Beukenlaan 137
5616 VD Eindhoven
The Netherlands
+31 85064 55 82
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.
Featured Articles
Chronic low back pain: could an anaerobic infection be responsible?
Around 80% of people in Western countries experience low back pain at some point in their life; indeed during a single year up to half of the adult population will experience back pain. In the majority no single clear cause can be identified, and the condition is self limiting. However for the approximately 7% of patients who develop chronic low back pain, quality of life can be significantly impaired. Chronic low back pain also has a serious financial impact in terms of healthcare costs and lost working days; in most industrialized countries it is the most common reason for workplace absence.
Now researchers at the Spine Centre of Southern Denmark have published the results of two very interesting and potentially far-reaching studies. The first involved sixty-one patients who had been suffering from low back pain for more than six months. Lumbar disc herniation was confirmed by MRI and the patients underwent primary surgery for removal of nucleus material. When this material was cultured for micro-organisms, forty-six percent of patients had positive cultures, predominantly with the normally commensal anaerobic bacterium Propionibacterium acnes. A significantly higher number of patients with anaerobic infections developed new Modic changes (MC), MRI-visible bone edema associated with low back pain, in vertebrae adjacent to the previous disc herniation compared with patients with negative cultures or cultures positive for aerobic organisms.
The second study was a double blind randomized controlled trial involving 162 patients who had been suffering from low back pain for more than six months and with MC in a disc adjacent to a previous disc herniation. Patients received either placebo or the antibiotic Bioclavid for a hundred days and were followed up at the end of treatment and after one year; outcome measures included both pain and workplace absence. Improvement was highly significant in the group treated with the antibiotic.
The authors suggest that when the lumbar disc is herniated, the anaerobic bacteria penetrate it and precipitate an insidious infection and chronic low back pain. Although they stress that antiseptic techniques were rigorously followed when the nucleus material was removed, it is surely still necessary to find a method of demonstrating anaerobic infection in patients who have low back pain and relevant MC but who have not had surgery. If this could be done many desperate chronic lower back pain sufferers might finally be able to stop taking analgesics or visiting osteopaths, chiropractors and acupuncturists, and get relief from a course of antibiotics instead.
DNA microarrays for SNP profiling in thrombosis and hemochromatosis
Specialized diagnostic DNA microarrays provide fast and reliable determination of factor V and factor II gene mutations associated with thrombosis or HFE gene defects linked to hereditary hemochromatosis. The simple microarray procedure includes fully automated data analysis and can be performed on whole blood samples, circumventing the need for preanalytical DNA isolation. Patient genotyping aids diagnosis in symptomatic individuals and risk assessment in healthy individuals, thus facilitating decision making in therapy and prevention.
by Dr Jacqueline Gosink
Laboratory analysis of genetic determinants is gaining momentum as ever increasing numbers of disease-associated alleles are discovered. With cutting edge diagnostic microarray technology, newly identified DNA parameters can progress rapidly from the research laboratory to routine diagnostics. Microarray platforms such as the EUROArray provide quick and easy determination of DNA mutations, enriching diagnosis and risk evaluation in a range of genetically linked diseases.
This article focuses on DNA microarray systems for genetic analysis in two common hereditary hematological disorders. The first detects single nucleotide polymorphisms (SNPs) in the factor V (FV) and/or factor II (FII) genes that lead to thrombosis and embolism. The second identifies up to four SNPs in the HFE (high iron) gene that contribute to hereditary hemochromatosis.
Thrombosis and embolism
Deep and superficial venous thrombosis and thromboembolism of the brain, lung and coronary vessels are among the most frequent causes of death, especially in western industrialized countries. These conditions result from a combination of genetic susceptibility and exogenous factors such as old age, immobility, smoking, diabetes mellitus, pregnancy, oral contraceptives or hormone replacement therapy. Notably, more than half of all cases can be attributed to genetic factors, particularly if the disease occurs before the age of 45 without any obvious external factors or at an atypical location.
The most important and most frequent genetic risk factors are the FV Leiden 1691G>A mutation (APC resistance) and the FII 20210G>A mutation in the prothrombin gene. These DNA mutations result in amino acid substitutions which disrupt the blood coagulation functions of FV and FII.
