Autoimmune diseases affect approximately 5 % of the population of developed countries with an increasing incidence. Analysis of disease-associated autoantibodies (AAb) plays a significant role in the differential diagnosis thereof. Indirect immunofluorescence (IIF) has been established as the gold standard for AAb screening in particular for systemic rheumatic diseases. In the recommended two-tier approach for antibody serology, confirmatory testing by molecular assay techniques such as ELISA is required to confirm positive findings by screening using IIF. To cope with the constantly increasing demand for AAb testing, new efficient diagnostic approaches are required. Thus, a new generation of IIF assays have been developed to combine screening and confirmatory testing on one platform for the simultaneous detection of AAb by cell-based and bead-based assays in one reaction environment. The multiplex analysis of antineutrophil cytoplasmic antibodies (ANCA) for the differential diagnosis of vasculitides will be discussed as a first application of this novel approach.
by Dr. Christina Fritz, Mandy Sowa and Dirk Roggenbuck
ANCA-associated vasculitis
Vasculitis is an inflammation affecting blood vessel walls and resulting in their damage, fibrinoid necrosis, tissue ischemia and necrosis, and finally vessel rupture with bleeding into the surrounding tissue [1, 2]. Due to etiological factors, systemic vasculitis is differentiated into primary and secondary vasculitis. Primary systemic vasculitis of particularly small vessels often has an autoimmune pathogenesis accompanied by the occurrence of ANCA [3,5-8]. Those so called ANCA-associated systemic vasculitides (AASV) comprise microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA or Churg-Strauss syndrome) or granulomatosis with polyangiitis (GPA or Wegener’s granulomatosis)[1, 2, 4]. In contrast, secondary vasculitis occurs in 5 – 10 % of patients with rheumatoid arthritis or with other autoimmune diseases (e.g., systemic lupus erythematosus [SLE], Sjögren’s syndrome). In addition, vasculitis can occur in patients suffering from infections such as HIV or hepatitis C.
In general, an acute AASV generally requires immunosuppressive treatment with high doses of cortisone. In severe cases, cyclophosphamide is recommended. Once remission is achieved, methotrexate, azathioprin, cotrimoxazol, leflunomid or mucophenolate mofetil are used as maintenance therapy.
Diagnosis of ANCA-associated vasculitis
According to the international consensus statement for the assessment of ANCA, IIF on ethanol-fixed human neutrophils (ethN) is followed by confirmation with antigen-specific molecular immunoassays [6-8]. IIF reveals two ANCA patterns sub-classifying ANCAs into cytoplasmic ANCA (cANCA) and perinuclear ANCA (pANCA). Regarding the autoantigenic target of ANCA, c and pANCA are directed against proteinase 3 (PR3) and myeloperoxidase (MPO), respectively. A positive cANCA pattern confirmed by the presence of PR3-ANCA is pathognomonic for GPA[5], whereas a positive pANCA pattern confirmed by MPO-ANCA is decisive for MPA and EGPA. Furthermore, the corresponding ANCA titres are strongly associated with activity of disease in patients suffering from GPA and MPA.
As a matter of fact, IIF is currently the only technique to provide a single reaction environment for the combined screening and confirmation of ANCA. Simultaneous detection of c and pANCA along with PR3- and MPO-ANCA would overcome time-consuming single parameter detection by different techniques [10].
The use of multiplexing bead-based IIF assays for the simultaneous detection of single ANCA reactivities provides the ideal reaction environment to be combined with ethN-based ANCA testing. The corresponding principle is based on a covalent surface immobilization of MPO and PR3 on microbeads coded by size and fluorescence. The differentiation in size and/or intensity of a red fluorescence dye filling entirely each microbead population generates a novel reaction environment for parallel analyte analysis [11] (figure 1).
Combination of cell-based and microbead based ANCA assessment by CytoBead assay
The CytoBead assay is a unique combination of a conventional cell-based immunofluorescence assay with multiplexing microbead technology in one reaction environment. A newly designed microscopic glass slide with triple parted wells is employed to fix ethN in the middle compartment and PR3- as well as MPO-coated microbeads in the right-hand compartment of the slide (figure 2). Thus, anti-PR3 antibody positive sera show a positive cytoplasmic fluorescence on ethN and a green fluorescence halo on the surface of PR3-coated microbeads (9 µm). In contrast, anti-MPO antibody positive sera demonstrate a perinuclear fluorescence pattern on the immobilized ethN and a fluorescence halo on the surface of MPO-coated microbeads (15 µm) (figure 2). A reference microbead population (12 µm) is integrated for particle differentiation. This assay set offers the possibility of classical evaluation by a simple fluorescence microscope as well as automated analysis by interpretation systems like the AKLIDES®.
