dependent on the IgA TTG Ab result. Leading to the question about why this test is not used alone if it is being used as a confirmatory check for the TTG Ab test. The EMA test is considered by some to be more expensive, as the laboratory has to have the correct equipment and staff that are competent at performing the test and reading immunofluorescence slides [10] and internal quality control is sometimes more difficult for general laboratories. Alternatively, the IgA TTG Ab assay is an easily automated test that is readily available in non-specialist laboratories on a number of assay platforms. Testing for both TTG and EMA initially in all patients will increase costs with little added benefit [1, 3, 5, 8, 9, 11]. A decision has to be made about which screening test is the most appropriate, both in performance and practicality, for each laboratory. This decision should be made based upon the IgA TTG Ab assay used, so cannot be generalized between laboratories until harmonization of the test is established [5, 9, 11, 12].
Genetic testing
Using HLA-DQ2/DQ8 screening has flaws because of a large false positive population. Individuals with these HLA types are at greater risk of developing celiac disease [3, 8]. Approximately 30% of the healthy Caucasian population have HLA-DQ2 and do not go on to develop the disease [10]. Implementing HLA testing as an initial screening test in asymptomatic children can lead to further testing in some patients that do not have, or will not develop celiac disease. It is also much more expensive than serological tests.
Duodenal biopsy
Duodenal biopsy is considered to be the ‘gold standard’ for diagnosis. However, this is an invasive procedure that is not without risk of complications. Current guidelines explicitly agree that the number of biopsies carried out should be minimized by only performing them in patients that are determined to be at a high risk of having celiac disease following serological tests. There are mixed opinions on whether a diagnosis can be made without the need for biopsy [5, 7, 8, 9, 12]. Duodenal biopsy is the most costly procedure performed during diagnosis of celiac disease. If the numbers of biopsies are reduced, cost savings will be made as well as preventing unnecessary harm in some patients [11].
Best testing strategy?
This depends on your local set-up and audit of outcomes. Current guidelines provide a starting point for determining which tests should be done and when. However the difference in performance between TTG Ab assays has not been adequately recognized. Clinical audit and local validation can help laboratories to decide if it is appropriate to follow the recommended pathways, with the assays that they currently use [5, 7, 8, 9, 11, 12]. This is the only method that can provide a true reflection of the sensitivity, specificity and positive or negative predictive values of the tests locally. This provides an evidence base for justification of the test strategy being used.
Conclusions
The guidelines provide recommendations for the best testing approach but this is not mandatory. A different strategy, for example, using EMA as the first-line screen, could be employed if there is sufficient evidence that this would work better in your laboratory, for your cohort and is economically justified. You must know how your assays perform and assess this using in-house clinical audit and discuss with your local clinicians to provide the best service locally.
The ultimate aim is to provide an approach that will benefit the patient by being the fastest and most reliable method for diagnosis. This relies on selection of the strategy with the best positive and negative predictive values, to avoid biopsies that are not required. Cost is also a major factor in the current economic climate that must be considered when deciding upon the test strategy. It is not currently possible to diagnose celiac disease on the basis of one test result. Choosing the most appropriate strategy for your laboratory can reduce the number of unnecessary referrals and biopsies [11], thereby reducing cost to the healthcare system without an impact on patient care.
References
1. National Institute of Clinical Excellence (NICE) guideline 86. Celiac disease: recognition and assessment of celiac disease, 2009. http://tinyurl.com/q3wspfp
2. Mubarak A, Wolters VM, Gmelig-Meyling FH, Ten Kate FJ, Houwen RH. Tissue transglutaminase levels above 100 U/mL and celiac disease: a prospective study. World J Gastroenterol. 2012; 18(32): 4399–4403.
3. Husby S, Koletzko S, Korponay-Szabó IR, Mearin ML, Phillips A, Shamir R, Troncone R, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. European Society for Paediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr. 2012; 54: 136–160.
4. Egner W, Shrimpton A, Sargur R, Patel D, Swallow K. ESPGHAN guidance on celiac disease 2012: multiples of ULN for decision making do not harmonise assay performance across centres. J Pediatr Gastroenterol Nutr. 2012; 55: 733–735.