Factor V and II mutations
In healthy individuals activated FV is normally prevented from triggering coagulation by proteolytic cleavage catalysed by activated protein C (APC) and its cofactor protein S. Persons with the FV Leiden mutation exhibit an altered form of FV resulting from an exchange of the amino acid at position 506 from arginine to glutamine. The modified structure of FV makes it resistant to inactivation by APC (APC resistance), which leads to hypercoagulability and an increased risk of thrombosis. More than 95% of cases of APC resistance are caused by the autosomal, dominant FV Leiden mutation. In Europe around 3-7% of the population is a heterozygous carrier. In these individuals the thrombosis risk is 3-8 times higher than in non-affected persons, and if oral contraceptives are taken up to 30 times higher. The homozygous FV Leiden mutation occurs in around 0.2% of the European population and is associated with a 50-100-fold increased risk of thrombosis.
The 20210G>A mutation in the FII gene leads to a raised plasma concentration of the coagulation factor prothrombin via an as yet unidentified mechanism. The resulting thrombosis can be venous or arterial. The heterozygous genotype is present in 1-3% of the population in Europe and is associated with a 3-fold higher risk of deep venous thrombosis. If oral contraceptives are taken, the risk of venous thrombosis is increased 16-fold and of brain venous thrombosis up to 150-fold.
The factor V and factor II gene defects have an additive effect, and thrombophilia patients who exhibit the FII 20210G>A mutation often also have the FV Leiden mutation. In these patients the risk of venous thrombosis is elevated by a factor of 20.
Genetic analysis of FV and FII mutations is of outstanding importance in individuals with a high thrombosis risk based on their personal or family history, as well as in patients with unexplained recurrent miscarriages, biochemically proven resistance to APC or proven protein C or protein S deficiency. Genetic risk determination should also be undertaken before prescribing oral contraceptives or hormone replacement therapy to women with a familial tendency to thrombosis, especially young smokers.
Hereditary hemochromatosis
Hereditary hemochromatosis is the most frequent autosomal, recessive inherited metabolic disorder and is characterized by increased resorption of iron in the upper small intestine. The augmented iron uptake leads to an increase in the total iron content in the body from around 2–6 g (normal value) to up to 80 g. Since the human body cannot excrete the excess iron, it is deposited in various organs such as the liver, pancreas, spleen, thyroid gland, pituitary gland, heart and joints. In untreated patients irreversible damage occurs, resulting in an increased risk of cardiomyopathy, arthropathy, diabetes mellitus, liver cirrhosis and liver and pancreas carcinoma. Most cases of hereditary hemochromatosis are caused by defects in the HFE gene, which lead to functional flaws in the encoded iron regulatory protein.
HFE mutations
There are four SNPs in the HFE gene that are associated with hereditary hemochromatosis. The two most frequent, representing 90% of cases, result in the amino acid substitutions C282Y or H63D which cause a loss or reduction of the physiological function of the Hfe protein. The penetrance of the mutations is dependent on age and gender. Thus, the disease does not necessarily manifest itself in all carriers of these mutations. The strongest disease association is observed in patients with a homozygous C282Y mutation, whereby the penetrance is much lower in young women than in men due to menstruation. While 80% of men under 40 with this gene defect develop hemochromatosis, less than 40% of women do so. The penetrance increases to 95% of men and 80% of women for the population group of over 40 year olds. The two further SNPs in the HFE gene that are associated with hereditary hemochromatosis are S65C, which results in an amino acid substitution in the Hfe protein, and E168X, which causes early termination of protein synthesis, whereby both of these mutations are rare.
Around 10% of the population in northern Europe is heterozygous for one of the disease-associated mutations in the HFE gene and 0.3–0.5% is homozygous. New studies show that 90–100% of hemochromatosis patients exhibit homozygous gene defects. However, even a mutation in one HFE allele is sufficient to cause at least minor abnormalities in iron metabolism. The early identification of HFE gene defects enables suitable preventative measures to be implemented, for example a reduction in the consumption of high-iron-containing foods.
