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C131 Gul M Mustafa figure1

Quantitative proteomics: closing the gap between biomarker discovery and validation – the rate-limiting step

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Many potential cancer biomarkers have been identified, however, the transfer of these biomarkers from discovery to clinical practice is still a process filled with pitfalls and limitations. To prove clinical utility and performance, these candidate biomarkers need to be reproducible, specific, sensitive, and validated using large sample cohorts, all of which require development of robust and highly sensitive multiplex protein assays.

by Dr G. M. Mustafa, Prof. J. R. Petersen, Prof. L. Denner Prof. F. A. Hussain and Prof. C. Elferink

Background
Although remarkable scientific and technological advances in medicine have been achieved, cancer incidences are still increasing worldwide with the ratio of deaths to new cancer cases remaining relatively unchanged at 49% [1]. The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancer at an early stage. When cancer develops in an area of the body, such as an organ, as long as it hasn’t spread (metastasized), it may respond well to treatment and in some cases a cure may be possible.  Many disease states, especially various types of cancer, can be better diagnosed by the aid of biomarkers, a key element of modern diagnostics and the value of which continuing to increase in modern medicine. As indicators of biological status, biomarkers, whether detected in blood, urine, or tissue, can be useful for the clinical management of various diseases. The changes in biomarker concentration have the potential to guide therapy in disease progression, prognosis and potentially be used to identify the stage of the cancer.  Overall, protein biomarkers are needed to help our understanding of cancer biology and to improve our ability to diagnose, monitor and treat cancers.

Biomarker research
One of the goals of biomarker research is to discover and validate markers that can be used in clinical research applications such as patient stratification, diagnosis and therapeutic monitoring, or in pharmaceutical development to fully characterize the behaviour and efficacy of candidate drugs. The identification of prognostic, predictive or pharmacodynamic biomarkers can be carried out using genomic or proteomic technologies. Proteomic profiling of body fluids, such as serum, has potential as a sensitive diagnostic tool for early cancer detection. Serum provides a rich sample for diagnostic analyses because the expression and release of proteins (potential biomarkers) into the bloodstream occurs in response to specific physiological states. As human plasma has a 1010 dynamic range of proteins [2] with 22 proteins being responsible for 99% of the proteins identified, one of the challenges working with such a complex biological fluid is the difficulty in identifying medium and low abundance proteins. Thus, sample enrichment is a very important step in the discovery phase.

Biomarker validation – the rate-limiting step
Discovery platforms typically result in an extensive hit-list of candidate biomarkers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Over the last several years, many proteins have been proposed as potential biomarkers for various cancers without further evaluation of their clinical utility.  The lack of follow-up is due, to a large extent, to the lack of an efficient technology for reproducible and accurate verification of these proteins as biomarkers in a specific disease state.  Without proper validation, the identification of biomarkers is of minimal utility. To show clinical utility, biomarkers must be validated using a reliable assay with an independent cohort in a prospective or longitudinal study. A search of the scientific literature clearly indicates that most published biomarkers are inadequate for replacing an existing clinical test or that they are only useful for detecting disease in an advanced stage, where treatment success and/or survival rates are low. Traditionally, validation has been performed with immunoassays which require antibodies that are often unavailable or of poor quality. In addition the immunoassays may not adequately identify or differentiate between post-translational modifications. The process of generating antibodies is also time consuming and costly and is therefore not a practical solution for screening the hit-list of proteins derived from discovery.