5. Swallow K, Wild G, Sargur R, Sanders DS, Aziz I, Hopper AD, Egner W. Quality not quantity for transglutaminase antibody 2: the performance of an endomysial and tissue transglutaminase test in screening celiac disease remains stable over time. Clin Exp Immunol; 2013; 171: 100–106.
6. Gidrewicz D, Lyon ME, Trevenen C, Butzner JD. How do the 2012 ESPGHAN celiac disease guidelines perform in a GI clinic. Gastroenterology 2013, 144(5)S1: S-14.
7. Bhardwaj M, Banoub H, Sumar N, Lawson M, Chong S. The impact of ESPGHAN guidelines on the investigations for celiac disease. Arch Dis Child. 2013; 98(Suppl 1): A92.
8. Klapp G, Masip E, Bolonio M, Donat E, Polo B, Ramos D, Ribes-Koninckx C. Celiac disease: the new proposed ESPGHAN diagnostic criteria do work well in a selected population. J Pediatr Gastroenterol Nutr. 2013; 56(3): 251–256.
9. Wolf J, Hasenclever D, Petroff D, Richter T, Uhlig HH, Laaβ MW, Hauer A, et al. Antibodies in the diagnosis of celiac disease: a biopsy controlled, international, multicentre study of 376 children with celiac disease and 695 controls. PLOS One 2014; 9(5): e97853 and Correction PLOS One 2014; 9(8): e105230.
10. Van Heel DA and West J. Recent advances in celiac disease. Gut 2006; 55(7): 1073–1046.
11. Hopper AD, Hadjivassiliou M, Hurlstone DP, Lobo AJ, McAlindon ME, Egner W, Wild G, Sanders DS. What is the role of serologic testing in celiac disease? A prospective, biopsy confirmed study with economic analysis. Clin Gastroenterol Hepatol. 2008; 6: 314–320.
12. Beltran L, Koenig M, Egner W, Howard M, Butt A, Austin MR, Patel D, et al. High titre circulating tissue transglutaminase-2 antibodies predict small bowel villous atrophy, but decision cut-off limits must be locally validated. Clin Expt Immunol. 2014; 176: 190–198.
The authors
Kirsty Swallow* BSc, MSc; Graeme Wild PhD; William Egner PhD, MD, FRCP, FRCPath; and Ravishankar Sargur MD, FRCP, FRCPath
Protein Reference Unit and Immunology Department, Northern General Hospital, Sheffield, UK.
*Corresponding author
E-mail: Kirsty.swallow@sth.nhs.uk
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, /in Featured Articles /by 3wmediaUltrasonic Rapid Tissue Processor: Histra-GT
, /in Featured Articles /by 3wmediaWhy is the current Ebola epidemic so difficult to control ?
, /in Featured Articles /by 3wmediaThe first recorded outbreaks of Ebola Virus Disease (EVD) occurred simultaneously in South Sudan (then The Sudan) and the Democratic Republic of Congo (DRC, then Zaire) nearly forty years ago. Although the causative agent and how it was transmitted were initially unknown to those with the task of containing these outbreaks, only 602 people became infected, 431 died and the outbreaks were controlled within three months. The latest WHO data record that the current epidemic, the first case of which occurred in Guinea in December 2013, has so far infected over four thousand people in five West African countries and caused over two thousand deaths, more than in all previous outbreaks put together. In a separate outbreak in the DRC there is a total of 24 suspected cases and 13 people have died so far. The obvious question is why, in spite of the initial ignorance about the disease in 1976, was the epidemic contained so promptly when all our current knowledge is now failing to stop the disease spreading exponentially?
Having established that the Ebola virus was spread via contact with patients’ body fluids, the teams investigating the 1976 outbreaks closed hospitals that were reusing their few needles, quarantined infected people and their contacts, and effectively disseminated information on simple barrier nursing of patients and safe burial practices. The 2014 outbreak, though, started in a much less isolated region where people regularly travel between countries using motor vehicles. Healthcare workers from outside were often distrusted, and local hospital staff had no previous experience of EVD. In addition Muslim law requires family members to wash their dead before burial. As the initial outbreak became an epidemic, control efforts were hampered by lack of suitable hospital facilities, basic equipment and staff, forcing patients to return home and infect others. And sadly some protective responses from the international community, such as closing airline routes, have greatly exacerbated the problem in West Africa by preventing medical experts and supplies, even food, from reaching affected areas.