Simple microarray analysis
DNA mutations associated with thrombosis and hemochromatosis can be reliably determined using DNA microarray systems such as EUROArray [1, 2]. This microarray system provides fast and efficient SNP detection with fully automated data analysis, and can easily be used by persons unfamiliar with molecular biology. A special feature of the thrombosis and hemochromatosis microarray procedures is the use of pretreated whole blood as sample material, which eliminates the need for a preanalytical DNA isolation step. The hands-on processing time for the direct procedure is thus reduced to as little as 1.5 minutes per sample.
In the microarray procedure [Figure 1], the sections of DNA containing the disease-associated alleles are amplified by multiplex polymerase chain reaction (PCR) using highly specific primers. During this process the PCR products are labelled with a fluorescent dye. The PCR mixture is then incubated with a microarray slide containing immobilized DNA probes [Figure 2]. The PCR products hybridize with their complimentary probes and are subsequently detected via the emission of fluorescence signals. The evaluation of the microarrays [Figure 3] proceeds quickly and objectively using the special microarray scanner and EUROArrayScan software. The software interprets the results, produces patient genotype reports, and archives all data and patient information [Figure 4]. It can be integrated seamlessly into existing laboratory software.
Reliable biochip technology
EUROArrays are based on proven biochip technology which has been adapted for DNA analysis. Each biochip is composed of DNA spots of wild type and mutant alleles and contains in addition integrated control sequences to verify correct performance of the test. The microarray slides are incubated using the established TITERPLANE technique, which provides standardized, parallel incubation of multiple samples. Up to five samples can be analysed per slide. The reproducibility and convenience of the analysis is further enhanced by ready-to-use PCR reagents and meticulously designed amplification primers and hybridisation probes. The entire procedure from sample arrival to report release is IVD validated and CE labelled.
In clinical evaluation using molecular genetically precharacterized samples, each microarray demonstrated a sensitivity of 100% and a specificity of 100% [Table 1]. Homozygous and heterozygous genotypes were reliably discriminated for every position.
Comprehensive microarray range
The thrombosis diagnostic microarray system is available in different constellations for separate or parallel analysis of the FV Leiden and FII 20210G>A mutations, while the hemochromatosis microarray system is available in two versions encompassing either just the two most frequent mutations C282Y and H63D or, for a more extensive analysis, the four disease-associated mutations C282Y, H63D, S65C and E168X.
In addition to the determination of FV/FII and HFE mutations, EUROArray technology can also be used to analyse further genetic risk factors such as HLA-DQ2/ DQ8 in celiac disease, HLA-Cw6 in psoriasis or HLA-B27 in ankylosing spondylitis. New parameters soon to be added to the platform include HLA-DR Shared Epitope in the diagnosis of rheumatoid arthritis and human papilloma virus detection and subtyping.
Summary
The current pace of genetic discoveries combined with advances in microarray technology is resulting in a plethora of novel DNA tests for the routine diagnostic laboratory. New DNA microarrays for rapid identification of thrombosis-associated mutations in the factor V/factor II genes and hemochromatosis-linked mutations in the HFE gene have greatly enhanced diagnosis and risk evaluation in susceptible individuals. Early awareness of a genetic predisposition enables individuals to adopt appropriate lifestyle or medical interventions to reduce the impact or even prevent development of these debilitating diseases.
References
1. Voss J. et al. to be presented at IFCC EuroMedLab, Milano, Italy (2013).
2. Axel K. et al. to be presented at IFCC EuroMedLab, Milano, Italy (2013).
The author
Jacqueline Gosink PhD
Euroimmun AG
Luebeck, Germany
Phage-displayed peptides as novel reagents for norovirus detection
Current methods for detecting noroviruses (NoVs) have significant limitations in sensitivity and feasibility for use in remote locations. Our group recently identified phage-displayed peptides with specific binding to NoVs and sensitivity comparable to that of existing antibodies. These reagents can be easily optimized by mutagenesis and represent promising diagnostic tools.