Targeted mass spectrometry for multiplexed protein quantitation
A targeted mass-spectrometry-based antibody-free platform for multiplexed protein quantitation fills the well-recognized gap between early biomarker discovery and final clinical assay. Stable-isotope-dilution multiple-reaction-monitoring mass spectrometry (SID-MRM-MS) is a novel technique whereby the quantification of a protein can be calculated from isotopically labelled peptide standards of known concentrations that correspond to the protein of interest [3]. With two to five unique peptides per protein, the concentration of the protein of interest can be accurately determined.  Further, the technique can be multiplexed to allow for the simultaneous measurement of many proteins. We have validated this approach using serum samples to identify biomarkers in hepatocellular carcinoma and are confident that it also has the potential for biomarker discovery in other cancers [4].  This powerful workflow opens up new possibilities for biomarker research that may lead to faster, more robust, and improved clinical assays. Targeted proteomics workflows based on SRM and MRM on triple quadruple mass spectrometry platforms show the potential for rapid verification of biomarker candidates in plasma by using heavy isotope-labeled internal standards. This approach has the selectivity, reproducibility, and sensitivity for a range of multiplexed protein assays and has the potential for quantifying protein isoforms in addition to posttranslational modifications for which good quality antibodies often do not exist.  The ability to rapidly quantify proteins in a highly multiplexed manner using MRM with internal standard peptides closes the gap between discovery and validation in the biomarker pipeline, which is the rate-limiting step in the biomarker world. Using a triple quadruple mass spectrometer, the first mass analyser (Q1) is set to only transmit the parent weight of a peptide from the target protein, the collision energy is then optimized to produce a diagnostic charged fragment of a peptide fragment in the second mass analyser (Q2), and the third mass analyser (Q3) is set to only transmit and identify this diagnostic peptide fragment (Fig. 1). The key strength of this work flow is that the protein assay development for hundreds of proteins can occur on the order of a month. Once identified, it just takes months to validate the low to sub-ng/ml concentration of candidate biomarkers in hundreds of blood samples. No target-specific reagents, such as antibodies, are needed and only a very small amount of sample (<500ng) is required for quantitation. In addition all potential biomarkers can be quantified in a single assay that is more accurate, rapid, and cost-effective. The capacity of SRM for multiplexed, high-throughput analysis, together with its sensitivity and quantitation, positions SRM as a promising application in medical screening [5, 6]. Summary
The type of clinical proteomics described here has important direct ‘bedside’ applications. We can foresee a future in which the physician will use these proteomic analyses at many points in the management of disease and drug discovery.

References
1. Yeom YI, Kim SY, Lee HG, et al. Cancer biomarkers in ’omics age. Biochip Journal 2008; 2(3): 160–174.
2. Anderson L.  Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J Physiol. 2005; 15: 23–60.
3. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008; 4: 222.
4. Mustafa MG, Petersen JR, Ju H, Cicalese L, et al. Biomarker discovery for early detection of hepatocellular carcinoma in hepatitis C-infected patients. Mol Cell Proteomics 2013; 12(12): 3640–3652
5. Keshishian H, Addona TA, Burgess M, et al. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics (2007); 6(12): 2212–2229
6. Zhao Y, Jia W, Sun W, et al. Combination of improved 18O incorporation and multiple reaction monitoring: a universal strategy for absolute quantitative verification of serum candidate biomarkers of liver cancer. J Proteome Res. (2010); 9(6): 3319–3327

The authors
Gul M. Mustafa*1 PhD, John R. Petersen2 PhD, Larry Denner3 PhD, Feroze A. Hussain4 MD, and Cornelis Elferink1 PhD
1 Department of Pharmacology, University of Texas Medical Branch, Galveston, Texas, USA
2 Victory Lakes Clinical Laboratory, Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA
3 Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA
4 Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA

*Corresponding author
E-mail: gmmustaf@utmb.edu

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C149 Euroimmun Fig1 PLA2R structure

Anti-PLA2R: the serological biomarker for primary MN

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Autoantibodies against phospholipase A2 receptors (PLA2R) are a new, highly specific diagnostic marker for primary membranous nephropathy (MN). Detection of anti-PLA2R using easy-to-perform and inexpensive serological assays can indicate primary MN in patients suffering from nephrotic syndrome and secure a differential diagnosis from secondary MN. Anti-PLA2R analysis is also useful for determining the disease activity, assessing the extent of treatment required and monitoring responses to therapy. Anti-PLA2R antibodies can be determined using innovative indirect immunofluorescence and ELISA test systems.

