Histopathological comparison of Sjögren-related features between paired labial and parotid salivary gland biopsies of sicca patients.

OBJECTIVES: To compare focus score and other histopathological features between paired labial and parotid salivary gland biopsies in a diagnostic cohort of suspected Sjögren's disease (SjD) patients. METHODS: Labial and parotid salivary gland biopsies were simultaneously obtained from patients with sicca complaints, suspected of having SjD. Biopsies were formalin fixed and paraffin embedded. Sections were stained with haematoxylin & eosin, and for CD3, CD20, CD45, cytokeratin, CD21, Bcl6, activation-induced deaminase (AID) and IgA/IgG. Focus score and other histopathological features characteristic for SjD were analysed. RESULTS: Based on the expert opinion of three experienced rheumatologists, 36 patients were diagnosed as SjD and 63 as non-SjD sicca patients. When taking all patients together, absolute agreement of various histopathological features between labial and parotid biopsies was high and varied between 80% (focus score) and 93% [(pre-)lymphoepithelial lesions (LELs)]. More labial gland biopsies had a focus score ≥1 compared with their parotid counterpart. Accordingly, the area of infiltrate was larger in labial gland biopsies. When considering only SjD patients, labial glands contained significantly fewer B-lymphocytes and germinal centres/mm2, and less severe LELs compared with parotid glands. CONCLUSION: Labial and parotid glands from SjD patients contain similar histopathological key features, and thus both glands can be used for diagnosis and classification of SjD. However, parotid salivary glands reveal more evident B-lymphocyte-related features, while labial glands exhibit more inflammation, which may be partially unrelated to SjD.

FDG-PET/CT discriminates between patients with and without lymphomas in primary Sjögren's syndrome.

OBJECTIVES: To assess the usefulness of [18F]-fluorodeoxyglucose (FDG)-PET/CT (i) to discriminate between primary SS (pSS) patients with and without lymphomas and (ii) to evaluate systemic disease activity in pSS. METHODS: ACR-EULAR-positive pSS patients who underwent FDG-PET/CT were included. Scans were visually evaluated and quantitative analysis was performed by measuring standardized uptake values (SUV) of salivary and lacrimal glands and systemic regions. Receiver operating characteristic curve analyses were performed to find SUV cut-off values to discriminate between lymphoma and non-lymphoma. RESULTS: Of the 70 included patients, 26 were diagnosed with a pSS-associated lymphoma, mostly of the mucosa-associated lymphoid tissue type (23/26). Lymphoma patients showed higher FDG uptake in the parotid and submandibular glands, and more frequently showed presence of nodular lung lesions, compared with non-lymphoma patients. The accuracy of the maximum SUV (SUVmax) in the parotid and submandibular gland to predict lymphoma diagnosis was good, with optimal cut-off points of 3.1 and 2.9. After combining these three visual and quantitative findings (nodular lung lesions, parotid SUVmax > 3.1 and submandibular SUVmax > 2.9), sensitivity was 92% when at least one of the three features were present, and specificity was 91% in case at least two features were present. Furthermore, FDG-PET/CT was able to detect systemic manifestations in pSS patients, mostly involving lymph nodes, entheses and lungs. CONCLUSIONS: FDG-PET/CT can assist in excluding pSS-associated lymphomas in patients without PET abnormalities, possibly leading to a decrease of invasive biopsies in suspected lymphoma patients. Furthermore, FDG-PET/CT is able to detect systemic manifestations in pSS and can guide to the best biopsy location.

Salivary gland 18F-FDG-PET/CT uptake patterns in Sjögren's syndrome and giant cell arteritis patients.