by Amy M. Hurwitz, Prof. Robert L. Atmar and Prof. Timothy G. Palzkill
Norovirus infection and diagnosis
Each year, norovirus (NoV) infections cause approximately 267 million new cases of gastroenteritis and 200,000 deaths worldwide [1]. Infection spreads rapidly in areas of close human contact, such as cruise ships and hospitals, and is treated only by rehydration, as no antiviral therapy currently exists. An infectious dose estimated to be as low as 18 virions and high environmental stability contributed to classification of NoVs as a category B biodefense agent in the U.S. Therefore, rapid, accurate and highly sensitive diagnosis is important for outbreak recognition and control, and also to guide physicians in patient management. The potential health and economic consequences that may be ameliorated by early NoV detection have led to a high demand for optimized detection reagents that can be used to develop reliable diagnostic assays with minimal requirements for expensive, bulky equipment or technical training.
NoVs are divided into six different genogroups (GI–GVI) based on the amino acid sequence of the major capsid protein (VP1). These are organized further into more than 30 genotypes, and finally into numerous strains or variants [2]. The VP1 protein assembles to form an icosahedral shell with an inner shell (S) domain and outer protruding (P) domain. The P domain is on the virus surface and is the most accessible, while the S domain has the highest sequence conservation across different strains. Given the ability of NoVs to evolve rapidly to result in novel or recombinant strains, continual optimization of detection reagents may be necessary in order to recognize the majority of human-infecting strains. Strains classified into GI and GII are most relevant for human infections, and thus the focus for diagnostic assay development efforts.
Current diagnostic methods and their limitations
Methods used currently for the diagnosis of norovirus infection are far from ideal as they exhibit several limitations that hinder their use for individual patient diagnoses or in rural and developing locations. The gold standard for diagnosis is reverse transcriptase (RT)-PCR, which requires multiple sets of primers to detect about 90% of human-infecting strains [3]. This method has significant equipment and expertise requirements, which are often not available outside of large institutions. Further, the expense of running multiple samples and the need for timely instrument accessibility limit the feasibility of applying RT-PCR as point-of-care applications or for preventing the rapid spread of an outbreak.
Other existing methods include immune electron microscopy (IEM) and enzyme immunoassays. IEM was the first method described for identifying NoVs and was used originally to classify viruses based on structural appearance. This method has limited sensitivity, and also requires expensive equipment and skilled expertise. Enzyme immunoassays, developed after the discovery of type-specific antibody epitopes on the NoV capsid, detect viral particles in human stool samples [4]. This method offers increased specificity and has led to the development of commercially available ELISA and lateral flow assays.
Currently, the only FDA-approved antigen detection assay is an ELISA called RIDASCREEN® (3rd Generation) produced by R-Biopharm, which uses an antibody cocktail with specificity for GI and GII NoVs [Fig. 1]. Due to limitations in sensitivity, this assay is only approved for use during outbreaks and takes several hours to produce results. Several companies, including R-Biopharm, have developed rapid diagnostic assays that use lateral flow technology and have also demonstrated strong specificity for NoV GI and GII strains. However, these have similar limitations with sensitivity and thus are only recommended for preliminary screening to be confirmed by RT-PCR, and are distributed primarily outside of the United States [5]. Overall, there is a clear need for improved diagnostic methods to detect norovirus rapidly with strong specificity, high sensitivity, and with minimal equipment and expertise requirements.
Novel diagnostic phage reagents
Recent studies in our laboratory have identified short, 12-mer peptide reagents with specific binding to the GI.1 NoV genotype [6]. The small size of these peptides displayed on phages offers the ability to access epitopes that may be buried in the capsid protein and not accessible to antibodies, and the potential for increased avidity through multiple linked peptide molecules. To identify peptides with specific binding to NoV, we used phage display technology to screen commercially available, large-scale libraries of randomized peptides that are fused to the gene III protein and expressed in five copies on one end of the phage. Rounds of biopanning were performed in which filamentous phage libraries were screened for phages displaying peptides that bind immobilized Norwalk (NV) GI.1 virus-like particles (VLPs). The phage libraries were added to VLPs and, after washing away non-binding phages, the phages displaying VLP-binding peptides were eluted with low pH [Fig. 2A]. Two to four subsequent rounds of biopanning using the resulting phage populations enriched for phages displaying peptides with the highest binding affinity for NV. DNA sequencing of individual phage clones recovered after multiple rounds of biopanning revealed three peptides, named NV-O-R5-3, NV-O-R5-6, and NV-N-R5-1, that occurred most commonly, and the phage clones displaying the peptides were further characterized for their NoV binding properties [6].
