Most of the colorectal cancer (CRC) screening programmes are based on fecal occult blood tests (FOBT). However, there is little information on the utility of FOBT in familial-risk CRC screening. A recently published study has evaluated the diagnostic accuracy of fecal immunochemical tests (FIT) for CRC and advanced colonic neoplasia detection in this population.
by Dr Inés Castro and Dr Joaquín Cubiella
Relevance of colorectal cancer screening
Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer deaths in developed countries [1]. Several factors have been related to the risk of developing CRC. Age, gender and CRC familial history, especially if there are first-degree relatives (FDR) with CRC, are the strongest associated risk factors [2]. Furthermore, CRC risk is directly related to the number of FDRs and inversely related to the age of youngest FDR [3, 4].
Evidence from several studies has shown that CRC screening is effective for CRC prevention in the average-risk population (asymptomatic individuals between 50 and 69 years) [2]. Indeed, both flexible sigmoidoscopy and fecal occult blood tests (FOBT) have been shown to reduce CRC-specific mortality and incidence in randomized controlled trials [2, 5, 6]. This reduction is based on CRC early detection and prevention through adenoma detection and endoscopic resection [2]. Accordingly, these two strategies, along with colonoscopy, have been universally accepted and recommended for CRC screening [2]. In Europe, annual or biennial FOBT is the most widespread CRC screening strategy.
Approximately 95% of CRCs develop on advanced adenomatous polyps (larger than 10 mm, with high-grade dysplasia or villous architecture). These lesions, and early CRC, are characterized by intermittent microscopic blood loss in the stool that can be detected with FOBTs before they are clinically apparent.
Advantages and disadvantages of the different FOBTs
FOBTs detect through blood or blood products (such as globin) in feces with different methods. Mainly, there are two types of FOBTs: chemical (cFOBT) or immunological (FIT).
Chemical fecal occult blood tests
cFOBTs are simple, qualitative tests that use different indicators such as guaiac resin (Hemoccults, Hemoccult II®, SmithKline Diagnostics, Sunnyvale, California, USA), orthotolidine (Hematest®, MilesLaboratories, Elkhart, Indiana, USA) or benzidine (Hemofec®, Med-Kjemi AS, Asker, Norway). By an oxidative reaction and in the presence of a developer solution, hemoglobin seudoperoxidase activity produces a colour change in the paper impregnated with guaiac resin, orthotolidine or benzidine.
However these tests have multiple drawbacks: first, they are not specific to human hemoglobin. Oxygenation reactions may occur with foods with peroxidase activity such as vegetables or uncooked red meat, so it is recommended to withdraw them from the diet for 3 days before sample collection to reduce false positive results. In addition, hemoglobin from the upper gastrointestinal digestive tract is also detected. So, gastrolesive drugs (NSAIDs or aspirin) should be interrupted 7 days before testing. Besides, two stool samples on three consecutive bowel movements are required, limiting adherence to screening programmes [7]. Furthermore, as it is a qualitative test, its result is subjected to interpretation. Finally, their sensitivity for CRC and advanced adenoma detection is low. This is associated with an interval CRC rate ranging between 30 and 50%.
Fecal immunochemical tests
FITs are based on the reaction of monoclonal or polyclonal antibodies specific for human hemoglobin, albumin or other fecal blood components. Thus, they require no dietary or pharmacological restriction, as long as they do not react with blood from upper digestive tract or with any food component. The greatest advantage is determined by its ability to detect and quantify fecal hemoglobin concentrations 7 to 15 times lower than those detected by chemical tests, significantly improving CRC and advanced adenoma detection sensitivity. Besides, FITs allows a reliable and accurate automated analysis, avoiding subjective interpretation. Up to 50 samples an hour can be processed, making this test ideal for population-based screening [7].
The most used and better evaluated methods nowadays for CRC screening are those based on latex agglutination (OC-Sensor®, OC-Micro®, Eiken Chemical Co., Ltd, Japan; FOB-Gold® Sentinel Diagnostics®, Milan) or magnetized gelatin particles (Magstream1000®, Fujirebio Inc.,Tokyo, Japan).
Effectiveness of FOBT-based CRC screening in the the average-risk population
As previously commented, multiple studies have demonstrated that CRC screening with cFOBT in the average-risk population significantly reduces CRC mortality [8]. So far, there are no data available on the effect of FIT in CRC mortality or incidence. However, several diagnostic test studies have compared cFOBT and FIT for CRC and advanced adenomas detection. These studies have shown that FIT is more sensitive and specific for CRC and advanced adenomas detection and is a cost effective screening test [9]. On this basis, CRC screening programmes are based mainly on FIT.
Efficacy of FOBT CRC screening in the familial-risk population
Evidence on the best screening strategy in this population is limited and its quality is low [10, 11]. At present, it is unclear which is the best screening strategy in individuals with a FDR with CRC [12]. Clinical practice guidelines recommend more aggressive screening than in the average-risk population, based on studies that have shown that endoscopic screening reduces CRC incidence and mortality in individuals with a FDR with CRC [2, 6]. Screening is recommended from 40 years or 10 years before the youngest proband. However, this approach has an empirical basis and no prospective controlled study has compared different screening strategies in this population. Furthermore, colonoscopy is an invasive technique that requires sedation, is associated with rare but serious complications (perforation or hemorrhage), requires a substantial financial investment in equipment and expertise and has a limited adherence.
Data on the utility of FOBT for CRC screening in this population are scarce. Our group has recently evaluated FIT diagnostic accuracy for advanced colorectal neoplasia (CRC or advanced adenoma) in asymptomatic individuals with at least one first-degree relative with CRC submitted to a colonoscopy [13]. Patients with an advanced neoplasia had a fecal hemoglobin concentration statistically higher than those without an advanced neoplasia. Fecal hemoglobin concentration was significantly higher among individuals with CRC or advanced adenomas when compared with subjects with non-advanced adenomas or without neoplasia. In contrast, no significant differences were found between individuals with CRC and advanced adenomas as shown in Figure 1. Diagnostic accuracy for CRC detection was high, reaching 100% at a fecal hemoglobin threshold below 115 ng/ml. On the other hand, depending on the number of determinations and the positivity threshold cut-off used sensitivity for advanced neoplasia detection ranged between 34.38 and 50% and specificity between 92.66 and 98.72%. In fact, analysing two samples did not improve diagnostic accuracy and, instead, increased the number of colonoscopies needed to detect a CRC or an advanced neoplasia and the costs per lesion detected [Fig. 2]. These data suggest that FIT is an adequate CRC screening test in this population. However, FIT as a screening method must be evaluated in prospective controlled studies designed to determine CRC mortality reduction in the long term.
Conclusion
The reduction in mortality in FOBT-based CRC screening programmes and the improved sensitivity of immunochemical tests for CRC and advanced adenomas detection makes FIT CRC screening programmes a cost effective strategy. Preliminary data show that FIT is equally effective in the familial-risk population. Thus, FIT could be an adequate CRC screening technique in this population.
References
1. Ferlay J, Shin H-R, Bray F, Forman D, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127(12): 2893–2917.
2. Levin B, Lieberman DA, McFarland B, Andrews KS, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008; 134: 1570–1595.
3. Baglietto L, Jenkins MA, Severi G, Giles GG, et al. Measures of familial aggregation depend on definition of family history: meta-analysis for colorectal cancer. J Clin Epidemiol. 2006; 59(2): 114–124.
4. Butterworth AS, Higgins JPT, Pharoah P. Relative and absolute risk of colorectal cancer for individuals with a family history: a meta-analysis. Eur J Cancer 2006; 42(2): 216–227.
5. Mandel JS, Church TR, Bond JH, Ederer F, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. NEJM 2000; 343(22): 1603–1607.
6. Atkin WS, Edwards R, Kralj-Hans I, Wooldrage K, et al. Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 2010; 375(9726): 1624–1633.
7. Quintero E. [Chemical or immunological tests for the detection of fecal occult blood in colorectal cancer screening?]. Gastroenterología y hepatología 2009; 32(8): 565–576.
8. Hewitson P, Glasziou P, Watson E, Towler B, Irwig L. Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am J Gastroenterol. 2008; 103(6): 1541–1549.
9. Guittet L, Bouvier V, Mariotte N, Vallee JP, et al. Comparison of a guaiac based and an immunochemical faecal occult blood test in screening for colorectal cancer in a general average risk population. Gut 2007; 56(2): 210–214.
10. Dove-Edwin I, Sasieni P, Adams J, Thomas HJW. Prevention of colorectal cancer by colonoscopic surveillance in individuals with a family history of colorectal cancer: 16 year, prospective, follow-up study. BMJ 2005; 331(7524): 1047.
11. Gimeno-García AZ, Quintero E, Nicolás-Pérez D, Hernández-Guerra M, et al. Screening for familial colorectal cancer with a sensitive immunochemical fecal occult blood test: a pilot study. Eur J Gastroenterol Hepatol. 2009; 21(9): 1062–1067.
