How to Assess Treatment Efficacy in Sjogren's Syndrome?
How to Assess Treatment Efficacy in Sjogren's Syndrome?
As mentioned in the introduction, the largest hurdle to be taken is to develop a reliable set of assessments by which the efficacy of a particular treatment approach can be assessed, meanwhile allowing evidence-based comparison of the various treatment approaches tested in pSS. Such a set of assessments probably also can be used to rate disease activity and disease progression in pSS.
Evidence-based therapy for Sjögren's syndrome is largely limited to treatments that improve sicca features. In addition, the variety of ad hoc outcome measures used to rate the effects of both symptomatic and disease-modifying treatments are mainly based on glandular symptoms. Therefore, validated activity indexes are needed to assess the effectiveness of (new) targeted therapies. For example, B-cell-targeted therapies have shown promising results for both systemic and glandular features, but, again, a variety of outcome measures has been used. To solve these short-comings, the European League against Rheumatism (EULAR) recently introduced a patient-administered questionnaire to assess patient symptoms (EULAR Sjögren's syndrome Patient Reported Index: ESSPRI) and a systemic activity index to assess systemic complications (EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) as there are for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).
With regard to pSS, studies are in progress to assess the reliability and sensitivity to change of the ESSDAI and ESSPRI. Such studies are important as the usefulness of instruments designed to measure change over time or after therapeutic intervention is not only dependent on their validity and reliability, but also on their potential to detect clinically relevant changes. Seror et al. retrospectively investigated the sensitivity to change of the ESSDAI over time in 96 patient profiles and reported that the internal and external responsiveness of ESSDAI was large, and changes over time were detected more accurately than with other known indices. Meiners et al. prospectively assessed the internal and external responsiveness to change of ESSPRI and ESSDAI in pSS patients treated with rituximab. The latter study revealed that ESSPRI and ESSDAI are, indeed, sensitive to measure the changes in disease activity after therapeutic intervention, which supports the usefulness of these indices for future clinical trials. The promising results from the retrospective analysis of Seror et al. and the prospective intervention study by Meiners et al. showed that, when validated, application of both instruments for outcome measurements in randomized controlled clinical trials should allow assessing all aspects of pSS (Fig. 1).
(Enlarge Image)
Figure 1.
Disease activity in primary Sjögren's syndrome patients treated with rituximab (median values). (a) ESSPRI and ESSDAI. (b) Disease activity for the ESSDAI domains showing the highest changes (percentage of patients with no disease activity in a particular domain is given). *Indicates significant (P<0.05) change compared with baseline.
Within the wide spectrum of clinical manifestations of Sjögren's syndrome, salivary gland dysfunction is considered to be a key manifestation. Dysfunction of the salivary glands results in changes of the amount and composition of the saliva. The autoimmune process itself is reflected by the presence of various components of the immune response, such as cytokines, chemokines, autoantibodies, in saliva. As such, sialometry is not only of diagnostic but also of prognostic importance. Moreover, as the amount and composition of saliva reflect the damage caused by the autoimmune process in the salivary glands, sialochemistry may be valuable in diagnosis, assessment of prognosis, and evaluation of treatment. Thus, sialometry and sialochemistry can be used as a diagnostic tool either by collecting whole saliva (the combined secretions of all salivary glands) or by collecting glandular saliva (gland-specific saliva). Although unstimulated whole saliva is a major criterion for the evaluation of salivary gland dysfunction in Sjögren's syndrome, when Sjögren's syndrome develops, not all major salivary glands may yet manifest dysfunction, rendering whole saliva less valuable as a parameter for evaluating progression of the disease or therapeutic intervention than glandular saliva (Fig. 2). In contrast to whole saliva, analysis of gland-specific saliva can reveal sequential involvement of particular glands, reflecting the ongoing autoimmune process in individual major salivary glands. By using glandular saliva, patients with Sjögren's syndrome may frequently be diagnosed at an earlier stage, and progression and effects of therapeutic intervention can be measured in a noninvasive way. For example, Pijpe et al. showed that regeneration of salivary gland tissue by rituximab was accompanied by an increase in salivary flow and normalization of salivary composition (Figs 3 and 4).