Phage-based ELISAs confirmed the binding specificity of phage-displayed peptides to NV VLPs. These affinity-binding assays used NV VLP captured by immobilized rabbit polyclonal anti-NV antibody in order to maintain the structural integrity of VLPs. Single phage clones were added to the captured VLPs and binding was detected using anti-M13 phage antibody that was conjugated to horseradish peroxidase to provide a signal for bound antibody [Fig. 2B]. Of the three peptide-displaying phage clones analysed, NV-N-R5-1 exhibited a dose-dependent response with decreasing NV VLP concentration and the highest sensitivity with a limit of detection at 1.56 ng NV VLP. Additional phage ELISAs indicated that NV-N-R5-1 binds to the P domain of the capsid protein, which extends the furthest out from the virus, and has comparable sensitivity for NV as existing antibodies used for diagnostics [6]. These results provide proof-of-concept and a strong lead reagent for developing novel phages displaying peptides as effective detection reagents for NoV. Further, the methods described establish a platform methodology for using phage display to identify antigen-specific binding reagents that may be applied to any pathogen with distinct surface epitopes.
Current status
To develop our lead phage-displayed peptide into a commercially viable tool, we are currently optimizing its binding affinity for other genogroups of NoV in order to broaden its diagnostic applications. Phage display technology provides a simple platform for constructing collections of new mutations in a lead peptide that can be used for additional rounds of biopanning to screen for variants with optimal affinity properties [Fig. 2C]. The three phage-displayed peptides discussed above share conserved amino acid sequence motifs that likely confer binding specificity for particular epitopes on the NV capsid protein. Directed evolution through mutagenesis of amino acids surrounding these consensus sequences can enable us to improve binding affinity to NV and alter binding specificities starting with the lead phage peptide, NV-N-R5-1. In particular, developing phage-displayed peptides with optimized binding affinity for the NoV GII.4 genotype, which accounts for >80% of NoV infections worldwide [1], and other GI and GII NoV genotypes will have the greatest relevance for diagnostic applications.
Future development of bacteriophage reagents
For decades, phages have been used to identify their target bacterial strains and species in order to diagnose the cause of infections by phage typing. More recent applications have begun to leverage synthetic biology and genomic engineering strategies to customize phage specificity and reporter signals to enable ‘near-real-time’ detection of a broader range of human pathogens [7]. Our recent work has established a methodology for the identification, characterization, and development of phage-based affinity reagents that may be applied to different pathogens and translated into diagnostic applications. The process outlined in Figure 2 demonstrates the progression from (A) identifying lead reagents against a target of interest, (B) characterizing binding affinity for the antigenic target, (C) optimizing leads through directed evolution or genomic engineering strategies, and finally (D) producing scalable quantities of reagent for commercial diagnostic applications. Zou and colleagues, for example, used a similar method to identify a phage-displayed peptide reagent with specific binding to transmittable gastroenteritis virus (TGEV) that also showed potential antiviral activity [8]. Several groups have also developed phage-based reagents to detect bacterial pathogens, such as Salmonella enterica and Escherichia coli [9, 10].
In summary, the use of phage-based reagents for microbial diagnostics offers many advantages in comparison to more commonly used detection reagents, such as antibodies. Phage display technology enables rapid identification and validation of candidate phage reagents with specificity for new or evolved pathogens through biopanning of commercial or custom made phage libraries (Fig. 2A, B). Phage manipulation through directed evolution facilitates development of reagents with optimized binding affinity and specificity to a target of interest (Fig. 2B). Finally, production of large quantities of phages is accomplished rapidly and inexpensively, as simple preparation methods can produce sufficient phage for hundreds of assays (Fig. 2D). As viral pathogens such as NoV continually evolve, the flexibility provided by phage-based reagents will be essential for developing next generation diagnostics for effective containment of outbreaks. A cocktail of phages, each of which binds to a specific target NoV genotype, may ultimately be the ideal strategy for producing an assay to detect the broadest possible range of NoVs without sacrificing specificity. Overall, phages have an enormous potential for use as detection reagents in clinical, agricultural, food, and environmental settings, and represent an underutilized resource for diagnostic development.
