The discovery of reliable biomarkers, which are eligible for the prediction of both disease progression and response to treatment, means a great challenge in the management of multiple sclerosis (MS), a devastating disease of the central nervous system. The results of recent proteomic findings from the cerebrospinal fluid of MS patients hold promise of finding ideal biomarkers in the near future.
by Dr J. Füvesi, Dr C. Rajda, Dr D. Zádori, Dr K. Bencsik, Prof. Dr L. Vécsei and Prof. Dr J. Bergquist
Multiple Sclerosis
Multiple sclerosis is a demyelinative disorder of the central nervous system that affects mainly young adults. It has a great impact on quality of life, social and family life, and the careers of the patients.
In the majority of cases the disease starts with a relapsing–remitting (RR) phase. After a variable period of time it turns into a secondary progressive (SP) phase characterized by the gradual accumulation of residual symptoms. In 10–15% of cases a continuous progression is observed from the very beginning, this is the primary progressive (PP) form. In very rare fulminant cases frequent relapses with incomplete remissions cause severe disability or even death in a short duration of time.
The diagnosis of multiple sclerosis is still mainly clinical, supported by MRI and cerebrospinal fluid (CSF) analysis findings. The revised McDonald Criteria [1] allow earlier diagnosis, especially in PP MS. The routine diagnostic CSF analysis in MS includes the detection of oligoclonal bands and quantitative IgG analysis. Isoelectric focusing (IEF) on agarose gels followed by immunoblotting is considered the ‘gold standard’ for detecting the presence of oligoclonal bands [2]. The sensitivity of the method is above 95% and the specificity is more than 86%. An increased IgG index and the presence of oligoclonal bands in the CSF support an MS diagnosis.
Although the diagnosis is quite straightforward in most cases, taking into account clinical findings and paraclinical tests, there are still no specific biomarkers to confirm the diagnosis nor do we have any validated prognostic markers to follow the progression of the disorder.
At the time of diagnosis, major problems include the identification of the different clinical forms of the disease and the identification of patients with a potential rapid progression before disability evolves; the differential diagnosis of clinically isolated syndrome (CIS) with optic neuritis as the presenting symptom, where neuromyelitis optica (NMO) spectrum disorder may be an alternative diagnosis. Markers of disease progression are needed to distinguish CIS patients with a high probability to develop clinically definite MS.
There is also a need for biomarkers of response to treatment and biomarkers for better understanding the underlying pathological processes of the disease. This is especially important with the growing variety of treatment options: now it is possible to change therapy in the case of an inadequate treatment response and to escalate MS treatment to more aggressive alternatives. In the near future individualized treatment choices need better classification of patient characteristics.
In order to discover new biomarkers in MS, one should analyse the whole protein content of body fluids, preferentially CSF. Because of its proximity to the central nervous system (CNS), CSF may reflect changes in the CNS that may help differentiate normal and pathological conditions.
Proteomics
Proteomics is the study of protein expression in an organism. There are excellent reviews on proteomic approaches [3–5], so we will discuss here only certain aspects of these methods relevant to multiple sclerosis biomarker research. Mass-spectrometry (MS in Italic to distinguish from multiple sclerosis in this paper) based protein identification strategies include whole-protein analysis (‘top-down’ proteomics) and analysis of enzymatically produced peptides (‘bottom-up’ proteomics) [4]. The latter is the standard for large-scale or high-throughput analysis of highly complex samples, and digestion with trypsin is the most common method. The separation of peptides and proteins is an important element of both approaches.
Mass spectrometry measures the mass-to-charge ratio (m/z) of ionized molecules, and, as multiple distinct peptides can have very similar or identical molecular masses, it can be difficult to identify the overlapping peptides [3]. The use of separation techniques, therefore, reduces the cases of coincident peptide masses simultaneously introduced into the mass spectrometer. One of the most commonly used separation techniques is high-performance liquid chromatography (HPLC) with a capillary column. Peptides of similar molecular mass but different hydrophobicity elute from the LC column and enter the mass spectrometer at different time points, no longer overlapping in the initial MS analysis. Liquid chromatography coupled to mass spectrometry reduces the complexity of the sample and allows more precise protein identification.