(Enlarge Image)
Figure 2.
Relation between disease duration, that is, the time from first complaints induced by or related to oral dryness until referral, and mean salivary flow rates (mean ± SEM). UWS, unstimulated whole saliva; SM/SL, submandibular/sublingual glands.
(Enlarge Image)
Figure 3.
Comparison of parotid biopsy specimens obtained from a pSS patient before rituximab therapy (A1–A4) and 12 weeks after therapy (B1–B4). A1, before treatment, double staining illustrates intense inflammation (arrows) with highly proliferating, large germinal centers (GS; red nuclear staining for Ki-67), fully developed lymphoepithelial lesions [LEL; brown staining for cytokeratin 14 (CK14)], and reduced glandular parenchyma (PAR). B1, After treatment, inflammation was reduced (arrows), with the absence of germinal centers and the presence of regular striated ducts (SD) devoid of LELs. A2, Before treatment, there was a dominance of B lymphocytes with germinal centers (CD20) in comparison with T lymphocytes (CD3; A3). B2, After treatment, the lymphoid infiltrate overall was reduced, with a slight dominance of T lymphocytes (CD3; B3) compared with B lymphocytes (CD20). A4, A higher magnification view shows fully developed LELs with many intraepithelial lymphocytes and increased basal cell proliferation (arrows), in contrast to the regular striated duct after therapy with CK14-positive basal cells (B4, arrows) with regular differentiation into luminal ductal cells devoid of intraepithelial lymphocytes (arrowheads) (original magnification 120x in 1A1 and 1B1; 100x in 1A2 and 1B2; 60x in 1A3 and 1B3; 200x in 1A4 and 1B4).
(Enlarge Image)
Figure 4.
Four out of the five patients included in the study of Pijpe et al. [36] (see also Fig. 3) showed a minor-to-moderate increase in the parotid salivary flow rate (mean increase 24%). The baseline sodium concentration in parotid saliva was increased in the saliva samples from these four patients (patient 5 had no parotid salivary flow at baseline). The sodium concentration decreased after treatment in all four of the above-mentioned patients, and values returned to near-normal in two of these four patients.
Although Kalk et al. suggested stimulated submandibular/sublingual flow rate in combination with parotid sodium and chloride concentration as a reliable diagnostic test for Sjögren's syndrome, recent progress in proteomics and genomics has shown that the proteomic and genomic profile reflecting the autoimmune process even might be more sensitive and specific in diagnosing Sjögren's syndrome. In contrast to most other diseases studied thus far, in which the majority of reports studying the same disease produced inconsistent results to studies on biomarkers, proteomic and genomic analyses of saliva in pSS were more consistent.
It has been studied whether biomarkers that differ between pSS patients and healthy individuals are indeed specific for pSS or are also commonly expressed in saliva of patients with other autoimmune diseases. As a first step to look into the specificity of biomarkers for diagnosing Sjögren's syndrome, the Sjögren's syndrome proteomic/genomic profile was compared with that of SLE patients (all not fulfilling criteria for secondary Sjögren's syndrome). It was shown that three protein biomarkers, namely, cathepsin D, alpha-enolase, and beta-2-microglobulin (B2M), and three mRNA biomarkers in saliva, namely, myeloid cell nuclear differentiation antigen (MNDA), guanylate-binding protein 2 (GIP2), and the low-affinity IIIb receptor for the Fc fragment of IgG (FCGR3B), were all significantly elevated in pSS patients compared with both SLE patients and healthy controls. The combination of cathepsin D, alpha-enolase, and B2M yielded a receiver operating characteristic (ROC) value of 99% in distinguishing pSS from healthy controls. The combination of the protein marker, B2M, and the mRNA biomarkers MNDA and GIP2 reached a ROC of 95% in discriminating pSS from SLE.