OBJECTIVES: Wide variety in salivary gland 18F-FDG-uptake is observed in the general population. A general consensus about the usefulness of 18F-FDG-PET/CT to detect salivary gland inflammatory conditions, such as in primary Sjögren's syndrome (pSS), is not yet clear. This study aimed to investigate whether there are differences in uptake of 18F-FDG in salivary glands among two autoimmune groups [pSS, giant cell arteritis (GCA)] and a non-autoimmune group (lung cancer). METHODS: PSS patients aged ≥50 years who underwent 18F-FDG-PET/CT were included and age-matched with GCA patients and a non-autoimmune control group (lung cancer patients). Scans were visually evaluated and quantitative analysis was performed by measuring standardised uptake values (SUV) within salivary glands and lacrimal glands. For GCA patients, arteries in the vicinity of the parotid and submandibular gland were assessed for positivity. RESULTS: PSS patients did not show increased 18F-FDG-uptake in the parotid or submandibular gland, compared to the other two groups. For the tubarial gland, significantly higher SUVmax was found in the pSS patient group. Interestingly, GCA patients had significantly higher SUVmax in the submandibular gland than the other two groups. Visual 18F-FDG-positivity of cranial arteries related to the parotid and submandibular glands was associated with significantly higher SUVmax in salivary glands of GCA patients. CONCLUSIONS: Although 18F-FDG-uptake was not increased in parotid and submandibular glands of pSS patients, increased 18F-FDG-uptake in tubarial glands of pSS patients might indicate a role for these glands in pSS. Furthermore, parotid and submandibular glands may be affected by local vasculitis in GCA.

Digital image analysis of intraepithelial B-lymphocytes to assess lymphoepithelial lesions in salivary glands of Sjögren's syndrome patients.

OBJECTIVE: Salivary glands of primary SS (pSS) patients characteristically harbour periductal infiltrates, in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. METHODS: A virtual triple-staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high-molecular-weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as having pSS or non-SS according to the ACR-EULAR classification criteria. RESULTS: T-lymphocytes were detected in almost all analysed ducts of pSS and non-SS sicca patients, whereas intraepithelial B-lymphocytes were present in 59-68% of labial and parotid gland biopsies of pSS patients, against only 2-3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, ∼25% of analysed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). CONCLUSION: The presence of intraepithelial B-lymphocytes in salivary gland biopsies of sicca patients is a clear indicator of pSS and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte-containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.

Histopathology, salivary flow and ultrasonography of the parotid gland: three complementary measurements in primary Sjögren's syndrome.

OBJECTIVE: The involvement of salivary glands in primary SS (pSS) can be assessed in different ways: histopathology, salivary flow and ultrasonography. To understand the relative value of these different approaches, it is crucial to understand the relationship between them. As we routinely perform these three modalities in the parotid gland for disease evaluation, our aim was to investigate the construct validity between these modalities in one and the same gland. METHODS: Consecutive sicca patients underwent a multidisciplinary diagnostic workup including parotid gland biopsy, collection of parotid gland-specific saliva and parotid gland ultrasonography. Patients who were classified as pSS according to the ACR-EULAR criteria were included. Construct validity was assessed using Spearman's correlation coefficients. RESULTS: The 41 included pSS patients completed a full workup within a mean time interval of 2.6 months. Correlations between histopathological features and stimulated parotid salivary flow were fair (ρ = -0.123 for focus score and ρ = -0.259 for percentage of CD45+ infiltrate). Likewise, poor correlations were observed between stimulated parotid salivary flow and parotid ultrasonography (ρ = -0.196). Moderate to good associations were found between the histopathological items focus score and the percentage of CD45+ infiltrate, with parotid US scores (total US score: ρ = 0.510 and ρ = 0.560; highest for homogeneity: ρ = 0.574 and ρ = 0.633). CONCLUSION: Although pSS-associated ultrasonographic findings did correlate with histopathological features, the three modalities that evaluate salivary gland involvement assess different (or at best partly related) constructs. Therefore histopathology, salivary flow and ultrasonography are complementary measurements and cannot directly replace each other in the workup of pSS.

Presence of intraepithelial B-lymphocytes is associated with the formation of lymphoepithelial lesions in salivary glands of primary Sjögren's syndrome patients.