In order to limit the risk of systematic errors and achieve a high sample throughput, labelling by means of isobaric tags for relative and absolute quantification (iTRAQ) may be used [6]. Multiple samples may be processed in parallel with this multiplexed approach. The main advantage is that the samples are analysed under exactly the same conditions. The relative abundance of labelled peptides indicates relative changes in protein expression.
LC-MS experiments generate an enormous amount of data, making data analysis one of the most challenging parts of proteomic analysis. Protein identification and quantification is achieved by database searching. Programs, such as Mascot etc., compare observed spectra to predicted spectra for candidate peptides from a protein database. In a recent study Schutzer et al. established a database of the normal human CSF proteome [7].
Proteomics in multiple sclerosis
In recent years a number of papers appeared describing proteomic analysis of CSF or brain tissue of multiple sclerosis patients [8–12]. The first papers in the field analysed pooled samples from a relatively small group of patients [8, 9]. Hammack et al. [8] reported the analysis of a pooled sample of three relapsing–remitting MS patients and a pooled sample of three patients with non-MS inflammatory CNS disorders using two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting. They identified four proteins in the gels containing MS CSF that were not reported previously in normal human CSF: CRTAC-1B (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein) and autotaxin t (a phosphodiesterase).
In the study of Dumont et al. [9] CSF samples from five MS patients (4 RR, one SP) were analysed by 2-DE followed by liquid chromatography tandem mass spectrometry. With this method 15 proteins have been identified that were not previously observed in non-multiple sclerosis CSF 2-DE gels. These proteins were: psoriasin, calmodulin-related protein NB-1, annexin 1, EWI-2, Niemann–Pick disease type C2 protein (NPC-2), semenogelin 1 (SEM1), semenogelin 2 (SEM2), complement factor H-related protein 1 (FHR-1), procollagen C-proteinase enhancer protein (PCPE), aldolase A, N-acetyllactosaminide β-1,3-N-acetylglucosaminyl-transferase, tetranectin, cystatin A, superoxide dismutase 3 and glutathione peroxidase.
Later, publications started to focus on the differentiation of the clinical forms of the disease. Lehmensiek et al. compared CSF samples from RR MS and clinically isolated syndrome (CIS) patients with controls using two-dimensional difference gel electrophoresis (2-D-DIGE) and matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry [10]. In RR MS Ig kappa chain NIG93 protein was increased in concentration, while transferrin isoforms, alpha 1 antitrypsin isoforms, alpha 2-HS glycoprotein, Apo E and transthyretin decreased. In a study of Stoop et al. [11] significant differences were observed comparing the peak lists of spectra from CSF of MS patients and patients with other neurological diseases (OND), and also clinically isolated syndrome (CIS) vs OND. Three differentially expressed proteins were identified in the CSF of MS patients compared to CSF of patients with OND: chromogranin A, clusterin and complement C3.
The same group compared proteome profiles of CSF from RR and PP multiple sclerosis and found that they overlap to a large extent [13]. The main detected difference was that protein jagged-1 was less abundant in PP MS compared to RR MS, whereas vitamin D-binding protein was only detected in the RR MS CSF samples. Ottervald et al. found an increased CSF level of vitamin-D-binding protein in SP MS compared to the control [14]. Recently, impaired vitamin D homeostasis has been linked to multiple sclerosis [15]: high serum levels of 25-hydroxyvitamin D correlated with a reduced risk of MS [16] and vitamin D supplementation was proposed as an add-on therapy [17].
Biomarkers of disease progression are emerging as new targets of proteomics. In our recently published paper we analysed the CSF of a rare fulminant case of MS and compared it with RR MS and control samples [18]. The aim of this study was to identify proteins related to rapid progression. The presented bottom-up strategy, based on isobaric tag labelling in conjunction with enzymatic digestion followed by nanoLC coupled off-line to MALDI TOF/TOF MS resulted in the identification of 78 proteins. Seven proteins were found to be upregulated in both fulminant MS samples but not in the relapsing–remitting case compared to the control. These proteins included Ig kappa and gamma-1 chain C region, complement C4-A, fibrinogen beta chain, serum amyloid A protein, neural cell adhesion molecule 1 and beta-2-glycoprotein 1. These proteins are involved in the immune response, blood coagulation, cell proliferation and cell adhesion.