12. Winawer S, Fletcher R, Rex D, Bond J, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003; 124(2): 544–560.
13. Castro I, Cubiella J, Rivera C, González-Mao C, et al. Fecal inmunochemical test accuracy for colorectal cancer and advanced neoplasia detection in familial risk colorectal cancer screening. Int J Cancer 2013; doi: 10.1002/ijc.28353 (Epub ahead of print).
The authors
Inés Castro MD and Joaquín Cubiella* PhD
Department of Gastroenterology, Complexo Hospitalario Universitario de Ourense, Ourense, Spain
*Corresponding author
E-mail: Joaquin.cubiella.fernandez@sergas.es
Sugar: a bad and good drug
, /in Featured Articles /by 3wmediaRecently a senior Dutch health official claimed that sugar is ‘the most dangerous drug of the times’ and called for cigarette packet-type warnings stating that ‘sugar is addictive and bad for health’ to be mandatory on the labels of products such as soft drinks and sweets.
A plethora of studies has examined the effects of overconsumption of sugar. Many are based on consumers reporting the amount of sugar in their diet; under-reporting is very common in such surveys, though a recently discovered biomarker based on the ratio of Carbon 12 and 13 can now measure long-term sugar intake from a single blood or hair sample. Other studies don’t distinguish between free monosaccharides and disaccharides added to food products and those occurring naturally in food. While recognising the limitations of many studies, most of us would accept that overconsumption of sugar is linked to obesity, dental caries, macular degeneration and Alzheimer’s disease in older age, cardiovascular disease and diabetes. And hypoglycemia (defined as a blood glucose level of < 2.5mmol/L), a frequent problem in diabetes patients receiving treatment, can also occur in non-diabetic subjects as a result of a diet that is too high in refined sugars and too low in complex carbohydrates. And the treatment for hypoglycemia is the sugar dextrose (= glucose), given orally or by intravenous drip depending on how low the glucose level is and how alert the patient.
Hypoglycemia is unfortunately becoming more common in neonates. Around one in three suffer from the condition in the West, reflecting the increase in gestational and maternal diabetes as well as the rising number of pre-term births. Careful management of the newborn is necessary to avoid seizures and serious brain injury, and this normally involves extra feeding with formula (in addition to breast milk, which often interrupts normal breastfeeding) and repeated blood glucose tests involving heel pricks. If a seriously low glucose level persists, babies are admitted to intensive care and intravenous dextrose is administered. However the good news is that a New Zealand study has just been published in ‘The Lancet‘ involving 514 neonates considered at high risk of hypoglycaemia. The babies diagnosed with the condition were randomly assigned to one of two groups. One hundred and eighteen were treated with six applications of 40% dextrose gel over two days, applied to the inside of the cheek, and 119 were treated with placebo gel. The blood glucose levels of the former group stabilised quicker, fewer babies needed extra formula feeds and fewer were admitted to intensive care. Sugar may be a ‘dangerous drug’ but it can also be invaluable!
Fecal immunochemical testing in colorectal cancer screening
, /in Featured Articles /by 3wmediaMost of the colorectal cancer (CRC) screening programmes are based on fecal occult blood tests (FOBT). However, there is little information on the utility of FOBT in familial-risk CRC screening. A recently published study has evaluated the diagnostic accuracy of fecal immunochemical tests (FIT) for CRC and advanced colonic neoplasia detection in this population.
by Dr Inés Castro and Dr Joaquín Cubiella
Relevance of colorectal cancer screening
Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer deaths in developed countries [1]. Several factors have been related to the risk of developing CRC. Age, gender and CRC familial history, especially if there are first-degree relatives (FDR) with CRC, are the strongest associated risk factors [2]. Furthermore, CRC risk is directly related to the number of FDRs and inversely related to the age of youngest FDR [3, 4].
Evidence from several studies has shown that CRC screening is effective for CRC prevention in the average-risk population (asymptomatic individuals between 50 and 69 years) [2]. Indeed, both flexible sigmoidoscopy and fecal occult blood tests (FOBT) have been shown to reduce CRC-specific mortality and incidence in randomized controlled trials [2, 5, 6]. This reduction is based on CRC early detection and prevention through adenoma detection and endoscopic resection [2]. Accordingly, these two strategies, along with colonoscopy, have been universally accepted and recommended for CRC screening [2]. In Europe, annual or biennial FOBT is the most widespread CRC screening strategy.
Approximately 95% of CRCs develop on advanced adenomatous polyps (larger than 10 mm, with high-grade dysplasia or villous architecture). These lesions, and early CRC, are characterized by intermittent microscopic blood loss in the stool that can be detected with FOBTs before they are clinically apparent.
Advantages and disadvantages of the different FOBTs
FOBTs detect through blood or blood products (such as globin) in feces with different methods. Mainly, there are two types of FOBTs: chemical (cFOBT) or immunological (FIT).
Chemical fecal occult blood tests
cFOBTs are simple, qualitative tests that use different indicators such as guaiac resin (Hemoccults, Hemoccult II®, SmithKline Diagnostics, Sunnyvale, California, USA), orthotolidine (Hematest®, MilesLaboratories, Elkhart, Indiana, USA) or benzidine (Hemofec®, Med-Kjemi AS, Asker, Norway). By an oxidative reaction and in the presence of a developer solution, hemoglobin seudoperoxidase activity produces a colour change in the paper impregnated with guaiac resin, orthotolidine or benzidine.
However these tests have multiple drawbacks: first, they are not specific to human hemoglobin. Oxygenation reactions may occur with foods with peroxidase activity such as vegetables or uncooked red meat, so it is recommended to withdraw them from the diet for 3 days before sample collection to reduce false positive results. In addition, hemoglobin from the upper gastrointestinal digestive tract is also detected. So, gastrolesive drugs (NSAIDs or aspirin) should be interrupted 7 days before testing. Besides, two stool samples on three consecutive bowel movements are required, limiting adherence to screening programmes [7]. Furthermore, as it is a qualitative test, its result is subjected to interpretation. Finally, their sensitivity for CRC and advanced adenoma detection is low. This is associated with an interval CRC rate ranging between 30 and 50%.
Fecal immunochemical tests
FITs are based on the reaction of monoclonal or polyclonal antibodies specific for human hemoglobin, albumin or other fecal blood components. Thus, they require no dietary or pharmacological restriction, as long as they do not react with blood from upper digestive tract or with any food component. The greatest advantage is determined by its ability to detect and quantify fecal hemoglobin concentrations 7 to 15 times lower than those detected by chemical tests, significantly improving CRC and advanced adenoma detection sensitivity. Besides, FITs allows a reliable and accurate automated analysis, avoiding subjective interpretation. Up to 50 samples an hour can be processed, making this test ideal for population-based screening [7].
The most used and better evaluated methods nowadays for CRC screening are those based on latex agglutination (OC-Sensor®, OC-Micro®, Eiken Chemical Co., Ltd, Japan; FOB-Gold® Sentinel Diagnostics®, Milan) or magnetized gelatin particles (Magstream1000®, Fujirebio Inc.,Tokyo, Japan).
Effectiveness of FOBT-based CRC screening in the the average-risk population
As previously commented, multiple studies have demonstrated that CRC screening with cFOBT in the average-risk population significantly reduces CRC mortality [8]. So far, there are no data available on the effect of FIT in CRC mortality or incidence. However, several diagnostic test studies have compared cFOBT and FIT for CRC and advanced adenomas detection. These studies have shown that FIT is more sensitive and specific for CRC and advanced adenomas detection and is a cost effective screening test [9]. On this basis, CRC screening programmes are based mainly on FIT.
Efficacy of FOBT CRC screening in the familial-risk population
Evidence on the best screening strategy in this population is limited and its quality is low [10, 11]. At present, it is unclear which is the best screening strategy in individuals with a FDR with CRC [12]. Clinical practice guidelines recommend more aggressive screening than in the average-risk population, based on studies that have shown that endoscopic screening reduces CRC incidence and mortality in individuals with a FDR with CRC [2, 6]. Screening is recommended from 40 years or 10 years before the youngest proband. However, this approach has an empirical basis and no prospective controlled study has compared different screening strategies in this population. Furthermore, colonoscopy is an invasive technique that requires sedation, is associated with rare but serious complications (perforation or hemorrhage), requires a substantial financial investment in equipment and expertise and has a limited adherence.