by Jacqueline Gosink, PhD

Primary membranous nephropathy
Primary MN, also known as primary membranous glomerulonephritis or primary MGN, is a chronic inflammatory autoimmune disease of the blood-filtering structures of the kidneys (glomeruli). It is accompanied by a progressive reduction in renal function. The disease manifests the complex nephrotic syndrome, which is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, edema and lipiduria. Primary MN is one of the leading causes of nephrotic syndrome in adults. As proteinuria increases, so does the long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications. Around a third of patients progress to end-stage renal disease, a third exhibit persistent proteinuria without progression to renal failure, and the remainder experience spontaneous remission. Primary MN is prevalent in all ethnic groups and in both genders, with men over 40 years old being the most frequently affected.

Diagnostic challenge
The diagnosis of primary MN is demanding, as the disease must be differentiated from other nephropathies, especially from secondary MN, which is triggered by an underlying cause such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as systemic lupus erythematosus or diabetes mellitus type 1. Of all MN cases, 20-30% are of secondary genesis, while the remaining 70–80% are classified as primary. Primary cases with no detectable anti-PLA2R antibodies are subclassified as idiopathic; it has been postulated that these patients may exhibit antibodies against other, as yet unidentified, target antigens. Reliable differentiation of primary and secondary forms of MN is critical because of different treatment regimes: primary MN is treated with immunosuppressants, while therapy for secondary MN is targeted at the underlying disease.

MN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to identify the characteristic glomerular immune deposits. To obtain a definite diagnosis of primary MN, secondary causes must be excluded, which involves additional time-consuming and often invasive procedures, for example tumour screening. Moreover, in some patients, MN appears before the secondary cause is even detectable, adding an extra layer of complexity to diagnosis and therapeutic decision-making. Primary MN must also be differentiated from other autoimmune diseases with kidney involvement, for example lupus nephritis, vasculitides associated with antibodies against neutrophil cytoplasm (ANCA) and Goodpasture’s syndrome. The availability of reliable serological tests to support the diagnosis of primary MN has been elusive until recently due to lack of knowledge about the target antigen.

New pathognomonic marker
Autoantibodies against PLA2R were first discovered and described in patients with primary MN in 2009 (1). PLA2R is a transmembrane glycoprotein (Figure 1) which is expressed in human glomeruli on the surface of podocytes and is involved in regulatory processes in the cell following phospholipase binding (Figure 2). Type M PLA2R has been identified as the major target antigen of autoantibodies. In patients with primary MN, antigen-antibody complexes form deposits in the glomerular basement membrane, where they trigger local complement activation with overproduction of collagen IV and laminin. This causes damage to the podocytes, via destruction of the cytoskeleton and broadening of the basement membrane. As a result protein enters the primary urine, giving rise to proteinuria and other symptoms.

Differential diagnosis
Autoantibodies of class IgG against PLA2R are present in the serum of up to 70-80% of patients with primary MN (1, 2), whereas they are not found in healthy blood donors or patients with secondary MN or other kidney diseases such as lupus nephritis (3) or IgA nephritis. The high predictive value of anti-PLA2R makes this parameter ideally suited as a diagnostic marker (4).

Disease evaluation
Anti-PLA2R antibodies are, moreover, very sensitive markers of clinical disease activity. They reflect the pathogenic immunological activity of the disease, which is responsible for the clinical expression in the form of proteinuria. High antibody titres indicate a severe disease course (2, 5), while low titres are associated with a decreased risk of renal failure and a greater rate of spontaneous remission (6). Thus, the anti-PLA2R titre also serves as a prognostic indicator.

Therapy monitoring
Treatment with immunosuppressants results in a drop in the anti-PLA2R titre, while in relapse the antibody titre increases again. Significantly, changes in the antibody titre typically precede changes in the proteinuria (6, 7). Thus, a titre increase is detectable before proteinuria appears, while a titre decrease is observed before a reduction in the proteinuria. Patients in remission exhibit residual proteinuria months after the anti-PLA2R titre becomes undetectable. Anti-PLA2R measurements are therefore extremely useful for early therapeutic decision-making and for long-term monitoring of responses to immunotherapy.