OBJECTIVES: Lymphoepithelial lesions (LELs) in salivary glands are associated with primary Sjögren's syndrome (pSS). LELs are composed of hyperplastic epithelium infiltrated with lymphocytes. The objective of this study was obtaining insight in the relative roles of intraepithelial B- and T-lymphocytes in the formation of LELs in salivary glands of pSS patients. METHODS: Parotid and labial salivary gland biopsies of pSS patients (n=15), non-SS sicca patients (n=5) and non-sicca controls (n=5) were analysed. Serial sections were stained with H & E and for cytokeratin, CD20 and CD3. Striated ducts with lymphocytes, but without hyperplasia, and striated ducts with LELs were identified in H & E and cytokeratin stained sections. LELs were classified in successive stages of severity based on the amount of hyperplasia (stage1-3). Numbers of B- and T-lymphocytes within striated ducts and LELs were counted in CD20 and CD3 stained sections. RESULTS: Lymphocyte-containing striated ducts of both salivary glands of all pSS and control patients harboured T-lymphocytes, scattered throughout the ductal epithelium. In contrast, B-lymphocytes were exclusively found in a small fraction (21%) of striated ducts without hyperplasia and in nearly all striated ducts with LELs of pSS patients, but not in controls. In striated ducts with LELs B-lymphocytes were mostly located in the areas of proliferating epithelium. Numbers of B-lymphocytes and B/T-ratios increased significantly with higher severity of LELs. This was even more pronounced in the parotid than in the labial gland. CONCLUSIONS: We conclude there is an association between presence of intraepithelial B-lymphocytes and the formation of LELs in salivary glands of pSS patients.

Serum immunoglobulin free light chains are sensitive biomarkers for monitoring disease activity and treatment response in primary Sjögren's syndrome.

Objectives: Serum immunoglobulin free light chains (FLCs) are frequently elevated in B-cell-mediated autoimmune diseases, including primary SS (pSS). The objective of this study was to assess if serum FLCs can contribute to classification, mucosa-associated lymphoid tissue (MALT) lymphoma detection, monitoring of disease activity and treatment response in pSS. Methods: Serum samples of 100 consecutive patients suspected of pSS were included. Forty-five patients fulfilled ACR-EULAR criteria for pSS. Additionally, samples of 17 pSS patients with MALT lymphoma and longitudinal samples of pSS patients treated with rituximab (n = 20), placebo (n = 10) or abatacept (n = 15) were included. Serum FLCκ/FLCλ was measured by nephelometry or turbidimetry. Results: At diagnosis, FLCκ and FLCλ serum levels were significantly higher in pSS compared with non-SS sicca patients. The FLCκ/FLCλ ratio was abnormal in 11% of pSS patients. In established MALT-pSS patients, without recent rituximab treatment (n = 12), 50% had abnormal FLCκ/FLCλ ratios. FLC measurement had no additional value for pSS classification, compared with IgG and anti-SSA. FLC levels correlated significantly with systemic disease activity, assessed by EULAR SS Disease Activity Index (ESSDAI) and clinical ESSDAI, both cross-sectionally and longitudinally following treatment. Treatment with rituximab or abatacept significantly lowered FLC levels. FLCs show a large sensitivity to change and relative changes induced by treatment were higher compared with IgG. Conclusion: Serum FLCs are elevated in pSS, and abnormal FLCκ/FLCλ ratios may be indicative for the presence of MALT lymhoma. FLC levels can be used as a biomarker for systemic disease activity and monitoring treatment responses. FLCs are sensitive to change and have more favorable kinetics than IgG.

Validation of the ACR-EULAR criteria for primary Sjögren's syndrome in a Dutch prospective diagnostic cohort.