Disease progression may be examined by analysing CSF samples from CIS patients who remain CIS and CIS patients who convert to clinically definite multiple sclerosis. Comabella et al. [19, 20] analysed pooled CSF samples with
isobaric labelling and mass spectrometry. They found that chitinase 3-like 1, ceruloplasmin and vitamin D-binding protein were upregulated in CSF of patients converted to clinically definite MS. In order to validate their results, the authors determined the levels of these selected proteins by enzyme-linked immunosorbent assay (ELISA) in individual CSF samples. Only chitinase 3-like 1 was validated. In a second validation step CSF chitinase 3-like 1 levels were measured in an independent CIS cohort and its level was again significantly increased in CIS patients who later converted to MS, compared to patients who remained as CIS. High CSF levels of this protein significantly correlated with the number of gadolinium enhancing and T2 lesions on baseline brain MRI scans and disability progression during follow-up.
The search for biomarkers that are able to identify patients at high risk of rapid progression becomes increasingly important with the appearance of more aggressive treatment possibilities. In another ongoing study we currently analyse LC-Fourier transform ion cyclotron resonance (FTICR) MS [20–22] data of a larger set of CSF samples from a variety of clinical forms of MS and matched controls.
Despite the increasing number of studies investigating potential biomarkers of MS disease progression and response to therapy, there is still no protein that is repeatedly identified and validated by different groups. This may be due to the relatively small sample sizes and the heterogeneity of the methods applied. Large scale multi-centre projects using standard methods for collecting, storing and analysing the samples are necessary to validate these preliminary results and integrate candidate biomarkers into the pathomechanism of the disease.
A great step in this direction is the BIOMS project, which aims a standardized sample collection, storage and processing during the preanalytical steps to rule out the differences occurred by sample preparation [23–25] and test the different methods and hypotheses on a great sample number in multiple centres to shed light on the sources of errors using different methods. One of these initiatives was the neurofilament validation study, which is a candidate biomarker in multiple sclerosis [26]. Another validation study tested two different methods of detecting the neutralizing antibodies against interferon-beta therapy, which is a biomarker of therapy in MS [27].
In the future multi-centre studies on standardized samples and methods can bring us closer to solve the questions regarding the pathological processes and the classification of patients to the most appropriate therapy.
Acknowledgement
TÁMOP-4.2.2.A-11/1KONV/-2012-0052 and The Swedish Research Council 621-2011-4423 are gratefully acknowledged for financial support.
References
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3. Karpievitch YV, et al. Liquid Chromatography Mass Spectrometry-Based Proteomics: Biological and Technological Aspects. Ann Appl Stat 2010; 4: 1797–1823.
4. Han X, et al. 3rd Mass spectrometry for proteomics. Curr Opin Chem Biol 2008; 12: 483–490.
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10. Lehmensiek V, et al. Cerebrospinal fluid proteome profile in multiple sclerosis. Mult Scler 2007; 13: 840–849.
11. Stoop MP, et al. Multiple sclerosis-related proteins identified in cerebrospinal fluid by advanced mass spectrometry. Proteomics 2008; 8: 1576–1585.
12. Han MH, et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 2008; 451: 1076–1081.
13. Stoop MP, et al. Proteomics comparison of cerebrospinal fluid of relapsing remitting and primary progressive multiple sclerosis. PLoS One 2010; 5: e12442.
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19. Comabella M, et al. Cerebrospinal fluid chitinase 3-like 1 levels are associated with conversion to multiple sclerosis. Brain 2010; 133: 1082–1093.
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27. Bertolotto A, et al. Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta. J Immunol Methods 2007; 321: 19–31.
The authors
Judit Füvesi1 PhD, MD; Cecilia Rajda1 PhD, MD; Dénes Zádori1 PhD, MD; Krisztina Bencsik1 PhD, MD; László Vécsei1,2 PhD, MD; and Jonas Bergquist3,4* PhD, MD
1 Department of Neurology, Faculty of Medicine, Albert Szent-Györgyi Clinical Center, University of Szeged, Szeged, Hungary
2 Neuroscience Research Group of Hungarian Academy of Sciences and University of Szeged, Szeged, Hungary
3 Analytical Chemistry, Department of Chemistry-Biomedical Center, Uppsala University, Uppsala, Sweden
4 Science for Life Laboratory (SciLife Lab), Uppsala University, Uppsala, Sweden
*Corresponding author
E-mail: jonas.bergquist@kemi.uu.se
Coronary artery disease has been linked to a hypercoagulable state of the blood, and the use of global hemostatic assays such as thromboelastography, thrombin generation or the overall hemostatic assay may allow for prediction of adverse events in these patients as well as targeted, individualized treatment.