Data on the utility of FOBT for CRC screening in this population are scarce. Our group has recently evaluated FIT diagnostic accuracy for advanced colorectal neoplasia (CRC or advanced adenoma) in asymptomatic individuals with at least one first-degree relative with CRC submitted to a colonoscopy [13]. Patients with an advanced neoplasia had a fecal hemoglobin concentration statistically higher than those without an advanced neoplasia. Fecal hemoglobin concentration was significantly higher among individuals with CRC or advanced adenomas when compared with subjects with non-advanced adenomas or without neoplasia. In contrast, no significant differences were found between individuals with CRC and advanced adenomas as shown in Figure 1. Diagnostic accuracy for CRC detection was high, reaching 100% at a fecal hemoglobin threshold below 115 ng/ml. On the other hand, depending on the number of determinations and the positivity threshold cut-off used sensitivity for advanced neoplasia detection ranged between 34.38 and 50% and specificity between 92.66 and 98.72%. In fact, analysing two samples did not improve diagnostic accuracy and, instead, increased the number of colonoscopies needed to detect a CRC or an advanced neoplasia and the costs per lesion detected [Fig. 2]. These data suggest that FIT is an adequate CRC screening test in this population. However, FIT as a screening method must be evaluated in prospective controlled studies designed to determine CRC mortality reduction in the long term.
Conclusion
The reduction in mortality in FOBT-based CRC screening programmes and the improved sensitivity of immunochemical tests for CRC and advanced adenomas detection makes FIT CRC screening programmes a cost effective strategy. Preliminary data show that FIT is equally effective in the familial-risk population. Thus, FIT could be an adequate CRC screening technique in this population.
References
1. Ferlay J, Shin H-R, Bray F, Forman D, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127(12): 2893–2917.
2. Levin B, Lieberman DA, McFarland B, Andrews KS, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008; 134: 1570–1595.
3. Baglietto L, Jenkins MA, Severi G, Giles GG, et al. Measures of familial aggregation depend on definition of family history: meta-analysis for colorectal cancer. J Clin Epidemiol. 2006; 59(2): 114–124.
4. Butterworth AS, Higgins JPT, Pharoah P. Relative and absolute risk of colorectal cancer for individuals with a family history: a meta-analysis. Eur J Cancer 2006; 42(2): 216–227.
5. Mandel JS, Church TR, Bond JH, Ederer F, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. NEJM 2000; 343(22): 1603–1607.
6. Atkin WS, Edwards R, Kralj-Hans I, Wooldrage K, et al. Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 2010; 375(9726): 1624–1633.
7. Quintero E. [Chemical or immunological tests for the detection of fecal occult blood in colorectal cancer screening?]. Gastroenterología y hepatología 2009; 32(8): 565–576.
8. Hewitson P, Glasziou P, Watson E, Towler B, Irwig L. Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am J Gastroenterol. 2008; 103(6): 1541–1549.
9. Guittet L, Bouvier V, Mariotte N, Vallee JP, et al. Comparison of a guaiac based and an immunochemical faecal occult blood test in screening for colorectal cancer in a general average risk population. Gut 2007; 56(2): 210–214.
10. Dove-Edwin I, Sasieni P, Adams J, Thomas HJW. Prevention of colorectal cancer by colonoscopic surveillance in individuals with a family history of colorectal cancer: 16 year, prospective, follow-up study. BMJ 2005; 331(7524): 1047.
11. Gimeno-García AZ, Quintero E, Nicolás-Pérez D, Hernández-Guerra M, et al. Screening for familial colorectal cancer with a sensitive immunochemical fecal occult blood test: a pilot study. Eur J Gastroenterol Hepatol. 2009; 21(9): 1062–1067.
12. Winawer S, Fletcher R, Rex D, Bond J, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003; 124(2): 544–560.
13. Castro I, Cubiella J, Rivera C, González-Mao C, et al. Fecal inmunochemical test accuracy for colorectal cancer and advanced neoplasia detection in familial risk colorectal cancer screening. Int J Cancer 2013; doi: 10.1002/ijc.28353 (Epub ahead of print).
The authors
Inés Castro MD and Joaquín Cubiella* PhD
Department of Gastroenterology, Complexo Hospitalario Universitario de Ourense, Ourense, Spain
*Corresponding author
E-mail: Joaquin.cubiella.fernandez@sergas.es
Progress in biomarkers of colorectal cancer
, /in Featured Articles /by 3wmediaColorectal cancer is one of the commonest types of cancer and contributes significantly to cancer-related mortality. Recent research has focused on the identification, development, validation of sensitive and specific biomarkers to improve early diagnosis, to assess disease outcome, accurately predict response to therapy or monitor disease status after treatment.
by Dr Caroline Coghlin and Professor Graeme I Murray
The requirement for biomarkers in colorectal cancer
Colorectal cancer (CRC) represents a heterogeneous disease with variable clinical presentations and equally variable outcomes observed between individual patients. At the molecular level, although several well-described pathways are known to exist in CRC tumorigenesis the reality is likely to be much more complex. In addition to genetic alterations, numerous interactions exist between multiple abnormal signalling networks and between the tumour cells and their microenvironment. Surgery is the mainstay of treatment in primary CRC but options for adjuvant or neoadjuvant chemotherapy are advancing. Given these choices, and the potential toxicity associated with some agents used, the need for validated and reliable biomarkers to aid in CRC diagnosis, management and predictive and prognostic stratification is growing [1].
Diagnostic, prognostic and predictive biomarkers
The detection of protein biomarkers using immunohistochemistry in fixed tissue is a reliable technique widely used in the histopathology laboratory setting. Accurate diagnosis of primary CRC is often clear from its site of origin detected colonoscopically or with imaging modalities and from the morphological features revealed on histology. However, diagnosis of CRC in secondary sites can be more difficult. CRC cells frequently express CK20 and CDX2 (an intestinal-specific transcription factor) and they are generally negative for CK7. Therefore, a targeted panel of immunohistochemical markers can aid in the diagnosis of metastatic CRC and so guide appropriate treatment [2].
Accurate prognosis in CRC currently relies on pathological and clinical staging including use of the tumour, node, metastasis (TNM) system. In the past, assay of plasma biomarkers such as carcinoembryonic antigen (CEA) and CA19.9 to aid in prognostic stratification was advocated but, while these markers may still have a role in disease monitoring after surgery, as diagnostic and prognostic tools such biomarkers lack sensitivity and specificity. A large body of research has focused on comparative proteomic analysis of primary site CRC, normal control tissue and metastatic tissue in attempts to identify clinically useful prognostic and predictive CRC biomarkers [3, 4]. Candidate markers have included the mismatch repair proteins, especially MLH1 and MSH2 (which may be linked with a better prognosis in early CRC but also with a poor response to some chemotherapeutic agents such, as 5-fluorouracil) along with many other molecules involved in diverse cellular processes such as matrix degradation, oxidative metabolism or protein folding [3, 4]. There is, however, often a deficiency in the consistent follow-up studies necessary for the validation of such potential biomarkers. This aspect of study remains crucial to enable the robust and reliable clinical application of biomarkers in CRC [Fig. 1].
Plasma/serum biomarkers in CRC
The goal of screening programmes for CRC is to decrease mortality and reduce morbidity associated with the disease by identifying cancers at an early stage when intervention is more likely to succeed. Fecal occult blood or DNA testing and targeted colonoscopy are methods commonly employed to screen for early lesions. Fecal testing has the disadvantage of poor patient acceptability and it also lacks sensitivity. Colonoscopy, while accurate, is invasive and associated with a small but significant risk to the patient. With this in mind, the ultimate aim of many recent studies has been to identify robust plasma-based biomarkers which represent an acceptable form of patient testing. Such biomarkers should ideally be able to identify early invasive or perhaps even pre-invasive CRC in a sensitive and specific manner.
Circulating tumour cells (CTCs) are malignant cells originating from a primary tumour or its metastases which have gained access to the bloodstream. Several methods have recently been developed to identify these cells which may be associated with a poorer prognosis or an increased risk of relapse after surgery. In addition, molecular genetic characterisation of CTCs has potential implications for targeted therapy and therefore predictive value. CTCs in CRC patients are more abundant in central blood compartments such as the mesenteric vessels but peripheral blood can also be a source providing enrichment techniques are employed. In patients with metastatic CRC identification of CTCs in peripheral blood is associated with an adverse prognosis [5]. In addition, post-operative measurement of CTC levels may be used to predict tumour recurrence after surgery [6]. However, attempts to link CTC levels in early stage CRC (stage 1 disease) with an adverse prognosis have shown less convincing results and therefore the utility of this method as a screening modality is currently unclear [7].
Increased levels of circulating cell-free DNA (cfDNA) in cancer patients have been studied as a potential biomarker of the disease [8]. The precise source of these cfDNA fragments in conditions such as CRC remains controversial but tumour cell apoptosis, necrosis, or possibly active secretion have been suggested as putative mechanisms. A recent study used quantitative real-time PCR to detect ALU repeats in the plasma or serum of operated or non-operated CRC patients and compared the levels of circulating cfDNA detected with normal healthy controls [9]. The results showed that circulating levels of cfDNA were significantly higher in non-operated CRC patients when compared with operated patients and controls. This study concluded that quantification of serum cfDNA could be important in detecting and monitoring CRC patients in both early and late stage disease. The predictive value of cfDNA analysis has also been suggested. A subset of patients with initial wild type KRAS status receiving monotherapy with epidermal growth factor receptor (EGFR) inhibitors have been shown to develop early KRAS mutations detectable in cfDNA in the serum months before imaging revealed disease progression [10]. Despite progress in the detection and monitoring of cfDNA there are no reproducible studies to date to show direct and consistent correlation between CRC stage and circulating cfDNA levels. As laboratories use different methods to detect, analyse and monitor cfDNA, future translation to clinical use will require further standardization to ensure consistency in analysis.