The next step has to be to further test the genomic and proteomic biomarkers in patients with pSS and RA, and to reveal biomarkers that can distinguish pSS from RA and from non-Sjögren's syndrome sicca patients. A validation study sponsored by National Institutes of Health (USA) is currently underway. Moreover, proteomic/genomic analysis of pSS, followed by gene ontology or functional pathway analysis, may also reveal molecular targets and related pathways associated with the disease pathogenesis. As such, these analyses may indicate the progression of the disease and serve as a measure for disease activity. In future, proteomic and genomic analysis also can be applied to rate whether therapeutic intervention has resulted in arresting pSS or regeneration of diseased tissues.
The focus of ultrasound is not only how to diagnose pSS, but also how to rate the progression of the disease and thus might evolve into a tool to objectively assess a treatment effect. Moreover, although the ultrasound image in major salivary glands affected by Sjögren's syndrome is rather characteristic, a current limitation is still that the image is characteristic for the advanced stages of pSS and not for the very early stages of pSS. The better performance of submandibular ultrasound in diagnosing Sjögren's syndrome is in agreement with the observation that submandibular/sublingual glands are earlier involved in Sjögren's syndrome than the parotid glands as based on sialometrical studies. Moreover, parotid glands seem to have a greater reserve capacity than submandibular glands as, on a histopathological level, changes can already be observed in the parotid glands when the salivary flow is not yet significantly affected. This is also in line with parotid ultrasound findings, as changes in parotid salivary composition precede changes in parotid flow showing that the ductal system is primarily affected by the disease process.
A widely accepted criterion for histological confirmation of Sjögren's syndrome is focal lymphocytic sialoadenitis in labial salivary glands. In a normal population, labial biopsy results in 6–9% false-positive diagnoses, and 18–40% of patients with clinically diagnosed Sjögren's syndrome have a negative labial biopsy. An alternative to a labial biopsy is an incision biopsy of the parotid gland. A major advantage of parotid biopsies over labial biopsies with regard to disease progression and assessing the effect of an intervention treatment is that parotid gland tissue can be harvested easily, repeated biopsies from the same parotid gland are possible, and the histopathological results can be compared with other diagnostic results derived from the same gland (e.g. secretory function, sialographic appearance, and ultrasound). There are, however, also some differences between labial and parotid biopsies as, for example, in contrast to labial glands, lymphoepithelial lesions (LELs) are often observed in parotid gland tissue of Sjögren's syndrome patients. Finally, by performing parotid biopsies as a routine diagnostic procedure for Sjögren's syndrome, lymphomas can be found at an earlier stage. However, very rare, B-cell MALT lymphomas are occasionally found in labial biopsies of Sjögren's syndrome patients. Recently, it has been shown that the presence of germinal center-like structures diagnostic of salivary biopsies for pSS might be a highly predictive and easy-to-obtain marker for lymphoma development. This observation allows the risk stratification of patients and the possibility to initiate preventive B-cell-directed therapy. It emphasizes the need to obtain salivary gland biopsies as a method to predict the risk of lymphoma.
Similarly to saliva, the amount and composition of tears are not only influenced by damage of the lacrimal glands, but also reflect the autoimmune response itself. Regarding the dry eye, the use of tear osmolarity in dry eye assessment has recently gained attention for diagnostics, monitoring disease progression, and treatment evaluation in pSS. Tear osmolarity values were shown to be greater in patients with dry eye syndrome related to pSS compared with control individuals, and positively correlated with the severity of dry eye. Measuring osmolarity is simple and probably will become an integral part of dry eye assessments in the clinical setting. Osmolarity has the advantage of functioning as a noninvasive and easily performed objective clinical biomarker for dry eye severity. Osmolarity values of greater than 308mOsms/l are generally indicative of dry eye disease (mild ≈308mOsm/l; moderate ≈320mOsm/l; severe >355mOsm/l).
