Medical laboratory in autoimmunity 2017

Immunopathological update to better understand the diagnostic lab

Currently, as the complexity of the immune system becomes exploitable using tools of systems biology we are witnessing a better view on the derangements which are at the origin of autoimmune disease. Four main advances are in focus:

Going to the medical laboratory

Suspicion of the presence of an autoimmune background for a patients’ presentation lets physicians search for lab assays bearing the potential for it to turn out to be pathological. The shortest link between clinical signs and symptoms and selection of the right assay places a bet which tries to avoid ordering from the lab an oversized array of possible assays and thus inadvertently contributing to the health costs; mediocre algorithms initiated with global assays within the all too detailed/disease-specific abnormality by AI might obtrude the approach. As a bad example, to start the algorithm with a search for single gene defect resulting in unusual susceptibility to autoimmunity would put the cart before the horse, although gene wide association studies of autoimmune diseases so far have mapped hundreds of susceptibility regions in the genome. Interpretation and clinical translation of cloning assays remain fraught with difficulty and ethical questions.

A holistic approach comprising lab assays classified as clinical chemistry, hematology, microbiology and immunology surrounded by genome wide screening using PCR technology is mandatory [28]. Thereafter, laboratory tests ordered to diagnose autoimmunity depend on the particular disorder the health practioner suspects a patient suffers from and usually includes blood tests of one or more autoantibodies as well as tests for inflammation, i.e. C-reactive protein, erythrocyte sedimentation rate and circulating immune complexes with or without complement sytem exploration (www.labtestsonline.org). Autoimmunity-specific assays are under development. A dabbler in science is seduced to directly jump to a large number of testable autoantibodies (Table 1). In view of the long list of identified autoantibody specificities currently available for the clinician, the search for so far unknown specificities and the search for new ones should not be neglected; the diagnostic value of antibodies in RA is completed by predictive, prognostic and follow-up value – follow-up reductions of their serum concentration are a good sign of treatment response for the patient. This is true for anti-citrullinated protein antibodies (ACPA) recognizing cyclic citrullinated peptides (CCP), mutated citrullinated vimentin and viral citrullinated peptides. It also applies for anti-cytosolic autoantibodies. Thus, posttranslational protein modifications as with citrullinated rheumatoid arthritis (RhFs) can be identified to enhance the meaning of RhF detection [29]. The European Autoimmunity Standardisation Initiative (EASI) or the forthcoming Dresden Symposium on Autoantibodies (Germany) (www.gfid-ev.de) make this topic a priority.

With new auto-antibody specificities relevant to clinicians at stake the laboratory is faced with GLP validation procedures of these new tests, and the generation of normal values by establishing reference intervals and day-to-day stability [30]; ISO 15189 standards need to be applied to autoantibody analysis wherever possible. To cite an example of a time consuming introduction into the routine laboratory, epitope recognition of domain I-specific anti-β2-glycoprotein-I antibodies allows estimating a pathognomonic index for antiphospholipd syndrome whereas the immunological profile for SS includes only anti-Ro autoantibodies → ANA and RhF anti-La being excluded in the diagnostic procedure due to lack of specificity [31, 32].

New 2016 ACR/EULAR ranking of primary SS includes simplified criteria. Firstly, either salivary gland biopsy or anti-Ro must be reactive in order to support the inflammatory and autoimmune nature of the disease. In addition, the diagnosis must confirm the systemic nature of SS: patients without salivary or ocular glandular symptoms, but with extraglandular manifestations and B cell activation markers also need to be treated.

It turns out, that ACPA-reactive, compared to non-reactive, represent distinct disease, grades the former with a higher rate of severe joint destruction, requiring more agressive treatment so clearly as to suggest a different genetic and environmental risk factor pattern [33].

Half of the patients with polymyositis or dermatomyositis express myositis-specific antibodies (MSA), so when the clinical exam suggests the possibility of these diseases, their presence becomes strong supporting evidence for the diagnosis. There are many mysositis-associated specific antibodies, the best-known are anti-aminoacyl-tRNA synthetases, anti-signal recognition particle (SRP), and anti-Mi-2: chromodomain helicase DNA binding protein 4; those associated with sclerodermia (anti-RNP, Ku and PmScl) are also found in myositis and present as an overlap. Several studies now purport that different serological types can be associated to differences in presentation and prognosis.