MicroRNAs (miRNAs) are small non-coding RNA strands that can post-transcriptionally regulate the expression of multiple target genes. They have been implicated in several steps in carcinogenesis and their measurement in the serum of CRC patients has shown early promise for disease detection and monitoring of cancer recurrence after surgery [8]. miRNA-29c is thought to suppress tumorigenesis by inhibiting cell proliferation and migration. Circulating miRNA-29c levels have been studied as a potential biomarker for both early and late recurrence following surgery in colorectal cancer [11]. The oncogenic miRNA, miRNA-21, which negatively regulates tumour suppressor genes, has shown promise as a possible diagnostic and prognostic marker in CRC. Levels of this miRNA were significantly raised in CRC patients and patients with colonic adenomas when compared with healthy controls [12]. Serum miRNA-21 levels also fell in those patients undergoing curative surgery. Increased levels of this miRNA in both tumour tissue and the patients’ serum were found to be significantly associated with tumour size, the presence of metastasis and reduced survival. A possible drawback of miRNA analysis in cancer patients is the variable extraction rates of these small molecules from patients’ plasma or serum. To date this has produced inconsistent results between different studies. Once again a standardized approach will be required.
Proteomic analysis of tissue samples in CRC has been proven a valuable technique for the identification of potential biomarkers. Given the issues surrounding patient acceptability with the use of faecal material or invasive techniques in CRC screening, recent studies have focused on identifying plasma or serum protein biomarkers which can aid in the early diagnosis of CRC. Choi et al. analysed plasma samples from patients with colorectal adenomas or invasive disease and identified a panel of proteins, including three cytokines, which were differentially expressed between the study groups [13]. Another group used combinations of serum CEA, cytokines and CA19.9 to try to differentiate adenoma-bearing patients from healthy controls or those with established CRC [14]. Multiplex protein arrays have been developed to analyse serum samples from CRC patients, adenoma-bearing patients and healthy controls. Initial results indicated that combinations of CEA and IL-8 or CEA and C-reactive protein showed the best screening performance for early CRC or adenoma detection [15]. However, although the overall specificity of the tests employed was relatively high, the sensitivity was much lower (particularly with regard to adenoma detection) and the authors concluded that clinical use of such novel systems could be made in combination with established techniques such as faecal testing, for screening purposes.
Conclusions
Although significant progress has been made recently in the development of biomarkers in CRC there is still a relative lack of consistent follow-up data available for the validation of such markers. This aspect of research needs to be addressed in order to facilitate the transition of putative biomarkers from the research stage into robust and reliable clinical applications. The search for acceptable plasma-based biomarkers to aid in screening is gaining momentum but perhaps in future a combination of techniques will be required to accurately guide early diagnosis and intervention in colorectal cancer.
References
1. De Wit M, Fijneman RJ, Verheul HM, et al. Proteomics in colorectal cancer translational research: biomarker discovery for clinical applications. Clin Biochem. 2013; 46: 466–479.
2. Coghlin C, Murray GI. Following the protein biomarker trail to colorectal cancer. Colorectal Cancer 2012; 1: 93–96.
3. Ralton LD, Murray GI. Biomarkers for colorectal cancer: identification through proteomics Curr Proteomics 2010; 7: 212–221.
4. O’Dwyer D, Ralton LD, O’Shea A, Murray GI. The proteomics of colorectal cancer: identification of a protein signature associated with prognosis. PLoS One 2011; 6: e27718.
5. Groot Koerkamp B, Rahbari NN, Büchler MW, Koch M, Weitz J. Circulating tumor cells and prognosis of patients with resectable colorectal liver metastases or widespread metastatic molorectal mancer: a meta-analysis. Ann Surg Oncol. 2013; 20: 2156–2165.
6. Galizia G, Gemei M, Orditura M, et al. Postoperative detection of circulating tumor cells predicts tumor recurrence in colorectal cancer patients. J Gastointest Surg. 2013; Epub ahead of print, doi: 10.1007/s11605-013-2258-6.
7. Linuma H, Watanabe T, Mimori K et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes’ stage B and C colorectal cancer. J Clin Oncol. 2011; 29: 1547–1555.
8. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011; 11: 426–437.
9. da Silva Filho BF, Gurgel AP, Neto MÁ, et al. Circulating cell-free DNA in serum as a biomarker of colorectal cancer. J Clin Pathol. 2013; 66: 775–778.
10. Misale S, Yaeger R, Hobor S et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486: 532–536.
11. Yang I-P, Tsai H-L, Huang C-W, et al. The functional significance of microRNA-29c in patients with colorectal cancer: A potential circulating biomarker for predicting early relapse. PLoS One 2013; 8: e66842.
12. Toiyama Y, Takahashi M, Hur K, et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. J Natl Cancer Inst. 2013; 105: 849–859.
13. Choi JW, Liu H, Shin D H, et al. Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients. Proteomics 2013; 13: 2361–2374.
14. Pengjun Z, Xinyu W, Feng G, et al. Multiplexed cytokine profiling of serum for detection of colorectal cancer. Future Oncol. 2013; 9: 1017–1027.
15. Bünger S, Haug U, Kelly M, et al. A novel multiplex-protein array for serum diagnostics of colon cancer: a case-control study. BMC Cancer 2012; 12: 393. doi: 10.1186/1471-2407-12-393.
The authors
Caroline Coghlin1 BA, BM Bch, DRCOphth, FRCPath; Graeme I. Murray2* MB ChB, PhD, DSc, FRCPath
1Department of Pathology, Aberdeen Royal Infirmary, NHS Grampian, Aberdeen, UK
2Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK
*Corresponding author
E-mail: g.i.murray@abdn.ac.uk
Lipocalin 2 and brain-derived neurotrophic growth factor: biomarkers that link colorectal cancer and obesity?
, /in Featured Articles /by 3wmediaColorectal cancer is one of the most prevalent types of cancer and is the fourth most common cause of cancer mortality. Identification of non-invasive biomarkers representative of disease heterogeneity is critical for diagnosis of early stage disease when the chance for cure is greatest. This article discusses two such biomarkers, brain-derived neurotrophic growth factor and lipocalin 2, which also reflect key independent risk factors for the disease obesity.
by Dr K. Y. C. Fung, Dr B. Tabor, Prof. P. Gibbs, Dr J. Tie, Dr P. McMurrick, Mr J. Moore, Prof. A. Ruszkiewicz, Prof. A. Burgess, Dr L. Cosgrove
Biomarkers for colorectal cancer: current status
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide where epidemiological studies have drawn strong correlations between its incidence and lifestyle factors [1, 2]. The incidence of CRC varies considerably with geographic region, where it is highest in affluent countries (e.g. in the USA, UK, Europe, Australia and New Zealand the incidence is approximately 20–45 per 100 000) and lowest in African and Asian countries (incidence of approximately 5–20 per 100 000) [2]. In countries with increasing industrialization such as Japan, Korea and Singapore, the incidence of CRC is rapidly approaching that of high risk countries with a longer history of affluence [2]. For most sporadic CRC, the transformation from normal colonic mucosa to carcinoma is believed to occur over 10–15 yrs [3]. This relatively long time frame for disease development enables implementation of population screening programmes for disease detection as early stage diagnosis and removal of premalignant (adenoma or polyp) or early stage malignant disease (stage I) can either prevent the occurrence of CRC or significantly increase the chance of a complete cure.
Ideally, diagnostic tests are robust and cost effective and biomarkers should have high sensitivity and specificity for the disease they are proposed to detect. Currently, colonoscopy is regarded as the ‘gold standard’ for CRC diagnosis (sensitivity and specificity greater than 95%) but it is expensive and invasive. Accordingly, low cost alternatives such as the fecal occult blood test (FOBT) and the fecal immune test (FIT) are currently in use in population screening programmes in a number of countries [4]. These tests detect the presence of blood in stool samples and have low specificity for CRC. Their low sensitivity also leads to high rates of false positive results and they do not reliably detect early stage disease [5, 6]. As a result, identification of suitable biomarker(s) with high sensitivity and specificity for CRC that can be included in a non-invasive test suitable for population screening is urgently required. Despite extensive research efforts, no single biomarker has been identified and it is becoming apparent that a panel of biomarkers panel reflecting the heterogeneity of the disease will be more effective.