Concentrations of autoimmune response parameters as anti-Ro/SSA antibody and anti-La/SSB antibody in serum are significantly higher in pSS patients with severe keratoconjunctivitis sicca than mild keratoconjunctivitis sicca. Also presence of anti-La/SSB antibody and mean tear IL-17 concentration were significantly higher in pSS patients with severe keratoconjunctivitis sicca. Thus, serum anti-Ro/SSA antibody, serum anti-La/SSB antibody, and tear IL-17 are likely to be strongly involved in the clinical severity of keratoconjunctivitis sicca in pSS patients and, thus, may have some potential as a disease progression and treatment outcome monitor in pSS.
Histological and functional changes of the lacrimal gland might be reflected in proteomic patterns in tear fluids. In a tear study, multiple protein changes were reproducibly detected in pSS patients, including 10 potential novel biomarkers. Thus, the ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technology using proteomic approaches and microfluidic homogeneous immunoassays. For example, when using the latter immunoassay, Karns and Herr were able to measure lactoferrin, a biomarker that is reduced in Sjögren's syndrome, in a very low volume of tears (<1 μl) which brings the use of tears to monitor Sjögren's syndrome disease activity and progression as well as to assess treatment outcome within reach for clinical application.
Serum may contain a variety of biomarkers related to the autoimmune process that can be used to diagnose Sjögren's syndrome, to characterize disease activity and progression in Sjögren's syndrome, to recognize Sjögren's syndrome patients who may develop extraglandular manifestations, to recognize Sjögren's syndrome patients with an increased risk to develop MALT lymphomas or non-Hodgkin lymphomas, and to evaluate the effect of an intervention treatment with biologicals.
Presence of autoantibodies against Ro (SSA) and La (SSB) in serum is commonly used as an accepted criterion for diagnosing Sjögren's syndrome. Also autoantibodies directed against alpha-phodrin and muscarinic acetylcholine receptors have been proposed as markers for diagnosis and progression of Sjögren's syndrome. Their role is still not clear as, for example, Potthoff et al. showed that neither the severity nor the progression of Sjögren's syndrome correlated with the presence of antialpha-phodrin antibodies in serum.
Increased serum levels of rheumatoid factor and circulating monoclonal immunoglobulins (in particular IgG), reduced levels of C4, and presence of cryoglobulins are frequently observed in Sjögren's syndrome patients and may be indicative for patients at risk of developing extraglandular manifestations and lymphomas and thus can be used as a monitor to early recognize potential development of a lymphoma.
Regarding the use of serum in rating and understanding the effect of intervention treatment with biologicals, the decrease and re-increase in rheumatoid factor, γ-globulins, IgG and β2-microglobulin following B-cell depletion therapy with rituximab in pSS patients might be a useful serum parameter for assessing treatment effects. Analysis of changes in immune activation markers, such as cytokines involved in lymphocyte activation and inflammation, following rituximab treatment might be indicative for response to treatment and, possibly, for recurrence of disease activity (Pollard et al., unpublished observation).
Application of unbiased proteomic technologies may be useful in further unraveling the mechanisms of autoimmune diseases. As discussed previously, first attempts have been made in proteomic and genomic analysis of saliva and tears in pSS, but no data are available yet about blood samples from pSS patients. However, recently it has been shown that interferon-α (IFN-α)-inducible protein 27 (IFI27) showed the most significant difference between Sjögren's syndrome patients and controls in aDNAmicroanalysis using a low-density DNA microarray system with 778 genes. IFI27 gene-expression level was increased in patients with Sjögren's syndrome compared with controls, and the level of IFI27 significantly correlated with serum IgG, β2-microglobulin, soluble interleukin-2 receptor, erythrocyte sedimentation rate, and antinuclear antibody titer. Thus, upregulation of IFN-inducible genes in Sjögren's syndrome patients seems to be a systemic phenomenon and is in line with the notion that pSS patients have a type I IFN signature. Expression levels of IFI27 could thus be an effective and specific biomarker associated with Sjögren's syndrome.