References
1. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunological Reviews 2008; 225(1): 190–211.
2. Kroneman A, Vega E, Vennema H, Vinjé J, White P, Hansman G, Green K, Martella V, Katayama K, Koopmans M. Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology 2013; doi:10.1007/s00705-013-1708-5.
3. Atmar RL, Estes MK. The epidemiologic and clinical importance of norovirus infection. Gastroenterology Clinics of North America 2006; 35(2): 275–290.
4. Parker TD, Kitamoto N, Tanaka T, Hutson AM, Estes, MK. Identification of Genogroup I and Genogroup II broadly reactive epitopes on the norovirus capsid. Journal of Virology 2005; 79(12): 7402–7409.
5. Ambert-Balay K, Pothier P. Evaluation of 4 immunochromatographic tests for rapid detection of norovirus in faecal samples. Journal of Clinical Virology 2013; 56(3): 194–198.
6. Rogers JD, Ajami NJ, Fryszczyn BG, Estes MK, Atmar RL, Palzkill TG. Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples. Journal of Clinical Microbiology 2013; 51(6): 1803–1808.
7. Lu TK, Bowers J, Koeris MS. Advancing bacteriophage-based microbial diagnostics with synthetic biology. Trends in Biotechnology 2013; 31(6): 325–327.
8. Zou H, Zarlenga DS, Sestak K, Suo S, Ren X. Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential. Antiviral Research 99(3): 383–390.
9. Schofield DA, Sharp NJ, Westwater C. Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage 2012; 2(2): 105–283.
10. Galikowska E, Kunikowska D, Tokarska-Pietrzak E, Dziadziuszko H, Loś JM, Golec P, Węgrzyn G, Loś M. Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents. European Journal of Clinical microbiology & Infectious Diseases 2011; 30(9): 1067–1073.
The authors
Amy M. Hurwitz1 BS, Robert L. Atmar2,3 MD, Timothy G. Palzkill*2,4 PhD
1 Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA
2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
3 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
4 Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
*Corresponding author
E-mail: timothyp@bcm.edu
Molecular techniques used in the diagnosis of cutaneous lymphoma
Cutaneous lymphomas are a heterogenic group of conditions often difficult to diagnose. The diagnosis requires careful correlation between clinical presentation pathology and molecular analysis. Molecular analysis includes inmunophenotyping, clonality assays and rarely chromosomal analysis. The importance of molecular analysis hinges on two main reasons: firstly to confirm the diagnosis and secondly to further characterize the nature of the lymphoma. In addition, molecular analysis may provide some further insight on the origin of the malignancy, for example if it is primarily cutaneous or if the skin is a secondary site of involvement.
by Dr Belén Rubio González and Dr Joan Guitart
Recently, cancer has been defined by unlimited growth of cells derived from a single mutated cell or a clonality expansion. The detection of a monoclonal population may help to distinguish a lymphoma from a reactive process. However, on the one hand, clonality by itself does not imply malignancy and, on the other hand, a negative clonality result does not rule out a malignant condition. During this process, genes encoding the antigen receptor immunoglobulin (Ig) for B cells and the T-cell receptor (TCR) for T cells are rearranged as commonly seen in primed lymphocytes, resulting in a wide diversity of unique antigen receptors providing high antigenic specificity.
The clonal nature of several skin conditions may help us recognize pre-malignant stages or the concept of cutaneous lymphoid dyscrasias (CLD) which has been recently introduced and includes parapsoriasis, pigmented purpuric dermatosis, idiopathic follicular mucinosis, pityriasis lichenoides, syringolymphoid hyperplasia with alopecia, and idiopathic generalized erythroderma (pre-Sézary). Although almost all of these conditions never progress to a frank malignancy, they have the potential risk of converting into cutaneous T-cell lymphoma (CTCL). The recognition of a T-cell clone may identify these dermatoses, which have been difficult to categorize in the past.