The immune-mediated necrotizing myopathy induced by statin medication over years [34] calls for such detection assays in specialized labs to identify anti-HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase) antibodies [35].

For certain groups of autoimmune diseases multiplex line/dot blots test sytems might have advantages, e.g. automation, numerical reading and reproducibility. The applicability is currently under scrutiny and multiplex autoantibody patterns to certain diseases, e.g. of the gastrointestinal tract, with autoimmune hepatitis, primary biliary cirrhosis or autoimmune gastritis are being tested in multicenter studies.

Multiplex immunoassays [36], introduced at the outset of the current century support the immediate identification of multiple autoantibodies from a single determination but to the best of the authors knowledge have not made their way to being much used.

When inefficiently designed medical software relates to non-evidence based medical practice, the result can create unnecessary costs and hurts the patient. We have ourselves scrutinized multiplex systems but with only two analyses at stake, both making sense for similar sexually transmitted infectious diseases [37]. We cannot set apart multiplexing with its large spectrum of autoantibodies currently offered by providers as a useful approach. However, we still lack clinical experience with the lab results stemming from such type of screening and clinical presentation of autoimmune disease would narrow the number of specificities involved in a given patient. The American College of Rheumatology and its ANA Task Force insists on the indirect immuno-fluorescence to remain the gold standard in antinuclear antibody (ANA) testing on human epithelial cells (Hep-2) now completed using computer-aided diagnosis [38].

Autoimmune disease diagnostics is incomplete using autoantibody measurements alone for two reasons:

1. Immunopathogenic mechanisms incompletely explored:

   The pathogenetic roles of autoantibodies largely remain elusive without a priori knowledge of disease-specific autoantigens. Reasonable endeavors to identify auto-epitopes come from quantitative proteogenomics: identification of full-length antibody variable heavy chain sequences are now possible. Recently, immune complexes from clinical specimens obtained from a patient with hepatitis C virus-induced cryoglobulinemia (HCV-CG) were taken as a source of antibodies. Reconstructed single-domain antibodies were reactive to both HCV antigens and potentially liver-derived human proteins [39]. Approaches like these may provide a proof-of-concept usage of autoantibodies to pinpoint the corresponding autoantigen which in turn is a candidate of a disease-specific expression. Highly epitope-restricted autoantibodies may then serve as novel biomarkers and for the development of antigen-specific immunotherapy against various autoantibody-related disorders. It is well known, that RhF non-reactive patients may nevertheless suffer from classical rheumatoir factor (RA) which underlines the sensitivity and specificity shortfalls of most autoantibodies described to date. Regarding the prognosis of a given autoimmune disorder, the presence of some autoantibodies may provide important information. Thus, antibodies against cyclic citrullinated peptides derived from filagrin (anti-CCP1), when considered in conjunction with RhF, are a criterion to evaluate the prognosis and development of erosions.

2. Non-autoantibody based lab analyses in autoimmune diseases:

   These abound and include complement system exploration, inflammatory parameters and hematologic manifestations due and/or secondary to autoimmune disorders. The addition of such types of analyses to the lab profile of a patient have been outlined recently [28]. The presence of circulating immune complexes (www.immune-complex.ch), cryoglobulins and reactive direct or indirect antiglobulin (DAT, IAT) assays on red blood cells and platelets are relatively rarely ordered lab assays but indispensable in some rare diseases [40]. Similarly, CD markers on lymphocytes, natural killer cells or monocytes macrophages are second-line assays.

Monoclonal antibodies (mAbs) in the therapy and diagnosis of autoimmune diseases and other disorders

Systematic reviews on efficacy of second-line biologic choice in RA, PsA, and ankylosing spondylitis (AS) are now available [41]. They extract data from randomized, controlled trials, national healthcare databases, post-marketing surveys and open-label observational studies. Much attention is directed to mAbs (Table 3). Infliximab (trade names Remicade among others) is a chimeric mAb biologic drug that works against tumor necrosis factor alpha (TNF-α) or its bio-similar (bs-IFX) allow choosing among different routes and frequency of administration used to treat autoimmune diseases. Approved for the treatment of Crohn’s disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and RA, infliximab is now widely used. It is used off-label outside its Federal Drug Administration (FDA) approval for Behçet’s disease and other conditions. Infliximab is administered by intravenous infusion, typically at 6–8-week intervals. It cannot be given by mouth because the digestive system would destroy the drug. Some variables in use during the last decade such as patients’ preference, treatments with anti-TNF monotherapy solely in potential childbearing women, and the intravenous route with dose titration in obese subjects were valid for all of the three rheumatic conditions. In RA, golimumab as a second-line biologic is now considered a good option in the case of anti-TNF failure with lab assays pointing the way. The switching strategy is preferable for responder patients who experience an adverse event, whereas more severe or class-specific side effects should be managed by the choice of a differently targeted drug. After two or more anti-TNF failures, swapping to a different mode of action may be necessary, especially in cases where RhF titers and excessive complement activation do not improve.