A recent clinical study with classical ANCA testing revealed a relative sensitivity and specificity of 98 % and 99.2 % for the novel CytoBead ANCA assay, respectively. Remarkably, the CytoBead ANCA assay showed a better discrimination of GPA and MPA patients in contrast to the classical anti-MPO and anti-PR3 ELISA. The detected cut-off values were determined on the basis of fluorescence intensity given in arbitrary units [AU] (personal communication).
Conclusion and future perspectives
The increasing demand for cost-effective autoimmune diagnostics requires new multiplexing technologies combining screening and confirmatory testing in one reaction environment. Thus, the novel CytoBead technology is a promising opportunity to accomplish this goal as demonstrated for the comprehensive assessment of ANCA. Automated digital immunofluorescence employed by recently established novel diagnostic interpretation system solutions such as Aklides even offers quantification and standardization of ANCA detection. The CytoBead technology provides an ideal reaction environment for the multiplexing of antinuclear antibody assessment and the simultaneous detection of celiac disease-specific antibodies.
References
1. Watt RA, Scott DG. Recent advances in classification and assessment of vasculitis. Best Pract Res Clin Rheumatol. 2009; 23: 429-443
2. Jeanette JC, Falk RJ. Small-vessel vasculitis. N Eng J Med. 1997; 337: 1512-23
3. Gross WL, Trabant A, Reinhold-Keller E. Diagnosis and evaluation of vasculitis. Rheumatology (Oxford). 2000; 39: 245-52
4. Waller R, Ahmed A, Patel I, Luqami R. Update on the classification of vasculitis. Best Pract Res Clin Rheumatol. 2013; 27: 3-17
5. Bosch X, Guilabert A, Font J: Antineutrophil cytoplasmic antibodies. Lancet 2006, 368:404-18
6. Jennette JC, Falk RJ, Bacon PA, Basu N, Ferrario F, Flores-Suarez LF, Gross WL, Guillevin L, Hagen EC, Hoffman GS, Jayne DR, Kallenberg CG, Lamprecht P, Langford CA, Lugmani RA, Mahr AD, Matteson EL, Merkel PA, Ozen S, Pusey CD, Rasmussen N, Rees AJ, Scott DG, Specks U, Stone JH, Takahashi K, Watts RA: 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitis. Arthritis Rheum. 2013, 65:1-11
7. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG: Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum 1994, 37:187-92
8. Savige JF, Gillis DF, Benson E, Davies DF, Esnault VF, Falk RJ, Hagen EC, Jayne D, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van der Woude F, Wieslander J, Wiik A: International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999, 111:507-13
9. Merkel PA, Polisson RP, Chang Y, Skates SJ, Niles JL: Prevalence of antineutrophil cytoplasmic antibodies in a large inception cohort of patients with connective tissue disease. Ann. Intern. Med. 1997, 126;866
10. Choi HK, Liu S, Merkel, PA, Colditz GA, Niles Jl: Diagnostic performance of antineutrophil cytoplasmic antibody tests for idiopathic vasculitides: metaanalysis with a focus on antimyeloperoxidase antibodies. J. Rheumatol. 2001, 28:1584
11. Grossmann K, Roggenbuck D, Schröder C, Conrad K, Schierack P, Sack U: Multiplex Assessment of Non-Organ-Specific Autoantibodies with a Novel Microbead-Based Immunoassay. 2011, Cytometry Part A! 79A: 118”125
Author
Dr. Christina Fritz*, Mandy Sowa and Dirk Roggenbuck
Medipan GmbH, Ludwig-Erhard-Ring 3,
15827 Dahlewitz,
Germany
*Corresponding author
E-mail: c.fritz@medipan.de
Which is the most common parasitic disease of the nervous system, which affection is the leading cause of seizures and acquired epilepsy in the developing world but still preventable? The answer: neurocysticercosis. An orphan disease suffering from the absence of a real ‘gold standard’ diagnosis. Meanwhile, many laboratories perform immunodiagnosis but what is its real value and what can it tell us?
by Dr Jean-François Carod
What is neurocysticercosis?