A recently published prospective study reinforced the value of anti-PLA2R antibodies as a marker of clinical outcome (8). In the study 133 patients with primary MN were tracked over a time period of 24 months. In all cases there was a clear correlation between proteinuria and anti-PLA2R levels. In patients who were given immunotherapy, a significant time lag was observed between the rapid fall in antibody levels and the protracted reduction in proteinuria. Moreover, remission of proteinuria occurred later in individuals with high antibody levels than in those with low levels. In patients who did not receive immunosuppressive therapy, spontaneous remission was also associated with a reduction in anti-PLA2R, while individuals who did not achieve remission showed continued elevated antibody levels. Thus, anti-PLA2R proved a reliable biomarker for immunological and clinical activity in primary MN.

Risk assessment
Up to 40% of patients with primary MN experience a relapse after kidney transplantation. The risk of recurrent primary MN is particularly high if anti-PLA2R antibodies are found prior to transplantation. In a study on a patient with primary MN, who exhibited high anti-PLA2R levels before and three months after transplantation (7), it could be shown that immunotherapy resulted in a drop in the antibody concentration and also the level of proteinuria. Other studies have shown that anti-PLA2R or PLA2R deposits are detected more often in transplant patients with recurrent MN than in those with de novo MN. A retrospective analysis of fifteen transplant patients revealed that a persistently positive anti-PLA2R activity at six months or later after transplantation was a significant risk factor for relapse, especially in patients on a weak immunosuppressive regimen (9). Thus, the anti-PLA2R antibody titre is useful for assessing the risk of relapse after transplantation and the extent of immunotherapy needed to prevent a recurrence.

Anti-PLA2R test systems
Anti-PLA2R autoantibodies can be determined easily and reliably using standardized indirect immunofluorescence test (IIFT) and ELISA systems. In the IIFT a BIOCHIP of transfected human cells expressing recombinant PLA2R is used as the antigenic substrate to provide monospecific antibody detection (Figure 3). A second BIOCHIP containing cells transfected with an empty vector serves as a control. The IIFT represents an established test for serodiagnostic screening, providing qualitative and semi-quantitative antibody analysis. The corresponding ELISA is based on purified recombinant PLA2R and shows the same high-quality characteristics as the IIFT. The ELISA is particularly useful for disease and therapy monitoring as it offers precise quantification of antibody levels in patient sera. The IIFT and ELISA are fast and simple to perform and are suitable for use in any diagnostic laboratory. Both procedures can be automated.

Clinical data
The performance characteristics of the Anti-PLA2R IIFT and ELISA have been assessed in a multitude of studies. In a retrospective clinical study (10) the Anti-PLA2R IIFT yielded a prevalence of 52% in a cohort of 100 patients with biopsy-proven primary MN, and a specificity of 100% with respect to healthy controls and patients with secondary MN or non-membranous glomerular injury. In a prospective clinical study (11) the sensitivity amounted to 82% in patients with biopsy-proven MN where no secondary cause could be found. The difference in sensitivities obtained in different study panels is most likely due to factors such as disease remission and the therapy status of the individuals, which can influence the antibody results, especially when studies are performed retrospectively.

Results obtained with the ELISA show a very good correlation with results from the IIFT (Figure 4). In a retrospective study with sera from 198 patients with primary MN and 836 healthy and disease controls, the ELISA showed a sensitivity of 96% with respect to the IIFT, and a specificity of 99.9% with borderline sera included (12). The few discrepant sera that were negative in the ELISA gave only low titres of 1:10 to 1:100 in the IIFT. All sera with titres of over 1:100 in IIFT were also positive in ELISA. 

Summary
Antibodies against PLA2R represent a landmark development in nephrological diagnostics. Their detection can secure a diagnosis of primary MN in patients with nephrotic syndrome, offering a convenient, non-invasive alternative to biopsy. Anti- PLA2R determination, moreover, yields information about the disease status and evolvement. Serial measurements are especially useful for monitoring therapy responses over the long term and guiding decision-making on the extent of treatment required for individual patients. Serological tests based on state-of-the-art IIFT and ELISA technology provide simple, quick and highly specific anti-PLA2R antibody detection.