Objectives: To validate the ACR-EULAR classification criteria for primary SS (pSS), and compare them to the American-European Consensus Group (AECG) and ACR criteria in a Dutch prospective diagnostic cohort. Methods: Consecutive patients (n = 129) referred for suspicion of pSS underwent a multidisciplinary evaluation, including a labial and/or parotid gland biopsy. Patients with an incomplete work-up (n = 8) or associated systemic auto-immune disease (n = 7) were excluded. The ACR-EULAR classification was compared with expert classification, AECG and ACR classification. Additionally, the accuracy of individual ACR-EULAR items in discriminating pSS from non-pSS was evaluated. The validity of criteria sets was described separately using parotid or labial gland biopsy results for classification. Results: Of the 114 evaluated patients, the expert panel classified 34 (30%) as pSS and 80 (70%) as non-pSS. Using labial gland biopsy results, ACR-EULAR classification showed 87% absolute agreement (κ = 0.73) with expert classification, with a sensitivity of 97% and specificity of 83%. Using the parotid gland biopsy results, the ACR-EULAR criteria performed excellently as well. Focus score, anti-SSA titre and ocular staining score showed good to excellent accuracy, whereas unstimulated whole saliva and Schirmer's test had poor accuracy. The ACR-EULAR and AECG criteria had equal validity. Compared with ACR classification, ACR-EULAR classification showed higher sensitivity but lower specificity. Conclusion: The ACR-EULAR criteria showed good agreement with expert classification, but some patients may be misclassified as pSS. Unstimulated whole saliva and Schirmer's test showed poor discriminative value. The ACR-EULAR criteria performed equally to the AECG criteria, and had higher sensitivity but lower specificity than the ACR criteria.

Increased Diagnostic Accuracy of the Labial Gland Biopsy in Primary Sjögren Syndrome When Multiple Histopathological Features Are Included.

OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy of the labial salivary gland biopsy based on multiple histopathological features in patients with suspected primary Sjögren syndrome (pSS). METHODS: Patients from a diagnostic sicca cohort with clinically suspected pSS who underwent a labial gland biopsy were included. Patients were categorized as having pSS or non-Sjögren syndrome sicca (non-SS sicca) based on vignettes scored by an expert panel. Labial gland biopsies were analyzed for the presence of four histopathological features: focus score (FS) ≥1, prelymphoepithelial and lymphoepithelial lesions, immunoglobulin G plasma cell shift, and germinal centers. Sensitivity and specificity of histologic features were calculated, and the optimal cutoff value for the number of histopathological features needed to diagnose pSS was determined with receiver operating curve analysis. RESULTS: A total of 38 patients were categorized as having pSS and 65 as having non-SS sicca. In labial gland biopsies of patients with pSS, the prevalence of FS ≥1 was 82%, followed by 68% for pre-lymphoepithelial and lymphoepithelial lesions, 63% for plasma cell shift, and 24% for germinal centers. Although FS ≥1 showed the highest sensitivity for patients with pSS (82%), specificity was higher for the other three features (98%-100%). The presence of two or more (of four) histopathological features had almost comparable sensitivity to FS alone, but specificity increased with 12% to 100%. For fulfillment of American College of Rheumatology/EULAR criteria, specificity increased from 84% to 95% when an abnormal biopsy was defined by the presence of two or more histopathological features instead of FS ≥1 only. CONCLUSION: The diagnostic accuracy of the labial gland biopsy increases when other histopathological features besides FS are taken into account, by reducing the number of false-positive biopsies.

More severe parotid gland histopathology in paediatric-onset than in adult-onset Sjögren's disease.