by Dr C. Reddel, Dr J. Curnow and Professor D. Brieger
Global hemostatic markers in coronary artery disease
Hemostasis is the process by which bleeding is stopped, involving blood coagulation and platelet aggregation. This process depends on the delicate balance of many pro- and anti-coagulant factors, and when hemostatic balance is disrupted, pathological clot formation may occur leading to potentially fatal venous or arterial thrombosis. Appropriate fibrinolysis, the breakdown of blood clots, is also essential to the process of hemostasis.
Coronary artery disease is considered an inflammatory disease in which patients are predisposed to arterial thrombosis, which can lead to myocardial infarction. Additionally, the presence of coronary artery disease can increase the risk of venous thrombosis [1]. This points to an overall hypercoagulable state of the blood in this disease. Although the use of antiplatelet and anticoagulant therapies is a common and necessary method of reducing this risk, this may unnecessarily expose patients to a risk of bleeding. There is a need to risk stratify patients and individually tailor thromboprophylaxis.
Imbalances in the hemostatic system can be assessed in citrated plasma samples from patients either by measuring individual coagulation and fibrinolytic factors, or by global coagulation assays. Such imbalances have been found to be associated with various pro-thrombotic states, such as cancer, pregnancy or trauma. In stable and acute coronary artery disease, there is evidence for links between prognosis and markers of coagulation and fibrinolysis, including prothrombin fragment 1+2, fibrinopeptide A, thrombin–antithrombin and plasmin–antiplasmin complexes, D-dimer, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator [2, 3]. However, measuring single factors does not reflect the overall hemostatic balance as other pro- or anti-coagulant, and pro- and anti-fibrinolytic factors may compensate for the deficient or elevated factor. Therefore measurement of the overall coagulable state of the blood may provide a more relevant picture.
Standard laboratory coagulation tests, such as prothrombin time (PT) or activated partial thromboplastin time (APTT), can be useful for patients with bleeding disorders, but do not reliably detect hypercoagulability in this context. Recently, there has been interest in global assays of coagulation and fibrinolysis as methods of assessing the overall potential of a patient’s blood to form or lyse a clot. These include assays of thrombin generation, thromboelastography and the overall hemostatic potential assay.
Thromboelastography
Thromboelastography is a method measuring clot formation and lysis in whole blood. A pin is suspended into a cuvette of whole blood heated to 37°C, and the cup and pin move relative to each other, so that when the clot forms the interference is detected by the pin. Thromboelastography (TEG, Haemonetics, Braintree, Massachusetts, USA) and Thromboelastometry (ROTEM, Tem International GmbH, Munich, Germany) are two commercial variants of the assay. The assay measures not only time to clot, but speed of clot formation, clot strength and elasticity, and can be modified to assess platelet function, fibrinogen, hyperfibrinolysis and effect of anticoagulant treatment. The use of whole blood means the role of the cell is incorporated into the assay, although this necessitates immediate use of the sample.
Thromboelastography is a point-of-care assay which is used to measure and characterize peri-operative bleeding. It may additionally be useful in monitoring antiplatelet therapy such as aspirin or clopidogrel. Recently, it has also been used to detect hypercoagulability in patients with coronary artery disease, and further, has been demonstrated to predict thrombotic events in patients who have undergone coronary stenting or coronary artery bypass grafting [4, 5].
Thrombin generation assay
The thrombin generation assay was first described in 1953, but has more recently been simplified, standardized and commercialized, including in the form of the Calibrated Automated Thrombogram (Thrombinoscope BV, Maastricht, The Netherlands) and Technothrombin (TGA, Technoclone, Vienna, Austria) [6]. In this assay, ex vivo potential for thrombin generation is measured in platelet-rich or platelet-poor plasma. In a 96-well plate, thrombin generation is triggered by addition of tissue factor, phospholipids and calcium at 37°C, and conversion of a substrate for thrombin measured over an hour by fluorescence.