Sporadic CRC is linked to multiple environmental risk factors, with obesity consistently demonstrated to be a significant and independent risk factor [1]. Brain-derived neurotrophic growth factor (BDNF) and lipocalin 2 (LCN2) are two protein biomarkers that have been implicated in both obesity and CRC. BDNF has been shown to have a key role in neural regulation of appetite and food intake control [7], where low BDNF levels in the hypothalamic region of the brain have been associated with decreased satiety and weight gain. There is also evidence indicating that serum BDNF levels are lower in patients with type 2 diabetes in comparison to controls [8]. Similarly, elevated levels of circulating LCN2 have been documented in obese men and women and in patients with metabolic syndrome [9]. With the aim of identifying a panel of biomarkers to identify individuals potentially at risk of developing CRC, we investigated the utility of BDNF and LCN2 as individual biomarkers and as a biomarker panel to determine if this combination provided higher sensitivity for CRC diagnosis.
BDNF and LCN2 as CRC biomarkers
We have previously reported on the utility of circulating BDNF and lipocalin as biomarkers for CRC [10, 11]. In these studies, enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of each biomarker in the sera of a cohort of CRC patients (n=97) and age/gender matched controls (n=99). In this cohort, the median BDNF concentration was found to be significantly lower (P<0.0001) in the control population (18.8 ng/mL, range 4.0–56.5 ng/mL) when compared to the CRC group (23.4 ng/mL, range 3.0–43.1 ng/mL). Conversely, in the same cohort, the median concentration of LCN2 was significantly higher (P<0.0001) in the CRC group (121.5 ng/mL, range 31.65–432.6 ng/mL) when compared to the control group (86.36 ng/mL, range 17.11–189.9 ng/mL). At 95% specificity, the sensitivity of BDNF was 18% [area under curve (AUC) 0.69, P<0.0001)] and the sensitivity of LCN2 was 31% (AUC 0.71, P<0.0001). Although both biomarkers performed equally well at separating CRC patients from the normal cohort (demonstrated by the AUC), neither biomarker when considered alone reached the desired sensitivity for clinical use as a diagnostic approach for CRC. Figure 1 shows the receiver operating characteristic (ROC) curve for BDNF, LCN2 and for BDNF and LCN2 in combination. Table 1 summarizes the sensitivity at 95% specificity for BDNF and LCN2 individually and as a biomarker combination for each disease stage. LCN2 had consistently higher sensitivity than BDNF for diagnosing CRC overall and at each Dukes’ stage, and the LCN2 and BDNF combination does not appear to improve diagnostic efficacy. For example, at 95% specificity, the sensitivity was 33% for the LCN2 and BDNF combination (compared with 32% for LCN2). Strategies for biomarker identification
Current strategies for CRC biomarker identification include identification of tumour specific biomarkers and biomarkers indicative of the disease process, such as inflammation, the immune response, angiogenesis, and metastasis. Investigators have also reported on the utility of biomarker combinations that include established tumour markers such as CEA and CA19-9 [12, 13]. These strategies have yielded many promising individual candidate markers and marker panels that have been tested in small cohort studies, but none has resulted in the sensitivity and specificity required for population based screening. This lack of success has been attributed to factors such as small sample size, over-representation of late stage disease in test cohorts leading to overestimation of biomarker sensitivity, and disease heterogeneity where CRC subsets with different genetic backgrounds have been characterized [14].
As part of our strategy, we have also considered biomarkers indicative of established risk factors such as obesity and type 2 diabetes. Inclusion of these biomarkers, or biomarkers that are indicative of other risk factors, should enable us to identify those individuals who may be at greater risk of developing the disease and hence improve our ability for earlier diagnosis. This is critical for reducing mortality and morbidity associated with CRC where the 5-year survival rate for patients with stage I disease is >90% in comparison to 5% at stage D. Currently, more than 50% of malignancies are detected at an advanced stage despite the implementation of screening programmes. Although the BDNF and LCN2 combination does not provide adequate sensitivity and specificity for use in a clinical setting, it is possible that a combination of (one of) these markers with a CRC tumour specific marker may yield the desired analytical performance.
Future directions
The lack of FDA approval for any biomarkers as a diagnostic for CRC highlights the challenges associated with discovery, verification and validation of biomarkers. While –omics technologies (e.g. genomics, transcriptomics and proteomics) have been, and continues to be, the primary tool for discovery of novel biomarkers, these efforts have largely focused on identification of tumour specific markers. Incorporation of biomarkers representative of other disease factors will likely improve our chances of identifying a panel of markers to successfully diagnose CRC. Furthermore, stratification of risk based on genotype or environmental/lifestyle factors together with a panel of molecular biomarkers may prove to be more successful than any one of these factors alone for early diagnosis.
Acknowledgements
We thank the Victorian Cancer Biobank (Melbourne, Victoria) for their assistance with sample collection and Ms Ilka Priebe for technical assistance with the ELISAs. This work was funded by the CSIRO Preventative Health National Research Flagship and the National Health and Medical Research Council (grant number 1017078).
References
1. World Cancer Research Fund / American Institute for Cancer Research. Food, nutrition, physical activity, and the prevention of cancer: a global perspective. Washington, DC: AICR 2007.
2. Jemal A, Bray F, et al. Global cancer statistics. CA Cancer J Clin. 2011; 61(2): 69–90.
3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990; 61(5): 759–767.
4. Hewitson P, Glasziou P, Watson E, Towler B, Irwig L. Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am J Gastroenterol. 2008; 103(6): 1541–1549.
5. Morikawa T, Kato J, Yamaji Y, Wada R, Mitsushima T, Shiratori Y. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology 2005; 129(2): 422–428.
6. Parra-Blanco A, Gimeno-García AZ, Quintero E, Nicolás D, et al. Diagnostic accuracy of immunochemical versus guaiac faecal occult blood tests for colorectal cancer screening. J Gastroenterol. 2010; 45(7): 703–712.
7. Vanevski F, Xu B. Molecular and neural bases underlying roles of BDNF in the control of body weight. Front Neurosci. 2013; 7: 37.
8. Fujinami A, Ohta K, Obayashi H, Fukui M, et al. Serum brain-derived neurotrophic factor in patients with type 2 diabetes mellitus: Relationship to glucose metabolism and biomarkers of insulin resistance. Clin Biochem. 2008; 41(10–11): 812–817.
9. Wang Y, Lam KS, Kraegen EW, Sweeney G, et al. Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans. Clin Chem. 2007; 53(1): 34–41.
10. Brierley GV, Priebe IK, Purins L, Fung KY, et al. Serum concentrations of brain-derived neurotrophic factor (BDNF) are decreased in colorectal cancer patients. Cancer Biomark. 2013; 13(2): 67–73.
11. Fung KY, Priebe I, Purins L, Tabor B, et al. Performance of serum lipocalin 2 as a diagnostic marker for colorectal cancer. Cancer Biomark. 2013; 13(2): 75–79.
12. Herszényi L, Farinati F, Cardin R, István G, et al. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer. BMC Cancer 2008; 8: 194.
13. Shimwell NJ, Wei W, Wilson S, Wakelam MJ, et al. Assessment of novel combinations of biomarkers for the detection of colorectal cancer. Cancer Biomark. 2010; 7(3): 123–132.
14. Tao S, Hundt S, Haug U, Brenner H. Sensitivity estimates of blood-based tests for colorectal cancer detection: impact of overrepresentation of advanced stage disease. Am J Gastroenterol. 2011; 106(2): 242–253.
The authors
Kim Y. C. Fung1* PhD; Bruce Tabor1 PhD; Peter Gibbs2 MBBS, MD, FRACP; Jeanne Tie2 MD; Paul McMurrick3 MBBS, FRACS; James Moore4 MBBS, MD, FRACS; Andrew Ruszkiewicz5 MD, FRCPA; Antony Burgess6 PhD; and Leah Cosgrove1 PhD
1CSIRO, Preventative Health National Research Flagship, Australia
2Royal Melbourne Hospital, Melbourne, Australia
3Cabrini Hospital, Melbourne, Australia
4Royal Adelaide Hospital, Adelaide, Australia
5SA Pathology, Adelaide, Australia
6Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
*Corresponding author
E-mail: Kim.fung@csiro.au
NGAL as a biomarker of acute kidney injury
, /in Featured Articles /by 3wmediaThe diagnosis of acute kidney injury (AKI) is often hindered by the reliance on serum creatinine as a marker of kidney function, which can delay detection. Neutrophil gelatinase-associated lipocalin is a promising biomarker which increases within hours of kidney damage and could therefore improve the early diagnosis of AKI.
by Dr Ashley Garner
Clinical background
Acute kidney injury (AKI) is a common condition associated with significant morbidity and mortality. It is currently diagnosed using serum creatinine and urinary output as markers of kidney function, as defined in the recent KDIGO criteria [Fig. 1][1]. However, these are relatively late markers of AKI since they mainly reflect a decrease in glomerular filtration rate and the time required for serum creatinine to accumulate can delay diagnosis. Biomarkers that can detect structural injury to the kidney rather than a loss of function may allow better and earlier detection of AKI. Earlier diagnosis of AKI could facilitate earlier intervention, potentially reduce the risk of irreversible kidney damage and improve patient outcomes. Much research in recent years has therefore focused on the discovery of improved biomarkers for AKI and neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising candidates [2].