OBJECTIVES: The aim of this study was to assess the histopathological features of the parotid glands in patients with paediatric-onset Sjögren's disease (pedSjD) in comparison to patients with adult-onset Sjögren's disease (adSjD). METHODS: This study was performed in Groningen, the Netherlands. Patients with pedSjD from a diagnostic paediatric cohort (n=19), patients with adSjD from a diagnostic adult cohort (n=32) and patients with adSjD who participated in a clinical trial (n=42) with a baseline parotid gland biopsy were included. Parotid gland biopsies were analysed after (immuno)histological staining for SjD-related histopathological markers and compared between groups. RESULTS: All characteristic histopathological features of adSjD were also observed in pedSjD. There were no significant differences in lymphoepithelial lesions or immunoglobulin A (IgA)/IgG plasma cell shift between the pedSjD and the adSjD cohorts. However, compared with the diagnostic adSjD cohort (with comparable total EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) scores), pedSjD showed more severe lymphocytic infiltration as reflected by a higher focus score (p=0.003), a higher relative surface area of CD45+ infiltrate (p=0.041), higher numbers of B and T lymphocytes/mm2 (p=0.004 and p=0.029, respectively), a higher B/T lymphocyte ratio (p=0.013), higher numbers of CD21+ follicular dendritic cell networks/mm2 (p=0.029) and germinal centres (GC)/mm2 (p=0.002). Compared with the trial adSjD cohort, with significant higher total ESSDAI scores (p=0.001), only the B/T lymphocyte ratio and numbers of GC/mm2 were significantly higher in the pedSjD cohort (p=0.023 and p=0.018, respectively). CONCLUSION: Patients with pedSjD exhibit more pronounced histopathological features compared with patients with adSjD at diagnosis. Notably, the histopathology of patients with pedSjD aligns more closely with that observed in an adSjD clinical trial cohort, with even stronger B lymphocyte involvement.

Incorporation of Salivary Gland Ultrasonography Into the American College of Rheumatology/European League Against Rheumatism Criteria for Primary Sjögren's Syndrome.

OBJECTIVE: To assess whether the addition of salivary gland ultrasonography (SGUS) or replacement of current criteria items by SGUS influences the performance of the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for primary Sjögren's syndrome. METHODS: Included were consecutive patients with complete data on all ACR/EULAR items (n = 243) who underwent SGUS in our primary Sjögren's syndrome expertise center. Clinical diagnosis by the treating physician was used as the gold standard. Separate analyses were performed for patients who underwent labial or parotid gland biopsies. The average score for hypoechogenic areas in 1 parotid and 1 submandibular gland was determined (range 0-3). Next, performance of the ACR/EULAR criteria was evaluated after addition of SGUS or replacement of current items by SGUS. RESULTS: Receiver operating characteristic analysis showed an optimal cutoff value of ≥1.5 for SGUS. The optimal weight for SGUS positivity was 1. Cutoff for ACR/EULAR fulfilment remained ≥4. In patients who underwent a labial gland biopsy (n = 124), the original criteria showed an area under the curve (AUC) of 0.965, sensitivity of 95.9%, and specificity of 92.2%. After the addition of SGUS, the AUC was 0.966, with a sensitivity of 97.3% and specificity of 90.2%. In patients who underwent a parotid gland biopsy (n = 198), similar results were found. Sensitivity of the criteria decreased substantially when SGUS replaced salivary gland biopsy or anti-SSA antibodies, while performance remained equal when SGUS replaced the ocular staining score, Schirmer's test, or unstimulated whole saliva flow. CONCLUSION: Validity of the ACR/EULAR criteria remains high after incorporation of SGUS. With SGUS, clinicians are offered a larger array of tests to evaluate fulfillment of the ACR/EULAR criteria.

Imaging in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by dysfunction and lymphocytic infiltration of the salivary and lacrimal glands. Besides the characteristic sicca complaints, pSS patients can present a spectrum of signs and symptoms, which challenges the diagnostic process. Various imaging techniques can be used to assist in the diagnostic work-up and follow-up of pSS patients. Developments in imaging techniques provide new opportunities and perspectives. In this descriptive review, we discuss imaging techniques that are used in pSS with a focus on the salivary glands. The emphasis is on the contribution of these techniques to the diagnosis of pSS, their potential in assessing disease activity and disease progression in pSS, and their contribution to diagnosing and staging of pSS-associated lymphomas. Imaging findings of the salivary glands will be linked to histopathological changes in the salivary glands of pSS patients.