References
1. Beck et al. N. Engl. J. Med. 2009: 361: 11-21
2. Hofstra et al. Clin. J. Am. Soc. Nephrol. 2011: 6: 1286-91
3. Gunnarsson et al. Am. J. Kid. Dis. 2012: 59 (4): 585-6
4. Schlumberger et al. Autoimmunity Reviews 2014: 13: 108-13
5. Kanigicherla et al. Kidney Int. 2013: 83: 940-948
6. Hofstra et al. J. Am. Soc. Nephrol. 2012: 23 (10): 1735-43
7. Stahl et al. N. Engl. J. Med. 2010: 363: 496-8
8. Hoxha et al. J. Am. Soc. Nephrol. 2014: 25: 1137-9
9. Seitz-Polski et al. Nephrol. Dial. Transplant. 2014: under revision
10. Hoxha et al. Nephrol. Dial. Transplant. 2011: 26 (8): 2526-32
11. Hoxha et al. Kidney Int. 2012: 82: 797-804
12. Daehnrich et al. Clin. Chem. Acta. 2013: 421C: 213-8

The author
Jacqueline Gosink PhD
EUROIMMUN AG
Seekamp 31
23560 Luebeck
Germany

E-Mail: j.gosink@euroimmun.de

C143 Figure 1

MALDI-TOF mass spectrometry for rapid identification and subtyping of H. cinaedi strains isolated from humans and animals

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Although Helicobacter cinaedi infection is now recognized as an increasingly important emerging disease in humans, it is difficult to identify particular isolates due to their unusual phenotypic profiles and similarity of 16S rRNA sequences among closely related helicobacters. MALDI-TOF MS resolved the present difficulties associated with the identification of H. cinaedi.

by Takako Taniguchi and Prof. Naoaki Misawa

Helicobacter cinaedi infection
Helicobacter cinaedi, was first recognized as a Campylobacter-like organism (CLO), is a Gram-negative, spiral-shaped, motile, microaerobic bacterium, and is now classified into enterohepatic Helicobacter species [1]. This organism was first isolated from homosexual men and was initially recognized as a rectal and intestinal pathogen among members of that population [1]. The first case of H. cinaedi bacteremia in Japan was in an HIV-negative patient, but was receiving immunosuppressive therapy after renal transplantation [2]. Moreover, a few cases of infection with H. cinaedi isolated from feces and blood from an apparently non-immunocompromised child and adult have been reported [3]. Since then, H. cinaedi has become thought of as an opportunistic pathogen that causes bacteremia, cellulitis, septicemia and enteritis in immunocompromised patients [4, 5], immunocompetent patients and even healthy individuals [6]. Kitamura et al. reported an outbreak of nosocomial H. cinaedi infections caused by direct person-to-person transmission [6]. Therefore, healthcare workers need to pay attention to H. cinaedi infection as an increasingly important emerging disease in humans.

Epidemiology
H. cinaedi-like organisms have also been isolated from non-human sources such as dogs, cats, monkeys, hamsters and other rodents [7–10], suggesting that the organism may be widespread in a broad range of animal species. As Gebhart et al. reported that H. cinaedi was found in 75% of the healthy hamsters used in their study [9], it was hypothesized that hamsters might be an important reservoir for human infection [7, 9]. However, no reliable epidemiological evidence of zoonosis has been demonstrated for human cases of H. cinaedi infection [3].

Diagnosis
To isolate H. cinaedi from blood, blood was usually collected in BACTEC culture bottles and incubated in a BACTEC 9050 blood culture system (Becton Dickinson, BD Biosciences) for at least 5 to 7 days. When the incubation time was less than 5 days or other culture systems were used, the organism was not often isolated. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods [2].