Thrombin is central to the process of hemostasis, and various pro-thrombotic states have been associated with variations in plasma potential to generate thrombin. Patients with stable coronary artery disease have elevated thrombin generation [Fig. 1] [7], and patients with acute coronary syndrome have still higher thrombin potential [8]. Antiplatelet therapies most likely do not affect the thrombin generation assay in platelet-poor plasma, but it may be possible to monitor the effect of anticoagulant drugs (including novel oral anticoagulants) using the assay, and preliminary assessment has suggested the assay can predict bleeding and ischemic events in patients with coronary artery disease [9].
Overall Hemostatic Potential (OHP) assay
The Overall Hemostatic Potential (OHP) assay is a test of fibrin generation and fibrinolysis first described in 1999 [10]. Similar to the thrombin generation assay, it is performed in citrated plasma in 96-well plates and triggered by tissue factor or thrombin and calcium at 37°C. It is a turbidometric assay, measuring the change in absorbance over an hour at 405nm, which allows for a kinetic analysis of fibrin clot formation. Tissue plasminogen activator is also added to half the wells, which triggers fibrinolysis. The assay measures coagulation potential and fibrinolytic potential, and is carried out on stored plasma samples.
A limitation of the plasma-based thrombin generation and OHP assays is the absence of cells. These assays have nonetheless identified differences between patients with pro-thrombotic states and healthy controls, and the use of plasma allows for samples to be stored and batch-tested, which is an advantage for screening large numbers of patients. The OHP assay additionally requires no specialized equipment, apart from a standard plate reader, and although not standardized, it is inexpensive. Unlike thromboelastography which is relatively insensitive to hypofibrinolysis, the OHP assay can detect and quantify hypofibrinolysis as well as hyperfibrinolysis.
Very recently the OHP assay has been used to show hypercoagulability and hypofibrinolysis in patients with acute and stable coronary artery disease [Fig. 2] [7, 11]. The observations in this latter population suggest the potential for this assay to predict future events, and prospective studies are required to determine its utility in this context.
Future trends and requirements
There is a growing body of evidence that ex vivo hypercoagulability of patients’ blood or plasma has prognostic value in arterial or venous thrombotic events. Global markers of hemostasis, including results of thromboelastography, the thrombin generation and OHP assays, may prove clinically relevant in identifying individual patients at risk of adverse event, and thus allow the tailoring of thromboprophylaxis. Further large-scale prospective trials are needed to directly address this.
References
1. Anandasundaram B, Lane DA, Apostolakis S, Lip GY. The impact of atherosclerotic vascular disease in predicting a stroke, thromboembolism and mortality in atrial fibrillation patients: a systematic review. J Thromb Haemost. 2013; 11: 975–987.
2. Stegnar M, Vene N, Bozic M. Do haemostasis activation markers that predict cardiovascular disease exist? Pathophysiol Haemost Thromb. 2003; 33: 302–308.
3. Gorog DA. Prognostic value of plasma fibrinolysis activation markers in cardiovascular disease. J Am Coll Cardiol. 2010; 55:2 701–709.
4. Hobson AR, Agarwala RA, Swallow RA, Dawkins KD, Curzen NP. Thrombelastography: current clinical applications and its potential role in interventional cardiology. Platelets 2006; 17: 509–518.
5. McCrath DJ, Cerboni E, Frumento RJ, Hirsh AL, Bennett-Guerrero E. Thromboelastography maximum amplitude predicts postoperative thrombotic complications including myocardial infarction. Anesth Analg. 2005; 100: 1576–1583.
6. Hemker HC, Giesen P, AlDieri R, Regnault V, de Smed E, Wagenvoord R, et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002; 32: 249–253.
7. Reddel CJ, Curnow JL, Voitl J, Rosenov A, Pennings GJ, Morel-Kopp MC, et al. Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay. Thromb Res. 2013; 131: 457–462.
8. Orbe J, Zudaire M, Serrano R, Coma-Canella I, Martinez de Sizarrondo S, Rodriguez JA, et al. Increased thrombin generation after acute versus chronic coronary disease as assessed by the thrombin generation test. Thromb Haemost. 2008; 99: 382–327.
9. Campo G, Pavasini R, Pollina A, Fileti L, Marchesini J, Tebaldi M, et al. Thrombin generation assay: a new tool to predict and optimize clinical outcome in cardiovascular patients? Blood Coag Fibrinolysis 2012; 23: 680-687.