Cysticercosis of the central nervous system (neurocysticercosis) is caused by the larval stage (cysticerci) of the pork tapeworm Taenia solium. When people eat undercooked pork containing viable cysticerci, they develop an intestinal tapeworm infection (Fig. 1). Humans can also become intermediate hosts, however, by directly ingesting T. solium eggs shed in the feces of human carriers of the parasite. These eggs then develop into cysticerci, which migrate mostly into muscle (causing cysticercosis) and into the central nervous system where the cysticerci can cause seizures and many other neurological symptoms, neurocysticercosis (NCC). NCC is a major cause of epilepsy in endemic countries. It is the most important neurological disease of parasitic origin in humans. The pathogenesis is unclear but symptoms seem to correlate with the stage of the cyst. Starting as a viable entity, the cyst then gradually degenerates and become calcified. Seizures seem to appear at the degenerating and calcified stage but treatment is effective on the living cysts. Human cysticercosis is endemic in the Andean area of South America, Brazil, Central America and Mexico; China, the Indian subcontinent, South-East Asia; and Sub-Saharan Africa including Madagascar.
Why do we need to diagnose it?
Diagnosing NCC is required in the event of unexplained encephalitic disorders such as first onset of seizures in countries where NCC is endemic or in patients travelling in countries where NCC is endemic and who may have been at risk of infection (e.g. exposed to NCC risk factors, such as inadequate hand and food hygiene).
How can it be diagnosed?
The diagnosis of cysticercosis of the central nervous system involves the interpretation of non-specific clinical manifestations, such as seizures, often with characteristic findings on computed tomography (CT) or magnetic resonance imaging (MRI) of the brain, and the use of specific serological tests (Fig. 2). Diagnostic criteria based on objective clinical, imaging, immunological and epidemiological data have been proposed but are not generally used in areas endemic for the disease [1].
Serology is indicated for the diagnosis of T. solium seropositivity. But from a positive serology to the assessment of NCC diagnosis, there is a huge gap. A positive T. solium serology is not predictive for a neurological localization and serology may remain positive years after the end of the infection.
No single test can lead to a definitive diagnosis of NCC. CT-scan or MRI may be performed on the presentation of clinical symptoms that could be attributed to NCC (first onset of seizure, unexplained headache…) for people who were exposed to NCC risk factors. Imaging may show typical ring lesions with or without inflammation and calcification. However, the image is not pathognomonic of NCC unless hooks (scolex) are visible inside the ring. Thus, serology may give the clue if positive. A positive serology (antibody) may be confirmed by Western-blot or electro-immuno transfer blot (EITB), which show the typical bands specific of T. solium glycoproteins. Antigen detection in the blood can also be performed. This test is specific for T. solium and does not require laboratory confirmation. Both antigen and antibody assays can be performed in the cerebrospinal fluid (CSF). The presence of antibody or antigen in the CSF may contribute towards the assessment of the neurological localization of the disease. In developing countries, the regions most affected by T. solium infection, CT-scan and, of course, MRI are unaffordable, if ever available.
What are the current laboratory tools?
The laboratory diagnosis of cysticercosis is basically the immunodiagnostic based firstly on antibody detection with ELISA (enzyme-linked immunosorbent assay) or immunoblot.
The detection of antibodies against T. solium is a common method of infection diagnosis, but presents many limitations as a single cyst carrier may not be easily detected. Commercially available tests include essentially ELISA and Western-blots. Western-blots are the ‘gold standard’ assays for the detection of specific antibodies against T. solium. The reference Western-blot assay remains the one developed at the Centers for Disease Control (CDC), Georgia, USA, by Tsang et al. [2]. It employs a specific fraction of T. solium cysts. Many of the components have been identified and cloned. The test is very specific for exposure and/or disease and to confirm the diagnosis. Both ELISA tests and Western-blot relay on antigens that have varied significantly throughout the years (Fig. 3) [3]. Historically, the first assays used crude soluble extracts, then purified proteins such as lentil lectin glycoproteins (LLGPs) Recent trends, though not yet commercialized, tend to emphasize the use of recombinant proteins. Designing recombinant antigens requires a proteinomic approach (Fig. 4) that is now frequently used in development units. Current studies propose the use of nanobodies for diagnostic purposes. These evolutions increased both the sensitivity and the specificity of the tests.