Progression and Disease Activity in Primary Sjögren's Syndrome
As mentioned in the introduction, the largest hurdle to be taken is to develop a reliable set of assessments by which the efficacy of a particular treatment approach can be assessed, meanwhile allowing evidence-based comparison of the various treatment approaches tested in pSS. Such a set of assessments probably also can be used to rate disease activity and disease progression in pSS.
EULAR Sjögren's Syndrome Disease Activity Index and Eular Sjögren's Syndrome Patient Related Index
Evidence-based therapy for Sjögren's syndrome is largely limited to treatments that improve sicca features. In addition, the variety of ad hoc outcome measures used to rate the effects of both symptomatic and disease-modifying treatments are mainly based on glandular symptoms. Therefore, validated activity indexes are needed to assess the effectiveness of (new) targeted therapies. For example, B-cell-targeted therapies have shown promising results for both systemic and glandular features, but, again, a variety of outcome measures has been used. To solve these short-comings, the European League against Rheumatism (EULAR) recently introduced a patient-administered questionnaire to assess patient symptoms (EULAR Sjögren's syndrome Patient Reported Index: ESSPRI) and a systemic activity index to assess systemic complications (EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) as there are for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).
With regard to pSS, studies are in progress to assess the reliability and sensitivity to change of the ESSDAI and ESSPRI. Such studies are important as the usefulness of instruments designed to measure change over time or after therapeutic intervention is not only dependent on their validity and reliability, but also on their potential to detect clinically relevant changes. Seror et al. retrospectively investigated the sensitivity to change of the ESSDAI over time in 96 patient profiles and reported that the internal and external responsiveness of ESSDAI was large, and changes over time were detected more accurately than with other known indices. Meiners et al. prospectively assessed the internal and external responsiveness to change of ESSPRI and ESSDAI in pSS patients treated with rituximab. The latter study revealed that ESSPRI and ESSDAI are, indeed, sensitive to measure the changes in disease activity after therapeutic intervention, which supports the usefulness of these indices for future clinical trials. The promising results from the retrospective analysis of Seror et al. and the prospective intervention study by Meiners et al. showed that, when validated, application of both instruments for outcome measurements in randomized controlled clinical trials should allow assessing all aspects of pSS (Fig. 1).
(Enlarge Image)
Figure 1.
Disease activity in primary Sjögren's syndrome patients treated with rituximab (median values). (a) ESSPRI and ESSDAI. (b) Disease activity for the ESSDAI domains showing the highest changes (percentage of patients with no disease activity in a particular domain is given). *Indicates significant (P<0.05) change compared with baseline.
Saliva
Within the wide spectrum of clinical manifestations of Sjögren's syndrome, salivary gland dysfunction is considered to be a key manifestation. Dysfunction of the salivary glands results in changes of the amount and composition of the saliva. The autoimmune process itself is reflected by the presence of various components of the immune response, such as cytokines, chemokines, autoantibodies, in saliva. As such, sialometry is not only of diagnostic but also of prognostic importance. Moreover, as the amount and composition of saliva reflect the damage caused by the autoimmune process in the salivary glands, sialochemistry may be valuable in diagnosis, assessment of prognosis, and evaluation of treatment. Thus, sialometry and sialochemistry can be used as a diagnostic tool either by collecting whole saliva (the combined secretions of all salivary glands) or by collecting glandular saliva (gland-specific saliva). Although unstimulated whole saliva is a major criterion for the evaluation of salivary gland dysfunction in Sjögren's syndrome, when Sjögren's syndrome develops, not all major salivary glands may yet manifest dysfunction, rendering whole saliva less valuable as a parameter for evaluating progression of the disease or therapeutic intervention than glandular saliva (Fig. 2). In contrast to whole saliva, analysis of gland-specific saliva can reveal sequential involvement of particular glands, reflecting the ongoing autoimmune process in individual major salivary glands. By using glandular saliva, patients with Sjögren's syndrome may frequently be diagnosed at an earlier stage, and progression and effects of therapeutic intervention can be measured in a noninvasive way. For example, Pijpe et al. showed that regeneration of salivary gland tissue by rituximab was accompanied by an increase in salivary flow and normalization of salivary composition (Figs 3 and 4).