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11. Leander K, Blomback M, Wallen H, He S. Impaired fibrinolytic capacity and increased fibrin formation associate with myocardial infarction. Thromb Haemost. 2012; 107: 1092–1099.
The authors
Caroline Reddel* PhD; Jennifer Curnow MBBS, PhD, FRACP, FRCPA; David Brieger MBBS, PhD, FRACP, FACC
ANZAC Research Institute, Concord Repatriation General Hospital, Concord NSW, 2139, Australia
*Corresponding author
E-mail: creddel@anzac.edu.au
As the appearance of circulating tumour cells in the peripheral blood of breast cancer patients is linked to a worse prognosis for overall survival and treatment efficiency, their detection and characterization will have a high impact on cancer therapy, opening roads to a more personalized treatment.
by Dr U. Andergassen, Dr A. C. Kölbl, Prof. K. Friese and Prof. U. Jeschke
Circulating tumour cells
Already in 1869 the occurrence of cancer cells in the peripheral blood of a metastatic cancer patient was described by Thomas Ashworth. Nowadays it is well known, that cells dissolve from primary epithelial tumours such as breast, lung, colon or prostate cancer, enter circulation and travel via the blood stream or lymphatic system throughout the whole body. If these cells [termed circulating tumour cells (CTCs)] leave circulation, they can settle at other sites in the body and are then considered to be the main reason for the generation of remote metastasis. Their appearance is linked to a poorer outcome of cancer therapy and to a worse prognosis for overall survival. Therefore, the detection of CTCs in peripheral blood [and of disseminated tumour cells (DTCs) in bone marrow] was already included into the international tumour staging systems.
Unfortunately the detection of CTCs is still a technical challenge, as the number of tumour cells in the blood stream is rather small (1 in 106–7 blood cells). To date, there is only one FDA-approved system for CTC detection, at least in the metastatic situation. This is the Cell Search® system (Veridex LLC.), which is based on immunomagnetic enrichment and simultaneous staining of tumour cells of epithelial surface markers, the cytokeratins. The huge disadvantage of this system is that it is rather expensive and, therefore, not yet routinely used in the clinic.
Real-time PCR in cancer cell detection
Another promising approach for CTC detection could be a real-time PCR-based method. The principle of this methodology is that breast-cancer CTCs are derived from an epithelial tumour, and, therefore, express a panel of epithelial cell genes. The surrounding blood cells in contrast are of mesenchymal origin, showing different gene expression profiles. Thus, it can be assumed that tumour cells are present in a given blood sample if the expression of epithelial genes is higher than in a negative control sample.
Real-time PCR measures gene expression levels by detecting an increase of fluorescence due to the incorporation of fluorescent reporter molecules into the newly synthesized DNA molecules during the PCR reaction. If a gene is highly expressed, a lot of mRNA of this gene is present, meaning plenty target for PCR reaction is available and thus influencing the fluorescence level measured at the end of each amplification cycle. The time point when fluorescence reaches a certain threshold is called the Ct-value, and this is the basis of the calculation of relative gene expression values by the 2-∆∆Ct-method [1]. In brief: the average Ct-value of a gene of interest is related to the average Ct-value of a reference gene. The resulting value is called the ΔCt-value. In the next step, this ΔCt-value is set in reference to the ΔCt-value of the same gene in the reference sample, rendering the so called ΔΔCt-value. The formula 2–ΔΔCt is then used to calculate relative quantification (RQ) values. RQ values >1 show an upregulation of the gene of interest, values <1 mean that the gene is downregulated.
Spiking experiments
The first step towards a real time PCR based quantitative cancer diagnosis is to create calibration curves for the used marker genes to evaluate the number of cancer cells exhibited at a certain level of gene expression in a blood sample. Therefore, blood samples of healthy donors, to which a certain number of cells from a breast-cancer cell line were added, were used to create standard curves. For this evaluation different breast-cancer cell lines were used (Cama-1, MCF-7, MDA-MB231 and ZR-75-1), and real-time PCR was carried out for Cytokeratin 8, 18 and 19 as marker genes [2, 3]. Cancer cells were added in rising numbers and calibration curves could be drawn [Fig. 1], showing an increase in gene expression level from 10 cells added to a blood sample upwards, meaning that even a small number of cancer cells in the blood (resembling the ‘real’ conditions, with 1 CTC per106–7 surrounding blood cells) can be detected by this methodology.