We previously isolated at least six different spiral-shaped organisms including H. cinaedi and H. bilis in a puppy with bloody diarrhoea [11]. These organisms were identified based on their morphology, biochemical traits, whole-cell protein profiles, and the results of molecular analysis of their 16S rDNA sequences. However, the biochemical identification of Helicobacter strains based on a limited number of tests is difficult because helicobacters frequently exhibit unusual phenotypic profiles, even in the same species [10, 12]. Furthermore, H. cinaedi cannot be clearly discriminated from H. bilis on the basis of 16S rRNA sequences because of the high level of sequence similarity (greater than 98%) [12].

Application of MALDI-TOF MS for rapid identification and subtyping of H. cinaedi strains
Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has made it possible to analyse the protein composition of a bacterial cell based on intact-cell mass spectrometry (ICMS) profiles as a new technique for species identification. The technique is simple, rapid and accurate for identifying microorganisms regardless of their characteristics, such as Gram-negative and Gram-positive bacteria, mycobacteria, anaerobes and yeast species [13, 14]. MALDI-TOF MS has an advantage in that it has a low-cost performance and is independent of the age of the culture, growth conditions or medium selected, making it applicable for routine bacterial identification in clinical laboratories. Although there is a commercially available library that includes more than three thousand kinds of microorganism such as bacteria, yeast and fungus for identification and phylogenic analysis (MALDI Biotyper Reference Library, Bruker Daltonics), we use a library created in-house.

Therefore, we considered that MALDI-TOF MS might resolve the present difficulties with identification of H. cinaedi. Furthermore, we examined whether H. cinaedi strains isolated from different animals could be differentiated or subtyped by their ICMS profiles [15].

As shown in Figure 1, although common peaks were detected in the H. cinaedi and H. bilis strains examined, the m/z 5200 and 10400 peaks were detected only in strains of H. cinaedi. These peaks showed good reproducibility regardless of the isolate origins, different media and passage numbers on the same medium. Therefore, the ICMS profile of H. cinaedi could be completely differentiated from those of H. bilis. Furthermore, the ICMS profile of H. cinaedi was also distinguishable from those of H. mustelae, H. pylori, H. fennelliae and H. canis, indicating that ICMS profiling using MALDI-TOF MS is applicable for the identification of H. cinaedi.

Cluster analysis of H. cinaedi strains based on the ICMS profiles
Several papers report that direct contact with pets may be a possible route of infection in humans [3–5]; however, details regarding the pathogenesis and epidemiological features, including routes of infection of animal isolates in the context of both humans and animals, are not fully understood. No reliable epidemiological evidence of zoonosis has been demonstrated for human infections caused by H. cinaedi. Therefore, ICMS profiles of H. cinaedi strains isolated from humans and animals were measured, and a phyloproteomic tree was constructed in order to analyse the relationships between the strains. As a result, these H. cinaedi strains were clearly divided into two groups. All of the strains isolated from humans belonged to Cluster 2. All the other animal-derived strains belonged to Cluster 1 (Fig. 2). Interestingly, the ICMS-based phyloproteomic tree agreed with the phylogenetic tree that had been based on the nucleotide sequences of the hsp60 gene. These H. cinaedi strains were also clearly divided into two groups by the hsp60-gene-based phyloproteomic tree. Thus, the data from phyloproteomic and phylogenetic analysis suggest that human strains of H. cinaedi may be distinct from animal strains. Kiehlbauch et al. also reported that there may be subgroups within H. cinaedi isolated from humans, dogs, cats and hamsters that correlate with the host source on the basis of DNA–DNA hybridization and ribotyping analyses [12]. The present study appears to support the hypothesis that H. cinaedi from different host sources may form subgroups, which may prompt a revision of the classification of H. cinaedi.

Conclusion
In conclusion, the construction of ICMS profiles using the MALDI-TOF MS approach may be a useful tool for H. cinaedi identification and subtyping. Further investigations will be required to analyse additional strains from a broader area to confirm whether human strains belong to a distinct subtype of H. cinaedi.