(Enlarge Image)
Figure 2.
Relation between disease duration, that is, the time from first complaints induced by or related to oral dryness until referral, and mean salivary flow rates (mean ± SEM). UWS, unstimulated whole saliva; SM/SL, submandibular/sublingual glands.
(Enlarge Image)
Figure 3.
Comparison of parotid biopsy specimens obtained from a pSS patient before rituximab therapy (A1–A4) and 12 weeks after therapy (B1–B4). A1, before treatment, double staining illustrates intense inflammation (arrows) with highly proliferating, large germinal centers (GS; red nuclear staining for Ki-67), fully developed lymphoepithelial lesions [LEL; brown staining for cytokeratin 14 (CK14)], and reduced glandular parenchyma (PAR). B1, After treatment, inflammation was reduced (arrows), with the absence of germinal centers and the presence of regular striated ducts (SD) devoid of LELs. A2, Before treatment, there was a dominance of B lymphocytes with germinal centers (CD20) in comparison with T lymphocytes (CD3; A3). B2, After treatment, the lymphoid infiltrate overall was reduced, with a slight dominance of T lymphocytes (CD3; B3) compared with B lymphocytes (CD20). A4, A higher magnification view shows fully developed LELs with many intraepithelial lymphocytes and increased basal cell proliferation (arrows), in contrast to the regular striated duct after therapy with CK14-positive basal cells (B4, arrows) with regular differentiation into luminal ductal cells devoid of intraepithelial lymphocytes (arrowheads) (original magnification 120x in 1A1 and 1B1; 100x in 1A2 and 1B2; 60x in 1A3 and 1B3; 200x in 1A4 and 1B4).
(Enlarge Image)
Figure 4.
Four out of the five patients included in the study of Pijpe et al. [36] (see also Fig. 3) showed a minor-to-moderate increase in the parotid salivary flow rate (mean increase 24%). The baseline sodium concentration in parotid saliva was increased in the saliva samples from these four patients (patient 5 had no parotid salivary flow at baseline). The sodium concentration decreased after treatment in all four of the above-mentioned patients, and values returned to near-normal in two of these four patients.
Biomarkers in Saliva
Although Kalk et al. suggested stimulated submandibular/sublingual flow rate in combination with parotid sodium and chloride concentration as a reliable diagnostic test for Sjögren's syndrome, recent progress in proteomics and genomics has shown that the proteomic and genomic profile reflecting the autoimmune process even might be more sensitive and specific in diagnosing Sjögren's syndrome. In contrast to most other diseases studied thus far, in which the majority of reports studying the same disease produced inconsistent results to studies on biomarkers, proteomic and genomic analyses of saliva in pSS were more consistent.
It has been studied whether biomarkers that differ between pSS patients and healthy individuals are indeed specific for pSS or are also commonly expressed in saliva of patients with other autoimmune diseases. As a first step to look into the specificity of biomarkers for diagnosing Sjögren's syndrome, the Sjögren's syndrome proteomic/genomic profile was compared with that of SLE patients (all not fulfilling criteria for secondary Sjögren's syndrome). It was shown that three protein biomarkers, namely, cathepsin D, alpha-enolase, and beta-2-microglobulin (B2M), and three mRNA biomarkers in saliva, namely, myeloid cell nuclear differentiation antigen (MNDA), guanylate-binding protein 2 (GIP2), and the low-affinity IIIb receptor for the Fc fragment of IgG (FCGR3B), were all significantly elevated in pSS patients compared with both SLE patients and healthy controls. The combination of cathepsin D, alpha-enolase, and B2M yielded a receiver operating characteristic (ROC) value of 99% in distinguishing pSS from healthy controls. The combination of the protein marker, B2M, and the mRNA biomarkers MNDA and GIP2 reached a ROC of 95% in discriminating pSS from SLE.