PCR marker genes for CTC detection
As CTCs in the blood are rare, PCR marker genes have to be selected as accurately as possible. The first choice are the Cytokeratin (CK) genes 8, 18 and 19, as they are also used in the routinely applicated APAAP-staining, which is a histochemical detection method for CTCs. The cytokeratin family members are characteristic epithelial cell markers and only weakly expressed in blood cells, rendering them potentially useful for PCR-based detection of CTCs.
Three other genes (BCSP, MGL, Her2) were selected and used in an approach to detect differences in gene expression between normal individuals and adjuvant and metastatic breast-cancer patients [4]. Mammaglobin (MGL) is a gene which is only expressed in the adult mammary gland and is known to be upregulated in breast cancer [5]. Breast cancer specific protein (BCSP) is highly expressed in advanced infiltrating breast cancer and is a marker for recurrence of the disease and formation of metastases [6], and c-erbB2 (Her2) was used, because it is over-expressed in 20% of breast cancers and is also responsible for the aggressiveness of the tumour [7].
These markers were comparatively analysed in blood samples withdrawn from adjuvant and metastatic breast-cancer patients during surgery. The gene expression levels of adjuvant as well as metastatic breast-cancer patients were normalized to levels in blood samples from 20 healthy donors, considered as a negative control group. Differences in gene expression between the three sample groups were detected [Fig. 2] and it was attempted to find a signature of marker genes for CTCs in breast cancer by real-time PCR.
From the experiments, it could be concluded that cytokeratin genes seem to be the most promising markers for the detection of CTCs from peripheral blood of breast-cancer patients with reverse-transcription real-time PCR. The most suitable marker of the cytokeratin array is apparently CK8, rendering most expression values >1.
MGL, BCSP, and Her2 mRNA show few expression values >1 as well in adjuvant as in metastatic patients. Altogether, higher amplitudes for these three genes were detected in the adjuvant setting. CTCs can be detected from peripheral blood by real-time-PCR, using the cytokeratin markers, especially cytokeratin 8.
In contrast to these findings are the results published by Obermayr et al. 2010 [8], who found an overexpression of MGL/hMAM in 39% of the examined advanced breast cancer cases. But they also conclude that using more marker genes for CTC detection results in a higher percentage of detected cancer cases. The same findings were obtained by [9], who also used a real-time PCR-based approach for CTC detection. They used CK19, SCGB2A2, MUC1, EPCAM, BIRC5 and Her-2 as marker genes and found a high sensitivity and specificity (56.3% and 100% respectively).
Additionally CK20 was identified as a promising marker gene [10] and seems to be correlated with the aggressiveness of the tumour. To further improve the detection of CTCs by real-time-PCR, more marker genes need to be tested; promising candidates are, for example, MMP13 [11], UBE2Q2 [12],
Nectin-4 [13], and ALDH [14].
Future directions for cancer therapy
Real-time PCR-based techniques were already used for solid tumour profiling and are considered to be objective, robust and cost-effective molecular techniques that could be used in routine cancer diagnosis. In future, a real-time PCR assay for the detection of circulating tumour cells from peripheral blood could find its way into modern medicine. This would be advantageous for the patient by limiting the number of invasive procedures, such as biopsies or bone marrow aspirations, that have to be undertaken to produce samples for analysis.
Furthermore by implication of more marker genes a characterization of tumour cells could be pursued, which already gives hints towards a cancer prognosis, as for example Bölke et al. described, that the expression of certain genes is correlated to advanced breast cancer stages [15]. A better knowledge of cancer properties in turn will help to apply a more personalized therapy, side effects can be reduced and treatment efficiency will strongly increase.
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The authors
Ulrich Andergassen* MD, Alexandra C. Kölbl PhD, Klaus Friese MD, and Udo Jeschke PhD
Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Ludwig Maximilian University of Munich, Munich, Germany
*Corresponding author
ulrich.andergassen@med.uni-muenchen.de
Leaders from the medical diagnostics, laboratory medicine, and healthcare fields convened in Houston, Texas, July 28 – August 1 for the AACC annual meeting, the world’s largest diagnostics conference and expo. Over 17,000 attendees took part in the event and the exhibit totalled more than 625 companies. A selection of research papers presented in Houston are summarized below.