Pathophysiology of NGAL
NGAL is a small 25kDa protein which belongs to the superfamily of lipocalins. It is expressed in many cells including neutrophils, hepatocytes and renal tubular cells and is induced in response to pathological stimuli including infection, inflammation, ischemia and malignancy. NGAL has a functional role in the innate immune system as a bacteriostatic agent, depleting iron-binding siderophores and thereby preventing bacterial iron acquisition. The iron-binding properties of NGAL are also proposed to provide protection from oxidative stress. There is growing evidence that NGAL also acts as a growth factor in some tissues including renal epithelial cells where it modulates cell proliferation, differentiation and apoptosis and may provide protection against renal tubular damage in AKI [3].
Animal studies of AKI induced by ischemia or nephrotoxicity have shown that NGAL is one of the most upregulated proteins in the kidney and is detectable in the urine within 2–3 h. It has been reported that urine NGAL concentrations increase 25–100 fold and plasma NGAL increases 7–16 fold following AKI. Unlike serum creatinine, NGAL is not increased when there is impaired glomerular filtration without renal tubular damage, often termed ‘pre-renal’ uraemia.
Low plasma concentrations of NGAL are found in health as it is expressed at a low constant rate from various tissues. NGAL is then freely filtered at the kidney and the majority is reabsorbed in the proximal tubule, resulting in low NGAL concentrations in the urine. Following AKI, NGAL is greatly upregulated in the cells lining the ascending loop of Henle and collecting ducts of the kidney and is then excreted in the urine. The origin of the increase in plasma NGAL following AKI is less clear and there is evidence to suggest that NGAL expression is increased in other organs such as the lungs and liver following kidney injury.
Since NGAL can be produced by different tissues in response to various stimuli, it is not specific to AKI. Other common conditions that can cause elevated NGAL, and therefore complicate the interpretation of results, include sepsis, heart failure, chronic kidney disease (CKD), malignancy and urinary tract infections.
NGAL assays
Commercial CE marked assays are available for measuring NGAL in plasma, whole blood and urine. It is not clear from the literature whether any of these sample types are preferred or have better diagnostic performance but there are limiting factors for each that may require consideration. Plasma and whole blood samples are invasive and may be contaminated by haemolysis releasing NGAL from neutrophils. Urine NGAL may theoretically be more sensitive for AKI due to greater induction in renal tubular cells but can be falsely elevated in urinary tract infections due to leukocyturia and it is still unclear whether the NGAL should be corrected for urine concentration effects or whether this is unnecessary or even misleading in AKI. Although non-invasive, urine samples may be more difficult to obtain especially at specific time points or if the patient has reduced urine output.
NGAL exists in monomeric, dimeric and heterodimeric or complexed forms. It has been reported that the monomer is the predominant form produced by renal tubular cells and the homodimer is predominantly released by neutrophils. The relevance of these different forms of NGAL will depend on the extent to which NGAL assays detect them and the sample type used. Even though the monomer form may be most relevant for urine NGAL the origin of plasma NGAL in AKI is less clear and may therefore include the other forms. This variation in NGAL assays and sample types makes it difficult to directly compare study results and derive clinically relevant cut-off values. Standardization of NGAL assays using an internationally approved reference material would greatly improve this variation but this is not currently available and would require agreement on what forms of NGAL should be measured.
Research from large heterogeneous populations suggests that urine NGAL concentrations are dependent on gender, age and ethnicity. Biological variation for urine NGAL has also been reported to be as high as 84%. These factors will need to be taken into account when establishing reference intervals for NGAL although they may not be clinically significant if a cut-off value is used to diagnose AKI, especially if it greatly exceeds the expected reference intervals in health.
Clinical utility of NGAL in AKI
There is evidence that NGAL could be useful as an early diagnostic and prognostic biomarker for AKI. Many studies have demonstrated that NGAL rises 24–72 h before creatinine in patients with AKI and is associated with poorer outcomes. It is difficult to determine the diagnostic performance of NGAL for AKI in terms of clinical sensitivity and specificity however, due to the limitations of using serum creatinine as the gold standard comparator. For example, the rise in creatinine caused by pre-renal uraemia will not be associated with a raised NGAL. In addition, a multicentre pooled analysis of prospective studies has shown that patients who have raised NGAL without increases in serum creatinine are at increased risk of adverse outcomes and suggests these patients have a condition termed ‘subclinical AKI’ where there may be tubular damage without glomerular impairment [Fig. 2][4].
The majority of studies assessing NGAL testing in AKI have focused on specific patient populations at high risk of AKI: namely post-cardiac surgery, post contrast infusion, intensive care and emergency admissions.
The advantage of using NGAL in post-surgery or post-contrast patients is that the time of insult is known and therefore NGAL can be measured at set time points for the early detection of AKI and timely intervention. It is more difficult to determine the best application of NGAL in ICU patients in regard to the timing and frequency of tests and it is less clear whether earlier detection can improve outcomes in these patients frequently complicated by multi-organ failure. In the emergency admissions setting NGAL has fewer advantages over serum creatinine since early detection (within hours) is less likely to be applicable. Also NGAL, like creatinine, can be raised in CKD so may require multiple measurements to detect AKI but more patients are likely to have had a previous creatinine result than an NGAL result.
Although there is an abundance of observational studies showing that AKI can be detected earlier using NGAL compared to serum creatinine there is an absence of randomized clinical trials to demonstrate that using NGAL instead of current practice will improve patient outcomes or provide cost benefits. This is probably the biggest barrier to the adoption of NGAL testing in routine practice and better treatments and interventions may be required to overcome it. This would suggest that one of the most important roles for NGAL and earlier biomarkers of AKI is in the discovery and development of effective interventions and therapeutics.
Another consideration regarding interventions for AKI is that NGAL only detects renal tubular damage, it does not distinguish between different causes. However, effective treatments may require the underlying cause to be determined and therefore further biomarkers may be needed to differentiate between causative factors and indicate the most appropriate intervention.
Conclusion
A large number of clinical studies suggest that NGAL may provide an early diagnostic and prognostic biomarker for AKI. However, further randomized clinical trials comparing the use of NGAL to standard practice are required to show cost benefits or improvements in patient outcomes. It seems that even if biomarkers like NGAL enable us to detect AKI earlier, this alone may not be sufficient to improve patient care but hopefully they will facilitate the development of better interventions that will eventually lead to improved outcomes for patients with AKI.
References
1. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int. Suppl. 2012; 2: 1–138.
2. ADQI Consensus on AKI Biomarkers and Cardiorenal Syndromes. Contrib Nephrol. Basel: Karger, 2013; 182: 13–29.
3. Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol. 2007; 18: 407–413.
4. Haase M, Devarajan P, Haase-Fielitz A, Bellomo R, Cruz DN, Wagener G, Krawczeski CD, Koyner JL, Murray P, Zappitelli M, Goldstein SL, Makris K, Ronco C, Martensson J, Martling CR, Venge P, Siew E, Ware LB, Ikizler TA, Mertens PR. The outcome of neutrophil gelatinase-associated lipocalin-positive subclinical acute kidney injury: a multicenter pooled analysis of prospective studies. J Am Coll Cardiol. 2011; 57(17): 1752–1761.
The author
Ashley Garner PhD
Department of Blood Sciences,
Leeds Teaching Hospitals Trust, Leeds, UK
E-mail: Ashley.Garner@leedsth.nhs.uk
Fibroblast growth factor-23: an emerging biomarker in chronic kidney disease
, /in Featured Articles /by 3wmediaFibroblast growth factor-23, a key regulator of phosphate and 1,25-dihydroxyvitamin D metabolism, appears to be an independent risk factor for mortality among chronic kidney disease patients. However, sample stability and poor analytical agreement between detection methods still need to be addressed for it to become a reliable biomarker.
by Dr A. Kumar, Dr W. Herrington, Dr S. Clark and Dr M. Hill
The function of fibroblast growth factor-23 and its role in chronic kidney disease
Fibroblast growth factor-23 (FGF-23) was identified as the key regulator of phosphate homeostasis from a study of renal phosphate-wasting condition autosomal dominant hypophosphatemic rickets [1]. It is secreted principally by bone-forming osteocytes and osteoblasts in response to increased dietary phosphate intake and abnormally elevated serum phosphate concentration (hyperphosphatemia). FGF-23 acts to correct raised phosphate levels by increasing urinary phosphate excretion, by direct inhibition of renal tubular phosphate reabsorption, and via reducing dietary phosphate absorption, by suppressing 1α-hydroxylation of 25-dihydroxyvitamin D to form active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Low 1,25(OH)2D production also provides a negative feedback signal in phosphate homeostasis by inhibiting further FGF-23 secretion (Fig.1)[2, 3].