Another available technique is based on the detection of circulating parasitic antigens using monoclonal antibodies [4]. This test is capable of detecting single cyst carriers and is more specific than available antibody ELISA tests. Its main advantage is its ability to monitor the response to cysticidal therapy.
Understanding the performance assessment of T. solium detection tests
Most commercially available ELISA tests have been evaluated by poor methodology. Assessing that a performance evaluation used the proper method means ensuring that the study used a serum bank of parasitologically-defined sera to assess test sensitivity. Defined cysticercosis sera should ideally include the following sera: two or more viable cysts, single viable cysts, degenerating cysts, calcified cysts.
Each series should be initially tested separately. A parasitologically-defined sera should correspond to the Del Brutto criteria [1]. In the absence of a true ‘gold standard’ for the diagnosis of neurocysticercosis, positive sera (cases) should be taken from patients with (1) absolute diagnosis of NCC, or (2) probable NCC diagnosis.
The test specificity should be carefully evaluated using defined negative and potentially cross-reactive sera. Negative sera (control) should be taken from the same area and if possible from people exposed to the same risk factors as the positive cases, with age and sex correlation. Negative cases are usually taken from blood donors of developed countries. Those people have not been in contact with many parasitic infections and the sensitivity of the test will not be accurate/reliable for use in developing countries. This is why specificity should not only be assessed on negative samples from Western countries but also on other parasitic infections from cysticercosis-free developing countries.
What are the new trends in laboratory tests?
If only immunodiagnostic tools based on antibody or antigen detection are currently commercialized, new approaches have been developed including molecular biology (gene amplification in CSF mostly) (Fig. 5). However, so far none constitutes a ‘gold standard’. Table 1 summarizes the pros and cons of NCC diagnosis tools.
Conclusions and future
A test is reliable and useful if it contributes to a care improvement; that is to say to an appropriate therapy for all the patients. As for NCC; the decision to treat is still subject to controversy. Furthermore, even basic serologies are unaffordable or unavailable in endemic countries, not to mention imaging. The key will be in developing a reliable rapid test able to screen infected patients and correlated to neurological lesions of cysticerci.
References
1. Del Brutto OH. Diagnostic criteria for neurocysticercosis, revisited. Pathog Glob Health 2012; 106(5): 299–304.
2. Tsang VC, Brand JA, Boyer AE. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J Infec Dis. 1989; 159(1): 50–59.
3. Esquivel-Velázquez M, Ostoa-Saloma P, Morales-Montor J, Hernández-Bello R, Larralde C. Immunodiagnosis of neurocysticercosis: ways to focus on the challenge. J Biomed Biotechnol. 2011; 2011: 516042. Doi:10.1155/2011/516042.
4. Garcia HH, Harrison LJ, Parkhouse RM, Montenegro T, Martinez SM, Tsang VC, Gilman RH. A specific antigen-detection ELISA for the diagnosis of human neurocysticercosis. The Cysticercosis Working Group in Peru. Trans R Soc Trop Med Hyg. 1998; 92(4): 411–414.
The author
Jean-François Carod Pharm D, MSc
Laboratoire de Biologie Médicale, GCS de l’ARC Jurassien, Centre Hospitalier Louis Jaillon, 2 Montée de l’hôpital, 39200 Saint-Claude, France.
E-mail: jean-francois.carod@ch-stclaude.fr
Colorectal 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
Prostate specific antigen (PSA) has significantly improved the early detection of prostate cancer (PCa), reducing the related mortality rate. However, PSA has a low specificity, being affected by many benign conditions. [-2]proPSA, a PSA precursor, is a more specific and accurate biomarker indicating prostate biopsy in men at real risk of PCa.
by Dr A. Abrate, Dr M. Lazzeri and Prof. G. Guazzoni
PSA as a marker for prostate cancer
Prostate specific antigen (PSA) is a serum marker widely used for the early detection of prostate cancer (PCa). Its introduction into clinical practice in the early 1990s had an extraordinary impact on the diagnosis and management of PCa. In fact, 20 years after its introduction, the PSA-based PCa opportunistic or systematic screening has resulted in a stage migration to more organ-confined tumours at the time of diagnosis, and consequently to a consistent reduction in PCa related mortality [1, 2]. However, PSA is not a perfect marker for the detection of PCa because of its low specificity and sensitivity. Its levels may increase as a result of benign conditions, such as benign prostatic hyperplasia (BPH) and chronic prostatitis. Moreover, PSA levels are also affected by biologic variability, which may be related to differences in androgen levels, prostate manipulation or ejaculation. Finally, alterations in PSA levels may be related to sample handling, laboratory processing, or assay standardization. All these factors made it difficult to find an appropriate PSA cut-off point diagnostic for PCa (for many years considered to be 4 ng/ml).