References
1. Quinn TC, Goodell SE, et al. Ann Intern Med. 1984; 101: 187–192.
2. Murakami H, Goto M, et al. J Infect Chemother. 2003; 9: 344–347.
3. Orlicek SL, Welch DF, Kuhls TL. J Clin Microbiol. 1993; 31: 569–571.
4. Kiehlbauch JA, Tauxe RV, et al. Ann Intern Med. 1994; 121: 90–93.
5. Matsumoto T, Goto M, et al. J Clin Microbiol. 2007; 45: 2853–2857.
6. Kitamura T, Kawamura Y, et al. J Clin Microbiol. 2007; 45: 31–38.
7. Comunian LB, Moura SB, et al. Curr Microbiol. 2006; 53: 370–373.
8. Fernandez KR, Hansen LM, et al. J Clin Microbiol. 2002; 40: 1908–1912.
9. Gebhart CJ, Fennell CL, et al. J Clin Microbiol. 1989; 27: 1692–1694.
10. Kiehlbauch JA, Brenner DJ, et al. J Clin Microbiol. 1995; 33: 2940–2947.
11. Misawa N, Kawashima K, et al. Vet Microbiol. 2002; 87: 353–364.
12. Vandamme P, Harrington CS, et al. J Clin Microbiol. 2000; 38:.2261–2266.
13. Saffert RT, Cunningham SA, et al. J Clin Microbiol. 2011; 49: 887–892.
14. Stevenson LG, Drake SK, et al. J Clin Microbiol. 2010; 48: 3482–3486.
15. Taniguchi T, Sekiya A, et al. J Clin Microbiol. 2014; 52: 95–102.

The authors
Takako Taniguchi1 MSc and Naoaki Misawa2* DVM, PhD
1Laboratory of Veterinary Public Health, Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
2Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan

*Corresponding author
E-mail: a0d901u@cc.miyazaki-u.ac.jp

p16 04

Serum B-cell maturation antigen: a novel marker for multiple myeloma

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B-cell maturation antigen (BCMA) was originally identified as a cell surface receptor expressed on late-stage B cells, plasma cells, and B-cell malignancies including multiple myeloma (MM). We recently discovered that BCMA is shed into the blood of MM patients, and, therefore, serum BCMA may serve as a new prognostic marker to track disease status and response to treatment.

by Eric Sanchez, Suzie Vardanyan, Mingjie Li, Cathy Wang, Dr Haiming Chen, and Dr James R. Berenson

Background
B-cell maturation protein (BCMA, also referred to as TNFRSF17 or CD269) is a receptor shown to be expressed in B lymphocytes and at increasing levels as these cells mature [1]. BCMA binds the ligands BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand) [2, 3]. Membrane-bound expression of BCMA has been demonstrated in human CD138-expressing cells from multiple myeloma (MM) bone marrow (BM) and MM cell lines [4]. It has also been shown to be expressed on malignant cells from Hodgkin’s lymphoma and Waldenstrom’s macroglobulinemia (WM) patients [5, 6]. Although the response to treatment of MM patients is traditionally followed with measurement of their monoclonal immunoglobulin (Ig) levels, some patients do not produce this marker; and, moreover, in others, it is lost during the course of their disease.