The WHO now predicts that it will take 600 million dollars and at least six to nine months to control the epidemic. The European Union, United States and the Gates Foundation have all committed funds for vaccine development and treatment, but according to Medecins Sans Frontieres the international response is still “lethally inadequate”. The imperative need now is for experts in biohazard containment.
Diagnosis of celiac disease
, /in Featured Articles /by 3wmediaPublished guidelines provide different approaches for laboratories to follow when investigating celiac disease. The aim is to minimize time to diagnosis and reduce unnecessary investigations. Variability between IgA tissue transglutaminase tests must be considered when implementing the local diagnostic strategy. Determining best practice depends on the assays used, expertise available, cost and local clinical audit of outcomes.
by K. Swallow, Dr G. Wild, Dr W. Egner and Dr R. Sargur
Background
Celiac disease is a common autoimmune condition affecting approximately 1 : 100 in the UK [1, 2]. Early diagnosis is key to appropriate management. Symptoms are eliminated by following a gluten-free diet after a confirmed diagnosis. Following best practice guidance can reduce repeated visits to GP practices and outpatient departments, and numerous requests for laboratory tests.
In recent years, guidelines for the diagnosis and management of celiac disease have been published by the National Institute of Health and Care Excellence (NICE) [1] and the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) [3]. The guidelines provide algorithms for laboratory testing strategies and recommendations about when a duodenal biopsy should be performed in both symptomatic and asymptomatic patients. There is a perceived need to avoid invasive procedures in children by using optimal in vitro testing.
Guidelines: recommendations for diagnostic testing in symptomatic patients
There are some similarities and several notable differences between the guidelines given for symptomatic patients. Both NICE and ESPGHAN guidance recommend IgA tissue transglutaminase antibodies (TTG Ab) as the first-line screening test. ESPGHAN also recommend testing all patients for IgA deficiency. NICE only recommend checking for IgA deficiency if the IgA TTG Ab result is negative. After this initial test the recommended pathways by the two groups differ; however, both base the strategies on the level of positivity of the IgA TTG Ab result.
NICE pathway after IgA TTG Ab testing
NICE recommend that if the TTG Ab test is positive the patient should be referred for confirmatory biopsy. If it is ‘equivocal’, IgA endomysial antibodies (EMA) should be tested in order to determine if the TTG Ab result is potentially false positive or if the patient should be referred to a gastroenterologist. A patient with negative IgA TTG Ab should be checked for IgA deficiency and IgG TTG/EMA performed if they are found to be deficient. The guidelines do not provide information regarding the definition of the ‘equivocal’ range for TTG Ab assays.
ESPGHAN pathway after IgA TTG Ab testing
For symptomatic patients the diagnosis can be made with or without the need for duodenal biopsy, dependent on the serological test results. As with NICE, all patients with positive IgA TTG Ab should be referred to a gastroenterologist. They also suggest using IgG TTG/EMA if the patient is IgA deficient.
ESPGHAN stratify the level of positivity based upon levels above the normal range or ‘upper limit of normal’ (ULN). If the TTG Ab result is >10× ULN the decision to go to biopsy is made after further testing EMA and HLA-DQ2/DQ8. If all tests (TTG Ab, EMA and HLA-DQ2/DQ8) are positive, a patient may be diagnosed without the need for duodenal biopsy. Biopsy is recommended if EMA and/or HLA typing is negative, or if TTG is positive but <10× ULN. Guidance for testing in asymptomatic patients (screening)
Several conditions, including IgA deficiency, autoimmune disease (type I diabetes, hypothyroidism, pernicious anemia), Down syndrome, or a having a first degree relative with celiac disease, are associated with an increased risk of the condition. Both sets of guidance recommend that screening should be considered in these groups. NICE testing follows the same pathway as recommended for symptomatic patients. ESPGHAN guidance takes a different approach, recommending HLA-DQ2/DQ8 as the first line test since virtually all celiac cases have these haplotypes. TTG Ab titres are then used to determine if EMA and/or biopsy are required. In this algorithm all patients would need a biopsy to confirm a diagnosis. In contrast NICE state that HLA typing should not be used in initial diagnosis, but can be of use to gastroenterologists in certain cases. Cost of testing will be a factor, as will the availability of an adequate testing strategy for HLA typing.