New biomarkers for prostate cancer
Dimitra Georganopoulou, PhD, Ohmx Corporation, presented results of a pilot study to find a new biomarker for prostate cancer aggressiveness. The researchers measured the enzyme activity of prostate-specific antigen (PSA), termed the “aPSA”, in patient specimens that had been removed by radical prostatectomy. They wanted to determine if this activity level could be a clue to how aggressive the cancer was. The team found that there was a significant negative correlation between prostate cancer progression and the aPSA in prostatic fluid. Patients with the least amount of aPSA (PSA activity) had the most aggressive prostate cancer. Tests for an “aggressiveness biomarker” would provide critical information for making decisions about when clinical treatment should occur or when it could be postponed. Many men might be able to avoid radical treatments if their cancer was known to be non-aggressive. Likewise, men whose cancer was too aggressive to employ the “active surveillance” or “watchful waiting” approach would have more information to help them make meaningful personal decisions with the help of their doctors about what level of treatment was right for them. The findings from this study could lead to the development of a new tool to use along with existing screening tests.
PSA Enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness.
Dimitra Georganopoulou, PhD, OHMX Corporation, Evanston, Ill., U.S.A.
Diagnosing cystic fibrosis at the point of care
Xuan Mu from Peking Union Medical College presented test results from cystic fibrosis patients using an exciting new point-of-care method. Microfluidics and colour changes within a Band-Aid type of adhesive strip on the skin allow the new device to rapidly, accurately, and quantitatively diagnose cystic fibrosis in a small amount of sweat. Detecting sweat chloride has been the gold standard in diagnosing cystic fibrosis for more than 50 years. The new test detects increased chloride in sweat using a colour change in paper on an adhesive strip when a very small amount of sweat is absorbed. The intensity of changed colour is recorded with a cell phone camera, and is then measured against a colour model. Cystic fibrosis in an inherited disease of the body’s mucus glands. Technically a rare disease, the incidence of cystic fibrosis varies around the world and by ethnic group. Different mutations in the CFTR gene cause the severity and symptoms of CF to vary considerably. Respiratory and digestive systems are affected, as well as sweat glands and reproductive systems. The new point-of-care test device can distinguish healthy people from cystic fibrosis samples and conveniently integrates the many separate steps of current sweat chloride tests whose results take several hours to obtain. Treatment advances have increased the life expectancy of cystic fibrosis patients over the past several decades from the mid-teens in the 1970s to more than 36 years today in the U.S. An early diagnosis and a comprehensive treatment plan can improve both survival and quality of life of patients. This new method demonstrates a fast and cost-effective opportunity in diagnosing cystic fibrosis.
On-site colorimetric detection of sweat chloride ion for diagnosing cystic fibrosis.
Xuan Mu, Peking Union Medical College, Beijing, China
Determining the safety of olanzapine for schizophrenia and bipolar disorder
AACC member Werner Steimer from Munich, Germany presented the results of research showing that study patients who carried a specific genetic variation in an antipsychotic-metabolizing enzyme developed significantly higher serum concentrations of the drug olanzapine. The increased drug concentrations were still noteworthy even when researchers accounted for differences in the patient’s age, sex, weight, and other medications that they used. This is the first study to demonstrate that this polymorphism influences serum levels of olanzapine, and the study is extremely timely in the context of the recent FDA safety alert on the injectable form of olanzapine, an “atypical” or second generation antipsychotic medication. Under investigation are two unexplained deaths of patients who received an intramuscular injection of Zyprexa Relprevv (olanzapine pamoate) and showed very high blood levels of the drug, although they had received appropriate doses. They died 3-4 days after injection. Olanzapine is approved by the U.S. FDA for treating schizophrenia and bipolar disorder in adults and children older than 13, and is one of the most widely prescribed of the atypical antipsychotics. Olanzapine is available in tablet, injectable, and long-acting “depot” formulations. Long acting medications can be more tolerable to some patients and help them adhere to treatment. Olanzapine is metabolized in the liver by specific cytochrome P450 enzymes. Some individuals have genetic variations – polymorphisms – of cytochrome enzymes. These can impact the way that drugs are broken down and distributed throughout the body and sometimes even the strength or effectiveness of treatment.
The CYP1A2*1D Polymorphism has a significant impact on Olanzapine serum concentrations.
Werner Steimer, MD, Klinikum Rechts der Isar – Technische Universität München, Munich, Germany.
November 2024
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