Chronic kidney disease (CKD) commonly causes a fall in glomerular filtration rate resulting in a reduced capacity for phosphate excretion [4]. FGF-23 levels increase early in CKD, often before any detectable rise in phosphate concentration [5] and those with the severest form of CKD, end-stage renal disease (ESRD), have FGF-23 levels that are 100 to 10,000-fold higher than healthy controls [4, 6]. Sustained suppression of renal 1,25(OH)2D synthesis by high FGF-23 levels contributes to lower serum calcium concentration, a stimulant of parathyroid hormone (PTH) secretion. PTH maintains normal serum calcium concentration by promoting reabsorption of the calcium from its reservoir in bone. The abnormal elevated levels of FGF-23 seen in CKD thus results in a disruption of the homeostatic balance of calcium and phosphate and this may impact on many normal processes including bone mineral metabolism and cardiovascular function. The consequences of prolonged derangement of bone mineral metabolism (known as CKD-mineral bone disease; CKD-MBD) is bone pain and increased fracture risk. CKD-MBD may also accelerate calcification of the vascular tree, a process that may explain some of the significantly increased cardiovascular risk in those with CKD [7]. Indeed, among CKD patients, several studies have shown FGF-23 to be an independent risk factor for mortality [8] and among those not on dialysis it appears to also predict CKD progression [4, 9]. Treatments that might positively impact on FGF-23 levels, for example, reducing dietary phosphate absorption with phosphate binders may therefore have beneficial effects on bone, renal and cardiovascular outcomes in those with CKD (both in those with hyperphosphatemia and those with high-normal serum phosphate concentration).
Methods for FGF-23 assessment
In vitro studies have shown that some of the FGF-23 synthesized by the osteocytes is cleaved between amino acid 179 and 180 by furin (a type I precursor convertase) releasing a C terminal fragment. Current immunometric methods detect either ‘intact’ FGF-23 (iFGF-23, ~32 KDa) or ‘C-terminal’ fragments (cFGF-23, ~14 KDa) in plasma or serum. The cFGF-23 assays recognize two epitopes in the C-terminus, thereby recognizing both iFGF-23 and cFGF-23 fragments. The intact assays recognize only the iFGF-23 because the epitopes flank the cleavage site [2]. At present, there is no reference method, or consensus to indicate which assay type is the most suitable for measuring circulating FGF-23. If all circulating FGF-23 is intact and biologically stable, concentrations detected by the intact and C-terminal assays should be comparable [10]. However, there is a paucity of data confirming this and so caution should be used when comparing studies using the different methods.
Several studies have assessed the performance of commercially available enzyme-linked immunosorbent assays (ELISAs) for FGF-23. Heijboer and co-workers evaluated the performance of one cFGF-23 assay (Immutopics, USA) and two iFGF-23 assays (Immutopics and Kainos Laboratories Inc., Japan) using samples from healthy volunteers and patients with expected high levels of FGF-23 [11]. Intra- and inter-assay variations were assessed in approximately 100 samples with low, normal and high FGF-23 concentrations providing <20% CV for the cFGF-23 Immutopics and iFGF-23 Kainos assays. A high intra-assay variation (22–61%) was observed for the Immutopic intact assay which may be due to lot-to-lot variation [11]. A potential difficulty observed with the Kainos intact assay is poor assay performance when using an automated plate washer, as directed in their protocol. Heijboer and co-workers found acceptable results were only obtained when the wells were washed manually, which made this method impractical for measurement of large numbers of samples [11]. However, a later version of the Kainos assay protocol (from October 2010 onwards) includes an improved wash instruction. Using plasma samples from patients with renal impairment, Devaraj and co-workers also found good inter- and intra assay precision for cFGF-23 assay (CV between 4–10.5%), however, the CVs for iFGF-23 Immutopic assay were found to be poor (6–37.5%) [12]. Poor analytical agreement exists between the commercially available FGF-23 assays, due principally to the lack of a reference method. The performance of four commercially available methods [iFGF-23 assays from Immutopics, Kainos and Millipore (USA) and a cFGF-23 assay from Immutopics] were recently compared using plasma from 31 healthy adults and 36 patients undergoing hemodialysis [13]. A broad range of FGF-23 values were obtained: whereas the patient ranges fell between 154–2561 pg/mL and 447–2063 pg/mL for iFGF-23 and cFGF-23 assay respectively, the levels for healthy adults ranged from 9.9–62 pg/mL for the two assay types. Poor analytical agreement was observed between the assays particularly in the patient group. No agreement of test results was found between the iFGF-23 and cFGF-23 assays and this was more evident at physiological concentrations than in the haemodialysis group [13]. The lack of analytical agreement between these commercially available FGF-23 methods emphasizes that they cannot be used interchangeably and that a comparison of findings from different assays requires careful interpretation. The above evaluation study was performed with plasma samples [13]. A further consideration is some assays are restricted to the sample type that can be used. The iFGF-23 Kainos assay is suitable for both plasma and serum; however, the cFGF-23 Immutopics assay is established only for plasma [10, 13] providing lower or undetectable results in serum [10, 12]. Further method comparisons, ideally on larger numbers of samples and in different patient groups, would provide a valuable insight in this area and help identify which assay type is the most suitable for measuring circulating FGF-23. Nevertheless, studies measuring either intact or C-terminal FGF-23 have reported associations with mortality risk and decline in renal function [4, 14]. Stability of FGF-23: implication for large scale epidemiological studies
Limited evidence exists for the short-term stability of FGF-23 in collected blood samples (6 hours or less) and no information is available for its long-term stability in stored samples. Smith and co-workers investigated the short-term pre-analytical stability of FGF-23, measured using iFGF-23 and cFGF-23 ELISAs from Immutopics, by performing a number of timed experiments with blood taken from 15 patients with mild CKD [6]. The effect of aprotinin, a serine protease inhibitor, and a commercially available protease inhibitor cocktail to preserve FGF-23 after blood collection was also investigated [6]. In the absence of any preservative or inhibitor, iFGF-23 degraded by approximately 40% within 2 hours of collection even when the blood samples were separated into plasma. Conversely, 2 hours after blood collection the FGF-23 concentrations had increased by approximately 35% using the cFGF-23 assay. However, with the addition of the protease inhibitor cocktail the stability of both iFGF-23 and cFGF-23 in the samples extended up to 4 hours (less than 10% change). Based on this evidence, it appears that FGF-23 cannot be measured reliably in blood samples collected without the use of any preservative or inhibitors. This could be a serious limitation for large-scale epidemiological studies, particularly if samples have already been collected and are in storage and for blood collection methods that need to be simple in order to be cost-effective and feasible.
Preliminary findings from our laboratory using samples from 54 CKD patients suggest that FGF-23 (measured by both the iFGF-23 Kainos ELISA and the cFGF-23 Immutopics ELISA) remains stable in whole blood stored for up to 96 hours without the use of a preservative [15]. The apparent lack of agreement with the results from Smith et al. may be explained by differences in the methods of sample collection. Smith et al. used a single K2-EDTA blood tube for each participant which was re-sampled at each time-point whereas we collected separate blood tubes corresponding to each time-point.
Large-scale epidemiological studies often involve the long-term frozen storage of samples prior to biomarker analyses, particularly nested case-control designed studies where it may take several years for sufficient incident cases to materialize. Limited information is available to help understand the impact of long-term frozen storage on the stability of many biomarkers, including FGF-23. Studies investigating the stability of biomarkers in different sample types stored at various temperatures (for example, −40 °C, −80 °C and in liquid nitrogen vapour) will have immense value, particularly in support of long-term blood based prospective studies and biobanks.
Summary
FGF-23 is a key regulator of phosphate homeostasis and has emerged as an important biomarker in patients with CKD. Despite an increasing amount of literature, there are still unanswered questions related to FGF-23 sample stability and the availability of robust reliable methods for measuring FGF-23. Further studies into these areas will improve the quality of clinical research into the use of FGF-23 as a potential early biomarker in CKD.
References
1. ADHR Consortium. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet. 2000; 26: 345–348.
2. Wolf M. Forging forward with 10 burning questions on FGF23 in kidney disease. J Am Soc Nephrol. 2010; 21: 1427–1435.
3. Quarles LD. Role of FGF23 in vitamin D and phosphate metabolism: implications in chronic kidney disease. Exp Cell Res. 2012; 318: 1040–1048.