Laboratory testing for celiac disease: things to consider
Serological tests
The IgA TTG Ab test is an integral part of both published guidelines. This test does not have an international reference preparation, therefore kits available from different manufacturers all perform in a slightly different manner. Monitoring performance of different TTG Ab assays via the UK National External Quality Assessment Service (NEQAS) external quality assurance scheme [4] provided evidence of the lack of consensus between assays from different manufacturers and between laboratories using the same assay. The same sample can generate a range of results when measured as ULNs, with the potential for different pathways being followed depending on the laboratory performing the test. This emphasizes that currently a generic statement about the level of positivity cannot easily be used across the board without local validation of outcomes.
Clinical audit plays an important role in determining whether published guidelines work when there is poor standardization of assays. Audit of your cohort using your assays should be performed. Data published about our experience of serological assay performance when following different testing strategies highlights that false positive IgA TTG Ab results, even at very high titres, can be problematic (Fig. 1) [5]. Other centres have also noted false positive IgA TTG Abs at high levels [6, 7]. Conversely, there are a number of reports that show following ESPGHAN guidance works well [2, 8, 9] and that avoiding biopsy could be reasonably justified in children with high IgA TTG Ab titres.
In all guidelines the EMA test by immunofluorescence is used as a secondary test dependent on the IgA TTG Ab result. Leading to the question about why this test is not used alone if it is being used as a confirmatory check for the TTG Ab test. The EMA test is considered by some to be more expensive, as the laboratory has to have the correct equipment and staff that are competent at performing the test and reading immunofluorescence slides [10] and internal quality control is sometimes more difficult for general laboratories. Alternatively, the IgA TTG Ab assay is an easily automated test that is readily available in non-specialist laboratories on a number of assay platforms. Testing for both TTG and EMA initially in all patients will increase costs with little added benefit [1, 3, 5, 8, 9, 11]. A decision has to be made about which screening test is the most appropriate, both in performance and practicality, for each laboratory. This decision should be made based upon the IgA TTG Ab assay used, so cannot be generalized between laboratories until harmonization of the test is established [5, 9, 11, 12].
Genetic testing
Using HLA-DQ2/DQ8 screening has flaws because of a large false positive population. Individuals with these HLA types are at greater risk of developing celiac disease [3, 8]. Approximately 30% of the healthy Caucasian population have HLA-DQ2 and do not go on to develop the disease [10]. Implementing HLA testing as an initial screening test in asymptomatic children can lead to further testing in some patients that do not have, or will not develop celiac disease. It is also much more expensive than serological tests.
Duodenal biopsy
Duodenal biopsy is considered to be the ‘gold standard’ for diagnosis. However, this is an invasive procedure that is not without risk of complications. Current guidelines explicitly agree that the number of biopsies carried out should be minimized by only performing them in patients that are determined to be at a high risk of having celiac disease following serological tests. There are mixed opinions on whether a diagnosis can be made without the need for biopsy [5, 7, 8, 9, 12]. Duodenal biopsy is the most costly procedure performed during diagnosis of celiac disease. If the numbers of biopsies are reduced, cost savings will be made as well as preventing unnecessary harm in some patients [11].
Best testing strategy?
This depends on your local set-up and audit of outcomes. Current guidelines provide a starting point for determining which tests should be done and when. However the difference in performance between TTG Ab assays has not been adequately recognized. Clinical audit and local validation can help laboratories to decide if it is appropriate to follow the recommended pathways, with the assays that they currently use [5, 7, 8, 9, 11, 12]. This is the only method that can provide a true reflection of the sensitivity, specificity and positive or negative predictive values of the tests locally. This provides an evidence base for justification of the test strategy being used.
Conclusions
The guidelines provide recommendations for the best testing approach but this is not mandatory. A different strategy, for example, using EMA as the first-line screen, could be employed if there is sufficient evidence that this would work better in your laboratory, for your cohort and is economically justified. You must know how your assays perform and assess this using in-house clinical audit and discuss with your local clinicians to provide the best service locally.