Thus, prostate biopsy is still mandatory to confirm the diagnosis. However, this is positive in only approximately 30% of patients [3], and the European Association of Urology suggests a repeat biopsy if PSA is persistently elevated, the digital rectal examination (DRE) is suspicious, or there is a pathological diagnosis of atypical small acinar proliferation (ASAP) or high-grade prostatic intraepithelial neoplasia (HG-PIN) [4]. Finally, PCa (also high-grade cancer) is not rare (approximately 15.2%) among men with PSA levels lower than 4 ng/ml, the previously widely accepted cut-off point [5].
Considering all these observations, it is clear that PSA is an organ-specific rather than an ideal cancer-specific marker.
The introduction into clinical practice of measuring the levels of several derivatives of PSA (free PSA, percentage of free PSA, PSA density, PSA velocity) improved the accuracy of total PSA (tPSA) in detecting PCa. Recently, free PSA (fPSA) has been found to include several subforms, such as proPSA. In particular [-2]proPSA seems to be specific for PCa, opening new ways for early cancer detection.
Biological basis of proPSA
The currently measurable serum tPSA consists of either a complexed form (cPSA, 70–90%), bound by protease inhibitors (primarily alpha1-antichymotrypsin), and a non-complexed form (fPSA). Recently fPSA has been discovered to exist in at least three molecular forms: proPSA, benign PSA (BPSA), and inactive intact PSA (iPSA), covering approximately 33%, 28%, and 39% of fPSA respectively (Fig. 1) [6]. In particular, proPSA is a proenzyme (precursor) of PSA, which is associated with PCa [7].
PSA is synthesized with a 17-amino acid leader sequence (preproPSA) that is cleaved co-translationally to generate an inactive 244-amino acid precursor protein (proPSA, with seven additional amino acids compared to mature PSA). proPSA is normally secreted from the prostate luminal epithelial cells. Immediately after its release into the lumen, the pro-leader part is removed, creating the active form, by the effect of human kallikrein (hK)-2 and hK-4, which have a trypsin-like activity and are expressed predominantly by prostate secretory epithelium. Other kallikreins, localized in the prostate, such as hK216 or prostin17, are involved in the conversion and activation of proPSA. Cleavage of the N-terminal seven amino acids from proPSA generates the active enzyme, which has a mass of 33 kDa.
The partial removal of this leader sequence leads to other truncated forms of proPSA. Thus, theoretically seven isoforms of proPSA could exist, although only [-1], [-2], [-4], [-5], [-7]proPSA were found; there is still no evidence of [-3], [-6]proPSA. However, all forms of proPSA are enzymatically inactive [8]. It is possible to detect three truncated forms of proPSA in serum: [-5/-7], [-4] and [-2]proPSA, which is the most stable form (Fig. 1).
Notably, in vitro experiments showed that the [-2]proPSA form cannot be activated by either hK2 or trypsin; thus, once it is formed, [-2]proPSA is resistant to activation into the mature PSA form and consequently this is the most reliable test.
Mikolajczyk et al. [7], using a monoclonal antibody recognizing [-2]proPSA, found increased staining in the secretions from malignant prostate glands. In particular [-2]proPSA is differentially elevated in peripheral gland cancer tissue; conversely transition zone tissue contains little or no proPSA.
The increased serum tPSA concentrations in patients with PCa do not result from increased expression but rather from an increased release of PSA into the bloodstream, due to disruption of the epithelial architecture. fPSA is catalytically inactive because of internal cleavages, occurring in seminal plasma, and does not form complexes with protease inhibitors or other proteins: in PCa %fPSA is lower presumably because, consequently to an increased release of PSA into the bloodstream, a very low part is still degraded into the ducts.