The next step has to be to further test the genomic and proteomic biomarkers in patients with pSS and RA, and to reveal biomarkers that can distinguish pSS from RA and from non-Sjögren's syndrome sicca patients. A validation study sponsored by National Institutes of Health (USA) is currently underway. Moreover, proteomic/genomic analysis of pSS, followed by gene ontology or functional pathway analysis, may also reveal molecular targets and related pathways associated with the disease pathogenesis. As such, these analyses may indicate the progression of the disease and serve as a measure for disease activity. In future, proteomic and genomic analysis also can be applied to rate whether therapeutic intervention has resulted in arresting pSS or regeneration of diseased tissues.
Ultrasound
The focus of ultrasound is not only how to diagnose pSS, but also how to rate the progression of the disease and thus might evolve into a tool to objectively assess a treatment effect. Moreover, although the ultrasound image in major salivary glands affected by Sjögren's syndrome is rather characteristic, a current limitation is still that the image is characteristic for the advanced stages of pSS and not for the very early stages of pSS. The better performance of submandibular ultrasound in diagnosing Sjögren's syndrome is in agreement with the observation that submandibular/sublingual glands are earlier involved in Sjögren's syndrome than the parotid glands as based on sialometrical studies. Moreover, parotid glands seem to have a greater reserve capacity than submandibular glands as, on a histopathological level, changes can already be observed in the parotid glands when the salivary flow is not yet significantly affected. This is also in line with parotid ultrasound findings, as changes in parotid salivary composition precede changes in parotid flow showing that the ductal system is primarily affected by the disease process.
Salivary Gland Biopsy
A widely accepted criterion for histological confirmation of Sjögren's syndrome is focal lymphocytic sialoadenitis in labial salivary glands. In a normal population, labial biopsy results in 6–9% false-positive diagnoses, and 18–40% of patients with clinically diagnosed Sjögren's syndrome have a negative labial biopsy. An alternative to a labial biopsy is an incision biopsy of the parotid gland. A major advantage of parotid biopsies over labial biopsies with regard to disease progression and assessing the effect of an intervention treatment is that parotid gland tissue can be harvested easily, repeated biopsies from the same parotid gland are possible, and the histopathological results can be compared with other diagnostic results derived from the same gland (e.g. secretory function, sialographic appearance, and ultrasound). There are, however, also some differences between labial and parotid biopsies as, for example, in contrast to labial glands, lymphoepithelial lesions (LELs) are often observed in parotid gland tissue of Sjögren's syndrome patients. Finally, by performing parotid biopsies as a routine diagnostic procedure for Sjögren's syndrome, lymphomas can be found at an earlier stage. However, very rare, B-cell MALT lymphomas are occasionally found in labial biopsies of Sjögren's syndrome patients. Recently, it has been shown that the presence of germinal center-like structures diagnostic of salivary biopsies for pSS might be a highly predictive and easy-to-obtain marker for lymphoma development. This observation allows the risk stratification of patients and the possibility to initiate preventive B-cell-directed therapy. It emphasizes the need to obtain salivary gland biopsies as a method to predict the risk of lymphoma.
Tears
Similarly to saliva, the amount and composition of tears are not only influenced by damage of the lacrimal glands, but also reflect the autoimmune response itself. Regarding the dry eye, the use of tear osmolarity in dry eye assessment has recently gained attention for diagnostics, monitoring disease progression, and treatment evaluation in pSS. Tear osmolarity values were shown to be greater in patients with dry eye syndrome related to pSS compared with control individuals, and positively correlated with the severity of dry eye. Measuring osmolarity is simple and probably will become an integral part of dry eye assessments in the clinical setting. Osmolarity has the advantage of functioning as a noninvasive and easily performed objective clinical biomarker for dry eye severity. Osmolarity values of greater than 308mOsms/l are generally indicative of dry eye disease (mild ≈308mOsm/l; moderate ≈320mOsm/l; severe >355mOsm/l).