Along these lines, the therapeutic mAb market received a boost when chimeric, humanized or fully humanized antibodies became approved by health authorities around the world. The second wave of approval and ongoing phase III clinical studies is due, in addition to better tolerance/less unexpected side effects, to the behavior of the target antigens/epitopes which are sensitive to pharmaceutical modulation of their functional expression, in this case upon binding to mAbs. Close to 100 mAbs can now be listed – Table 3 selects a few, prescription of which substantially depends on the results of laboratory assays. Lack of response to mAbs has been associated with inadequate mAb serum concentrations. TDM of mAbs has the potential to guide a more effective dosing in individual patients. The interpatient variability of mAb pharmacokinetics can be monitored by interval-timed lab assays. With mAbs being macromolecules, ligand-binding assays are at the forefront of reproducible test procedures still constrained by a substantial variation in detection and quantification. The optimal avoidance strategy towards formation of anti-drug, i.e. anti-mAb antibodies by patients adaptive immune response is not yet fully mapped out [43, 44].

The use of immune checkpoint inhibitors to treat cancer is expanding. Case-per-case success using anti-CTLA-4 (cytotoxic T lymphocyte antigen) monoclonal Abs (ipilimumab) used to treat advanced melanoma has fostered usage of this mAb, especially mAbs to target programmed death 1 protein (PD-1) and its ligand PD-L1, to also treat other cancer types: non-small cell-type of the lung, bladder, Hodgkin lymphoma, renal and squamous cell carcinoma of the head and neck. With somtimes spectacular remissions, approval by authorities was faster than usual and side effects now become known using postmerketing surveillance registers.

In fact, immune checkpoint inhibitors may elicit the irAE (immune related adverse events): rash, colitis, hepatitis, endocrinopathies. Nivolumab and iplimumab can cause deadly myocarditis or induce neurologic syndromes [45, 46].

To trace the drug upon administration, assessment of serum or tissue appearance of the administered mAb or its outcome on various lab assays likely reflect their administration. For example, mAbs specific for B-lymphocytic antigen CD20 (rituximab) can be followed beyond clinical efficacy by lab assays on CD19+ cell counts or by IgG serum concentrations [47]. Indeed, elimination half-lives of anti-CD20 varied as widely as between 1 and 17 days when measured by a two-compartment model assessing first-order distribution.

In a cohort of 64 patients suffering from RA the rituximab-elimination rate increased in speed with the number of CD19+ cell counts and increasing IgG serum concentrations which might be explained, at least in part, by different target-antigen loads in the patients. T cell activation can be identified using a calcium flux assay by flow cytometry – an original and progress promising approach [48]. Adverse events with adjuvant therapeutic measures, e.g. with glucocorticoids, in this context, would follow their own evaluations [49]. The same is true to estimate the therapeutic efficacy of IVIG – next to subtle exploration of the action mechanisms [50] inflammatory mediators such as TFN-α can be assessed in the diagnostic laboratory to estimate potency of IVIG treatment protocols [51].

Post-marketing surveillance programs with mAb therapeutic attempts are mandatory the more so as clinical studies designed for authority-approval may have included insufficient numbers of patients for a wise judgment of the unexpected side-effect ratio [52]. TDM of mAbs is used for personalized medical protocols and dosage schedules in individual patients. Interpatient variability of therapeutic mAb have been observed mainly in the treatment of autoimmune, inflammatory and malignant disease [42]. Therefore, individual adjustment of dosage with biopharmaceuticals is a must – whilst the problem is well recognized, only few randomized controlled TDM studies are available.

In a nutshell, the ongoing progress in diagnosis involving fast and scalable AI protocols and treatment of autoimmune disease is closely followed and depends on reliable lab assays.

Beyond diagnosis, the analyses help to direct treatment protocols and to explain action mechanisms as well as to adjust therapeutic protocols in personalized medicine.