4. Isakova T, Xie H, Yang W, et al. Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA 2011; 305: 2432–2439.
5. Russo D, Battaglia Y. Clinical significance of FGF-23 in patients with CKD. Int J Nephrol. 2011; 2011: 364890.
6. Smith ER, Ford ML, Tomlinson LA, Weaving G, Rocks BF, Rajkumar C, Holt SG. Instability of fibroblast growth factor-23 (FGF-23): implications for clinical studies. Clin Chim Acta 2011; 412: 1008–1011.
7. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.
8. Jean G, Terrat JC, Vanel T, Hurot JM, Lorriaux C, Mayor B, Chazot C. High levels of serum fibroblast growth factor (FGF)-23 are associated with increased mortality in long haemodialysis patients. Nephrol Dial Transplant. 2009; 24: 2792–2796.
9. Titan SM, Zatz R, Graciolli FG, dos Reis LM, Barros RT, Jorgetti V, Moyses RM. FGF-23 as a predictor of renal outcome in diabetic nephropathy. Clin J Am Soc Nephrol. 2011; 6: 241–247.
10. Shimada T, Urakawa I, Isakova T, Yamazaki Y, Epstein M, Wesseling-Perry K, Wolf M, Salusky IB, Jüppner H. Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active. J Clin Endocrinol Metab. 2010; 95: 578–585.
11. Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee, PM, Dijstelbloem HM, Blankenstein MA. Determination of fibroblast growth factor 23. Ann Clin Biochem. 2009; 46: 338–340.
12. Devaraj S, Duncan-Staley C, Jialal I. Evaluation of a method for fibroblast growth factor-23: a novel biomarker of adverse outcomes in patients with renal disease. Met Syndr Relat Disord. 2010; 8: 477–482.
13. Smith ER, McMahon LP, Holt SG. Method-specific differences in plasma fibroblast growth factor 23 measurement using four commercial ELISAs. Clin Chem Lab Med. 2013; 51: 1971–1981.
14. Fliser D, Kollerits B, Neyer U, Ankerst DP, Lhotta K, Lingenhel A, Ritz E, Kronenberg F, Kuen E, Konig P, Kraatz G, Mann JF, Muller GA, Kohler H, Riegler P. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study. J Am Soc Nephrol. 2007; 18: 2600–2608.
15. Illingworth N, Edmans M, Clark S, Kumar A, Sutherland S, Herrington W, Hill M. Investigation of FGF-23 sample & assay suitability for large scale epidemiological studies. Ann Clin Biochem. 2013; 50: 73–74
The authors
Aishwarya Kumar1* PhD; Will Herrington2 MBBS, MRCP; Sarah Clark1 PhD; and Michael Hill1 PhD
1Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU), University of Oxford, Oxford, UK
2Oxford Kidney Unit, Oxford University Hospitals, Oxford, UK
*Corresponding author
E-mail: Aishwarya.Kumar@ctsu.ox.ac.uk
Facilitating kidney function testing through the use of reagents applicable to a variety of automated systems
, /in Featured Articles /by 3wmediaThe kidneys play an important role in homeostasis, they regulate the amount of water and salts present in the body by filtering blood through the nephrons. Waste products are filtered out and eliminated from the body in the urine, which is made up of the excess water, salts and waste products.
When the kidneys are not functioning efficiently, waste products and fluids begin to accumulate instead of being excreted which can cause serious health problems. Furthermore, kidney disorders can often develop and advance over a period of time without showing any signs; alternatively, symptoms are not recognized as being associated with kidney problems. Kidney function testing is therefore relevant for diagnosing and monitoring disease and assists in the development of appropriate treatment plans. Laboratory automation facilitates the efficiency and productivity of clinical laboratories. The determination of parameters related to kidney function by using tests incorporating reagents applicable to a variety of automated analysers facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
Kidney function assessment
Many conditions can affect the ability of the kidneys to carry out their vital functions. Some conditions can lead to a rapid (acute) decline in kidney function; other conditions lead to a gradual (chronic) decline. A number of clinical laboratory tests in blood and urine can be used to assess renal function. The unit measure of kidney function is the glomerular filtration rate (GFR), which can be defined as the volume of plasma cleared of an ideal substance –freely filtered at the glomerulus and neither secreted nor reabsorbed by the renal tubules- per unit of time. The normal range is 80-120 ml/min. Measuring this rate is a laborious process. Creatinine is the closest to an ideal endogenous substance for measuring GFR.[1,2] Creatinine is derived from creatine and creatine phosphate in muscle tissue and is defined as a nitrogenous waste product. Creatinine is not reutilized but is excreted from the body in the urine via the kidney. As a consequence of the way in which creatinine is excreted by the kidney, its measurement is used almost exclusively in the assessment of kidney function.
Urea, a byproduct of protein metabolism, is produced in the liver and then is filtered from the blood and excreted in the urine by the kidneys. The blood urea nitrogen test (BUN) measures the amount of nitrogen contained in the urea, high levels can indicate kidney dysfunction. As these levels are also affected by protein intake and liver function, this test is usually done together with a blood creatinine test.
Cystatin C is a small cysteine proteinase inhibitor that is steadily produced by all nucleated cells. The small molecular weight of cystatin C allows it to be freely filtered by the glomerular membrane and therefore cystatin C levels in the blood are indicative of a normal or impaired GFR. Levels of cystatin C in serum/plasma are almost entirely dependent on GFR.[3]
Other tests for the measurement of other parameters regulated in part by the kidneys can also be useful for the evaluation of kidney function; these tests include electrolytes (sodium, potassium, chloride, bicarbonate), protein, uric acid and glucose:
Application of kidney function tests to automated systems
In clinical settings the application of tests for the determination of parameters related to kidney function to automated systems, facilitates clinical effectiveness and productivity. There are currently tests available for the determination of creatinine, BUN, cystatin C, electrolytes, protein, uric acid and glucose among others. If a variety of these tests could be applied to one system, the result output for each system would increase, which would maximize efficiency. The use of tests incorporating reagents applicable to a variety of automated analysers is beneficial as it increases the testing capacity of one system. This is further enhanced by the analyser’s capability to employ different methodologies with different reagents. The combination of automation and the use of stable, high performance reagents, lead to optimal analytical performance, extensive measuring ranges to ensure detection of abnormal values and reduced interference to produce more accurate results. For instance, a study using a creatinine test reported no interference with bilirubin and metamizol.[6]
The application of other kidney function related tests to studies in patients with nephrotic syndrome, chronic liver diseases and diabetes have also been reported.[7-8]
The automation of laboratory testing still requires qualified laboratory professionals for the evaluation of the results but reduces errors, staffing concerns and safety issues. This facilitates the diagnosis and the monitoring of kidney function, which is of great importance in clinical practice and in research.
Conclusion
The kidneys are the body’s natural filtration system and perform many vital functions. Kidney function tests is a collective term for a variety of individual tests and procedures for the evaluation of how well kidneys are functioning. The determination of parameters related to kidney function (i.e.creatinine, BUN, cystatin C, electrolytes, protein, uric acid, glucose) by using tests incorporating reagents applicable to a variety of automated analysers, increases the testing capacity of the systems and facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
References
1. Berger A. Renal function – and how to assess it. BMJ. 2000; 321: 1444.
2. Traynor J, Mactier R, Geddes CC, Fox JG. How to measure renal function in clinical practice. BMJ. 2006; 333 (7571): 733-737.
3. Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin. Chem. 2002; 48(5): 699-707.
4. Kirby M. Screening for microalbuminuria. The British Journal of Diabetes and Vascular Disease. 2002; 2(2): 106-109.
5. Sechi LA, Catena C, Zingaro L., Melis A, De Marchi S. Abnormalities of glucose metabolism in patients with early renal failure. Diabetes. 2002; 51: 1226-1232.
6. Harmonien AP. Bilirubin and metamizol do not interfere with the Randox enzymatic creatinine test. An evaluation of a new enzymatic creatinine determination method. Eur. J. Clin. Chem. Clin. Biochem. 1996; 34(12): 975-976.
7. Mula-Abed W-AS and Hanna BE. Measurement of serum fructosamine as an index of glycated protein in patients with nephrotic syndrome and chronic liver diseases. Bahrain Medical Bulletin 2001; 23(4).
8. Hirnerova E, Krahulec B, Strbova L, Stecova A, Dekret J, Hajovska A, Ch A Dukat A. Effect of vitamin E supplementation on microalbuminuria, lipid peroxidation and blood prostaglandins in diabetic patients. Bratisl. Lek. Listy 2004; 105(12): 408-413.
Author
María Luz Rodríguez
Randox Laboratories Limited,
55 Diamond Road, Crumlin,
County Antrim, N. Ireland, BT29 4QY,
United Kingdom
IMMULEX S. pneumoniae
, /in Featured Articles /by 3wmediaPocket-size blood analyzers for hemoglobin, lactate and ketones
, /in Featured Articles /by 3wmediaSodium in ‘enzymatic’ format
, /in Featured Articles /by 3wmedia