The ultimate aim is to provide an approach that will benefit the patient by being the fastest and most reliable method for diagnosis. This relies on selection of the strategy with the best positive and negative predictive values, to avoid biopsies that are not required. Cost is also a major factor in the current economic climate that must be considered when deciding upon the test strategy. It is not currently possible to diagnose celiac disease on the basis of one test result. Choosing the most appropriate strategy for your laboratory can reduce the number of unnecessary referrals and biopsies [11], thereby reducing cost to the healthcare system without an impact on patient care.
References
1. National Institute of Clinical Excellence (NICE) guideline 86. Celiac disease: recognition and assessment of celiac disease, 2009. http://tinyurl.com/q3wspfp
2. Mubarak A, Wolters VM, Gmelig-Meyling FH, Ten Kate FJ, Houwen RH. Tissue transglutaminase levels above 100 U/mL and celiac disease: a prospective study. World J Gastroenterol. 2012; 18(32): 4399–4403.
3. Husby S, Koletzko S, Korponay-Szabó IR, Mearin ML, Phillips A, Shamir R, Troncone R, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. European Society for Paediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr. 2012; 54: 136–160.
4. Egner W, Shrimpton A, Sargur R, Patel D, Swallow K. ESPGHAN guidance on celiac disease 2012: multiples of ULN for decision making do not harmonise assay performance across centres. J Pediatr Gastroenterol Nutr. 2012; 55: 733–735.
5. Swallow K, Wild G, Sargur R, Sanders DS, Aziz I, Hopper AD, Egner W. Quality not quantity for transglutaminase antibody 2: the performance of an endomysial and tissue transglutaminase test in screening celiac disease remains stable over time. Clin Exp Immunol; 2013; 171: 100–106.
6. Gidrewicz D, Lyon ME, Trevenen C, Butzner JD. How do the 2012 ESPGHAN celiac disease guidelines perform in a GI clinic. Gastroenterology 2013, 144(5)S1: S-14.
7. Bhardwaj M, Banoub H, Sumar N, Lawson M, Chong S. The impact of ESPGHAN guidelines on the investigations for celiac disease. Arch Dis Child. 2013; 98(Suppl 1): A92.
8. Klapp G, Masip E, Bolonio M, Donat E, Polo B, Ramos D, Ribes-Koninckx C. Celiac disease: the new proposed ESPGHAN diagnostic criteria do work well in a selected population. J Pediatr Gastroenterol Nutr. 2013; 56(3): 251–256.
9. Wolf J, Hasenclever D, Petroff D, Richter T, Uhlig HH, Laaβ MW, Hauer A, et al. Antibodies in the diagnosis of celiac disease: a biopsy controlled, international, multicentre study of 376 children with celiac disease and 695 controls. PLOS One 2014; 9(5): e97853 and Correction PLOS One 2014; 9(8): e105230.
10. Van Heel DA and West J. Recent advances in celiac disease. Gut 2006; 55(7): 1073–1046.
11. Hopper AD, Hadjivassiliou M, Hurlstone DP, Lobo AJ, McAlindon ME, Egner W, Wild G, Sanders DS. What is the role of serologic testing in celiac disease? A prospective, biopsy confirmed study with economic analysis. Clin Gastroenterol Hepatol. 2008; 6: 314–320.
12. Beltran L, Koenig M, Egner W, Howard M, Butt A, Austin MR, Patel D, et al. High titre circulating tissue transglutaminase-2 antibodies predict small bowel villous atrophy, but decision cut-off limits must be locally validated. Clin Expt Immunol. 2014; 176: 190–198.
The authors
Kirsty Swallow* BSc, MSc; Graeme Wild PhD; William Egner PhD, MD, FRCP, FRCPath; and Ravishankar Sargur MD, FRCP, FRCPath
Protein Reference Unit and Immunology Department, Northern General Hospital, Sheffield, UK.
*Corresponding author
E-mail: Kirsty.swallow@sth.nhs.uk
Call for more physician awareness as prevalence of celiac disease leaps
, /in Featured Articles /by 3wmediaAlthough 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.
Blood-based tests for colorectal cancer screening
, /in Featured Articles /by 3wmediaWorldwide, 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
Tumour markers for the diagnosis of mucinous ovarian cancer
, /in Featured Articles /by 3wmediaThe 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
FUS & Microscopy Comparison
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