Clonality methods
T-cell clonality studies are based on the detection of specific T-cell receptor gene rearrangements (TCR-GR) by Southern blot analysis (SBA) or polymerase chain reaction (PCR). We should expect that the tumour cells contain identical TCR-GRs, reflecting a monoclonal T-cell population.
SBA used to be the gold standard for detection of T-cell clonality, but the procedure is laborious and lengthy. Furthermore, fresh or frozen tissue and radioactive probes are required. If this method is used, the clonal population must represent at least 5% of the total DNA extracted, which includes cells other than T-cells decreasing the sensitivity of the test. For the reasons above, SBA has been gradually replaced by PCR techniques.
The overall sensitivity of PCR-based methods for detection of T cell clonality ranges between 70 and 90%, with specificity range depending on the sample population. The test amplifies extracted DNA using primers directed against the TCR beta, gamma and delta chains. The gamma chain gene is most commonly used because of the lower complexity of the gene. Adding probes to the beta TCR gene allows for a higher sensitivity and specificity of the clonal analysis.
In the case of B-cells, PCR uses primers for four conserved reliable targeted regions for immunoglobulin heavy-chain. In our experience the sensitivity of PCR for the immunoglobulin heavy chain is lower than for T-cell clonality. The detection of light chain restriction by immunophenotypic test (often referred as monotypical immunoglobulin expression) is also consistent with a clonal B-cell population. This can be accomplished with immunohistochemistry at the protein level or in situ hybridization at the RNA level. Monoclonality can also be demonstrated with flow cytometry targeting kappa and lambda light chain expression at the B-cell membrane. An international consensus on B- and T-cell clonality assays was established with the BIOMED-1 proposal.
In most of the conventional PCR methods monoclonality is defined by the presence of a band after high-resolution capillary gel electrophoresis of the PCR product. Using temperature- or chemical-gradient gel electrophoresis can enhance separation of DNA products. After that, fluorescent fragment analysis using consensus primers for the TCR gene and the fluorescence input is analysed by capillaroscopy. Furthermore, clonal definition should be confirmed using multiple PCR probes labelled with different fluorochromes.
The detection of a dominant T-cell clone, defined as the same PCR product at different sites (two skin biopsies, skin and blood, skin and lymph node, etc.) implies dissemination of a prevailing T-cell clone, and has been associated with a higher incidence of tumour progression. Clonal heterogeneity has been reported in patients with early stage or indolent mycosis fungoides (MF) and in CLD conditions without a malignant process.
The value of the detection of circulating clonal T-cells in peripheral blood has been debated. That is much more common in patients with erythrodermic MF (42%) compared to other lower stages (12.5%). It may also help in distinguishing a dominant CTCL clone from innocent cytotoxic T-cell clones, which are often detected in the blood of elderly patients.
In the context of palpable lymphadenopathy, detection of the same clone in the lymph node and the skin CTCL lesions may indicate a poor prognosis, similar to the identification of lymphoma by histology.
T-cell clonality and significance
TCR clonality should be tested for in skin and blood samples at the time of diagnosis when a cutaneous lymphoma is suspected. The detection of a dominant clone in both sites is important to confirm the diagnosis and for prognostic guidance. T-cell clonality is particularly helpful in the early stage of an MF which does not include sufficient clinical or microscopic evidence for the diagnosis. TCR gamma clonality was positive in 53% of the patch stage and in 100% of plaque or tumour stage in different series. An increased rate of clonality was observed in connection with more advanced cutaneous disease and higher histopathological diagnostic score.
False-positive monoclonal and oligoclonal bands may be identified in inflammatory dermatosis, where the T-cell infiltrate is sparse. Amplification of TCR-GRs from a few T-cells may result in a false-positive clone or ‘pseudomonoclonality’. A pseudoclone is infrequently associated with a malignant T-cell process. Repeating the analysis using the same DNA template or fresh DNA extraction may solve the problem because in reactive conditions, the predominant PCR product typically varies in repeated analysis of the same sample. In contrast, in lymphomas, dominant TCR clones are reproducible and should be routinely verified to confirm monoclonality.