In another later study [9], Mikolajczyk et al. found that [-2]proPSA was specifically higher in patients with PCa. Analysing a small number of patients with biopsy positive for PCa and tPSA between 6 and 24 ng/ml, they found that [-2]proPSA constituted a high fraction of fPSA (25% to 95%), which was greater than in patients with a negative biopsy. However, the molecular basis for the proPSA elevation in PCa is uncertain, although a decreased cleavage by hK2 could be the cause.
Clinical utility of proPSA
Sokoll et al. [10] were the first to study the role of proPSA in the early detection of PCa. The study involved archival serum from 119 men (31 PCa, 88 non-cancer), obtained before biopsy and in the tPSA range of 2.5–4.0 ng/ml. The serum levels of tPSA, fPSA, proPSA, and proPSA/fPSA ratio (%proPSA) were analysed: PSA and %fPSA values were similar between the non-cancer and PCa groups, and %proPSA was relatively higher in the PCa group (50.1±4.4%) compared to the non-cancer group (35.5±6.7%; P=0.07). Concerning the clinical utility, the area under the curve (AUC) for %proPSA was 0.688 compared to 0.567 for %fPSA. At fixed sensitivity of 75%, the specificity was significantly greater for %proPSA at 59% compared with %fPSA at 33% (P<0.0001).
Afterwards, the Prostate Health Index (PHI) has been proposed as a mathematical algorithm combining tPSA, fPSA and [-2]proPSA according to the formula: ([-2]proPSA/fPSA) × √tPSA.
A large American prospective trial [11], involving 892 men who had tPSA levels of 2–10 ng/ml and negative digital rectal examination results, showed that PHI had greater predictive accuracy for prostate biopsy outcome (AUC 0.703) than [-2]proPSA (AUC 0.557), %fPSA (AUC 0.648) and PSA (AUC 0.525), directly correlating with Gleason score (GS) (P=0.013), with an AUC of 0.724 for GS ≥4+3 disease. Moreover, men with PHI >55 had a 42% likelihood of being diagnosed with high-grade disease on biopsy compared to 26% of men with PHI 0–24.9.
Accordingly, an observational European multicenter cohort study involved 646 men with tPSA levels of 2–10 ng/ml, who had undergone prostate biopsy [12]. [-2]proPSA and PHI improved the predictive accuracy for the detection of overall PCa (and also GS ≥7 disease) compared to PSA and derivatives. In fact, at 90% sensitivity, the PHI cut-off of 27.6 could avoid 100 (15.5%) biopsies, missing 26 (9.8%) cancers (23 with GS 6, three with GS 3+4).
Moreover, a PHI based nomogram to predict PCa at extended prostate biopsy was developed and validated over 729 patients [13]. Including PHI in a multivariable logistic regression model, based on patient age, prostate volume, digital rectal examination and biopsy history, significantly increased predictive accuracy by 7% from 0.73 to 0.80 (P<0.001). Decision curve analysis showed that using the PHI based nomogram resulted in the highest net benefit.
Recently, it was demonstrated that PHI might have a role in screening patients at high risk of PCa [14]. Specifically, the study involved 158 men with a positive family history undergoing prostate biopsy within the multicentre European PROMEtheuS cohort. Similarly to previous studies in the general population, PHI outperformed tPSA and %fPSA for PCa detection on biopsy (AUC 0.73, 0.55 and 0.60, respectively). In addition, both [-2]proPSA and PHI were directly associated with GS in men with a positive family history. Overall, the authors reported that using a PHI cutoff value of 25.5 would have avoided 17.2% of biopsies while missing only two GS 7 cancers. On decision curve analysis, the addition of PHI to a base predictive model that included age, prostate volume, tPSA, fPSA and %fPSA resulted in net benefit at threshold probabilities of 35–65%. This result suggests that PHI should be incorporated into a multivariable risk assessment for high-risk patients because it offers improved performance for PCa detection.
Conclusions
[-2]proPSA and PHI are more accurate than the currently used tests (PSA and derivatives) in predicting the presence of PCa at biopsy. Their implementation in clinical practice has the potential to significantly increase physicians’ ability to detect PCa and avoid unnecessary biopsies. Further work is needed to confirm and generalize these data on wider populations.
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The author
Alberto Abrate* MD; Massimo Lazzeri MD, PhD; and Giorgio Guazzoni MD
Dept of Urology, Ospedale San Raffaele Turro, San Raffaele
Scientific Institute, Milan, Italy
*Corresponding author
E-mail: alberto.abrate@gmail.com
March 2026
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