Serum BCMA levels are elevated in MM patients
We measured serum BCMA by ELISA from patients with newly diagnosed MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy control subjects. Using the International Staging System, 30, 12 and 7 patients with stages 1, 2 and 3, respectively, and one with unknown staging were analysed. We found that the serum levels of BCMA in MM patients (13.87 ng/ml) were elevated when compared to healthy controls (2.57 ng/ml; P <0.0001) and MGUS individuals (5.30 ng/ml; P = 0.0157; Fig.1A). We then determined that serum BCMA levels correlated with the MM patient’s response to therapy. Patients responding to their treatment regime had lower serum BCMA levels than patients with progressive disease (P = 0.0038; Fig. 1B). To confirm that the BCMA found in the blood of MM patients came from cells within the BM, BM aspirates were obtained from MM patients and BM mononuclear cells (MCs) were isolated and cultured for 48 hours. MM patients showed high levels of BCMA in culture medium whereas healthy subjects lacked significant amounts of BCMA.  Moreover, serum and supernatant BCMA levels from MM patients were compared and showed a strong correlation (r = 0.82) between the levels in the serum and supernatants from cultured MM BMMCs (Fig. 2). To exclude the possibility that the BCMA detected in MM patient serum and from cultured MM BMMCs may have been derived from non-malignant cells, we evaluated human BCMA levels in our human MM xenografts growing in severe combined immunodeficient (SCID) mice.  Animals were implanted with the human MM tumour LAGκ-2 and analysed for human serum BCMA levels. SCID mice dosed with bortezomib (0.5 mg/kg, twice weekly) had a reduction in tumour volume compared to untreated mice (P = 0.0067; Fig. 3A), and human serum BCMA levels from these bortezomib-treated animals were also markedly lower compared to untreated mice (P = 0.0006; Fig. 3B). Similar results were obtained when using our other MM xenograft models (LAGλ-1, LAGκ-1A). Human BCMA was not detected in the serum of non-tumour-bearing mice (data not shown). A rise in serum BCMA levels from the possible release of membrane-bound protein from dead MM cells was not observed in mice following drug treatment. Thus, it can be concluded that the serum levels of BCMA in the mice are derived from live MM cells. Soluble BCMA has been shown to inhibit normal B-cell development through interference with the binding of its ligands (BAFF and/or APRIL) to membrane-bound BCMA on normal B cells. One group demonstrated that administration of BCMA–Ig fusion protein to normal mice, which inhibits the binding of BAFF to B cells, resulted in a dramatic reduction in B-cell numbers in the blood and peripheral lymphoid organs [2]. Splenic B-cell reductions were shown to occur in an in vitro mouse splenocyte proliferation assay following in vivo administration of BCMA–Fc fusion protein to normal mice [7]. Other investigators have shown that injecting soluble BCMA–Fc fusion protein, which binds BAFF and APRIL both as free and membrane-bound ligands, into nude mice bearing human colon or lung carcinoma cell lines resulted in inhibition of tumour growth [3]. The authors suggested that BCMA bound its ligand APRIL, and prevented the known stimulatory effect of APRIL on these cell lines. Additionally, investigators have shown in vitro the existence of BCMA–BAFF complexes [3, 7, 8]. In the context of cancer growth, one group demonstrated that BCMA injected into mice reduced the growth of tumour cells from human colon or lung carcinoma cell lines in vivo by binding its ligand APRIL [3]. Thus, we are currently conducting studies to determine if such complexes exist in vivo and may block the immune function of myeloma patients.

Conclusions and future directions
Measurement of MM tumour mass is indirect, given the location of this BM based malignancy. Thus, assessment of tumour burden in response to therapy is difficult but essential to effectively monitor patients with MM. Traditionally, changes in Ig levels have been used to follow disease progression and response to treatment. In fact, for three decades Ig was a prognostic factor used in the Durie-Salmon staging system [9], which, until recently, was the most widely used staging system [10].  However, assessment of this protein does not always accurately reflect changes in MM tumour burden [11–13]. Additionally, small subsets of MM patients do not produce this marker. Thus, additional markers are needed to assess response to therapy in these non-secretory patients and in MM patients as a whole.

We have now shown that BCMA is present in the serum of MM patients; and, moreover, its levels correlate with the patient’s response to therapy [14]. We have also previously shown that supernatant from cultured BMMCs from MM patients with active disease contain much higher levels of this protein in their culture medium than in healthy subjects and those with MGUS or indolent MM [14]. SCID mice bearing human MM xenografts also showed high levels of BCMA in their sera, and these levels decreased in response to anti-MM therapy. We believe that that serum BCMA will eventually be used in the clinic as a diagnostic and prognostic marker for MM patients.

References
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The authors
Eric Sanchez BA; Suzie Vardanyan BS; Mingjie Li BS; Cathy Wang BS; Haiming Chen MD, PhD; and James R. Berenson* MD
Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA

*Corresponding author
E-mail: Jberenson@imbcr.org

 

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