A correlation between TCR clonality by PCR methods and response to treatment has been suggested in several studies. The absence of a detectable clone in CTCL was associated with a higher rate of complete remission, but was not necessarily associated with improved survival.
Also immunophenotypic and immunogenotypic assays have been used to monitor the response of CTCL to therapy. The concept of minimal residual disease is defined as the persistence of the tumour T-cell clone in tissue or blood despite clinical complete remission status. Minimal residual disease as detected by deep sequencing methods may help identify patients at risk of relapse but the real prognosis is still uncertain. In the future, the presence or absence of the dominant or persistent clone may guide our therapeutic approach, aiming for more durable remissions while minimizing the adverse effects of therapy.
Other methods used in the olecular diagnosis of cutaneous lymphomas
Flow cytometry analysis
Blood flow cytometry analysis (FCA) is routinely performed in erythrodermic patients to rule out Sézary syndrome (SS). This method is based on the abnormal expression of various surface markers of malignant T-cells compared with normal T-cells. Other helpful findings are the demonstration of overwhelming dominance of specific T-cell subsets (clusters of differentiation CD4 vs CD8) and the loss of one or more pan-T-cell antigens (i.e. CD2, CD3, CD5, and CD7). A high CD4 : CD8 ratio of more than 10 : 1 and loss of CD7 and CD26 are the most reliable findings in SS. However, low CD7 expression has lower specificity because some inflammatory diseases also show the same deletion. The addition of CD26 to standard T-cell panels enhances the sensitivity of FCA in the diagnosis of SS.
Moreover, flow cytometry is able to detect a clonal population by using antibodies against different subsets of T-lymphocytes based on the expression of V beta family antibodies. This is used mainly as a research tool because the extensive panel of antibodies is expensive, incomplete and does not include the entire spectrum of V beta families.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) involves annealing of fluorescently labelled nucleic acid probes with complementary DNA or RNA sequences and the subsequent detection of these probes within fixed cells. FISH is used to detect major chromosomal gains or losses, as well as specific translocations, and requires a target specific probe. Although FISH is not routinely used in the diagnosis of cutaneous lymphomas, recent publications have shown its potential for future applications in various areas.
Genomic analysis by microarray assays or comparative genomic hybridization
Comparative genomic hybridization (CGH) allows the identification of chromosomal imbalances but it is not able to identify specific genes involved due to its measurement resolution. The microarray-based CGH is more precise, and chromosomal imbalances can be quantified and defined appositionally. A high frequency of gains in chromosomes 1, 7, 8, and 17 and losses of chromosomes 5, 9, and 13 was demonstrated using array-based CGH for identification of genomic differences between SS and MF.
Conclusion
Molecular diagnosis, in combination with a meaningful correlation with histological results and clinical presentations can provide an important tool in the evaluation of cutaneous lymphoid infiltrate. While PCR-based clonality techniques need to be interpreted with caution, modern capillaroscopy methods offer clone-specific data that allow us to improve the accuracy for diagnosis, prognosis and staging implication.
References
1. Deonizio JM, Guitart J. Semin Cutan Med Surg 2012; 31: 234–240.
2. Groenen PJ, Langerak AW, et al. J Hematop 2008; 1: 97–109.
3. Guitart J, Magro C. Arch Dermatol 2007; 143: 921–932.
4. Rübben A, Kempf W, et al. Exp Dermatol 2004; 13: 472–483.
5. Kulow BF, Cualing H, Steele P, et al. J Cutan Med Surg 2002; 6: 519–528.
6. Nihal M, Mikkola D, et al. Hum Pathol 2003; 34: 617–622.
7. Meyerson HJ. G Ital Dermatol Venereol 2008; 143:21–41.
8. Van Dongen JJ, Lamgerak AW, et al. Leukemia 2003; 17: 2257–2317.
9. Sandberg Y, Heule F, et al. Haematologica 2003; 88: 659–670.
10. Guitart J, Camisa C, et al. J Am Acad Dermatol 2003; 48: 775–779.
The authors
Belén Rubio González* MD and Joan Guitart MD
Northwestern Medical Hospital, Chicago, IL, USA
*Corresponding author
E-mail: rubiogonzalezbelen@gmail.com