Concentrations of autoimmune response parameters as anti-Ro/SSA antibody and anti-La/SSB antibody in serum are significantly higher in pSS patients with severe keratoconjunctivitis sicca than mild keratoconjunctivitis sicca. Also presence of anti-La/SSB antibody and mean tear IL-17 concentration were significantly higher in pSS patients with severe keratoconjunctivitis sicca. Thus, serum anti-Ro/SSA antibody, serum anti-La/SSB antibody, and tear IL-17 are likely to be strongly involved in the clinical severity of keratoconjunctivitis sicca in pSS patients and, thus, may have some potential as a disease progression and treatment outcome monitor in pSS.
Histological and functional changes of the lacrimal gland might be reflected in proteomic patterns in tear fluids. In a tear study, multiple protein changes were reproducibly detected in pSS patients, including 10 potential novel biomarkers. Thus, the ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technology using proteomic approaches and microfluidic homogeneous immunoassays. For example, when using the latter immunoassay, Karns and Herr were able to measure lactoferrin, a biomarker that is reduced in Sjögren's syndrome, in a very low volume of tears (<1 μl) which brings the use of tears to monitor Sjögren's syndrome disease activity and progression as well as to assess treatment outcome within reach for clinical application.
Serum
Serum may contain a variety of biomarkers related to the autoimmune process that can be used to diagnose Sjögren's syndrome, to characterize disease activity and progression in Sjögren's syndrome, to recognize Sjögren's syndrome patients who may develop extraglandular manifestations, to recognize Sjögren's syndrome patients with an increased risk to develop MALT lymphomas or non-Hodgkin lymphomas, and to evaluate the effect of an intervention treatment with biologicals.
Presence of autoantibodies against Ro (SSA) and La (SSB) in serum is commonly used as an accepted criterion for diagnosing Sjögren's syndrome. Also autoantibodies directed against alpha-phodrin and muscarinic acetylcholine receptors have been proposed as markers for diagnosis and progression of Sjögren's syndrome. Their role is still not clear as, for example, Potthoff et al. showed that neither the severity nor the progression of Sjögren's syndrome correlated with the presence of antialpha-phodrin antibodies in serum.
Increased serum levels of rheumatoid factor and circulating monoclonal immunoglobulins (in particular IgG), reduced levels of C4, and presence of cryoglobulins are frequently observed in Sjögren's syndrome patients and may be indicative for patients at risk of developing extraglandular manifestations and lymphomas and thus can be used as a monitor to early recognize potential development of a lymphoma.
Regarding the use of serum in rating and understanding the effect of intervention treatment with biologicals, the decrease and re-increase in rheumatoid factor, γ-globulins, IgG and β2-microglobulin following B-cell depletion therapy with rituximab in pSS patients might be a useful serum parameter for assessing treatment effects. Analysis of changes in immune activation markers, such as cytokines involved in lymphocyte activation and inflammation, following rituximab treatment might be indicative for response to treatment and, possibly, for recurrence of disease activity (Pollard et al., unpublished observation).
Application of unbiased proteomic technologies may be useful in further unraveling the mechanisms of autoimmune diseases. As discussed previously, first attempts have been made in proteomic and genomic analysis of saliva and tears in pSS, but no data are available yet about blood samples from pSS patients. However, recently it has been shown that interferon-α (IFN-α)-inducible protein 27 (IFI27) showed the most significant difference between Sjögren's syndrome patients and controls in aDNAmicroanalysis using a low-density DNA microarray system with 778 genes. IFI27 gene-expression level was increased in patients with Sjögren's syndrome compared with controls, and the level of IFI27 significantly correlated with serum IgG, β2-microglobulin, soluble interleukin-2 receptor, erythrocyte sedimentation rate, and antinuclear antibody titer. Thus, upregulation of IFN-inducible genes in Sjögren's syndrome patients seems to be a systemic phenomenon and is in line with the notion that pSS patients have a type I IFN signature. Expression levels of IFI27 could thus be an effective and specific biomarker associated with Sjögren's syndrome.
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