Introduction. Association of clinical and diagnostic findings is important in making correct diagnosis in patients with primary Sjogren’s syndrome. The aim of this study was to evaluate the correlation of minor salivary gland biopsy, serological findings and sialometry, as components used to classify primary Sjögren’s syndrome.
Materials and methods. Thirty-six patients with subjective symptoms of possible primary Sjögren’s syndrome underwent minor salivary gland biopsy and sialometry. Clinical and laboratory data were retrieved from the clinical files. We compared and correlated biopsy results, serological findings and salivary flow rate between Sjögren and non-Sjögren patients. Patients were classified into two groups, twenty-eight cases (77.8%) had a diagnosis of primary Sjögren’s syndrome and eight cases (22.2%) did not fulfill the classification criteria for diagnosis.
Results. Primary Sjögren’s syndrome diagnosis strongly correlated with “positive” biopsy (rho= 0.93, p<0.001), serological findings (rho= 0.38, p=0.023) and negatively correlated with saliva flow rate (rho= –0.51, p=0.002). “Positive” biopsy results were in negative correlation with saliva flow rate (rho= –0.41, p=0.012), but in a stronger correlation with patients below the diagnostic flow rate cutoff (≤0.1 ml/minute, rho= 0.46, p=0.005).
Conclusion. In conclusion, unstimulated whole salivary flow rate ≤0.1 ml/minute is highly predictive of “positive” biopsy and can be used as a supplemental method to biopsy in diagnosing the oral component of primary Sjögren’s syndrome.
Key words: Sjögren’s syndrome; Sialometry; Salivary; Gland; Biopsy; Classification
INTRODUCTION
Primary Sjögren’s syndrome (pSS) is a chronic, lymphoproliferative, autoimmune disease affecting exocrine glands. The main features of the disease are dry mouth (xerostomia) and dry eyes (xerophthalmia) resulting from immune-mediated damage and dysfunction of the salivary and lacrimal glands (1, 2). The disease can be of varying intensity, from milder forms involving oral and ocular dryness, fatigue and pain, to more severe systemic conditions affecting multiple organs (3). Patients are at increased risk of developing lymphoma, particularly non-Hodgkin’s lymphoma, compared with the general population (4). Thus, the appropiate and timely diagnosis is important due to clinical follow-up of the possible complications.
The disease occures in all age groups; however it mainly affects women, at ratio of 9:1 (5). The diagnosis of pSS commonly requires a multidisciplinary approach, including rheumatologists, oral medicine specialists, ophthalmologists and it relies on interpreting and integrating all aspects of the patient’s medical history, clinical and laboratory testing and clinical experience of the physician (6, 7). The optimal management of pSS is not yet clear and the current treatments are mainly symptomatic.
The development of classification criteria for use in clinical practice as well as studies have occupied the scientific literature for many decades. Misdiagnosis of pSS is common and numerous studies have shown great variation in the frequency of pSS. Approximately half of all patients are thought to be undiagnosed (8). That makes it one of the least understood inflammatory diseases in current clinical practice. Although various classification criteria for pSS have been proposed, a revised version of the European criteria proposed by the American-European Consensus Group (AECG) has been the most widely used classification set (9). The revised version includes six criteria, four of the six criteria are necessary to confirm the diagnosis of pSS, among which should be either a „positive“ histopathological finding or the presence of autoantibodies (anti-SSA and/or anti-SSB) (10). In daily clinical practice, the only reliable „gold“ standard for the diagnosis of pSS is the clinical judgement of an experienced physician confirmed with objective complementary tests.
Minor salivary gland biopsy (MSGB) is widely accepted and currently remains the best method for diagnosing the salivary gland damage (11), but it is not a sine qua non criterion for diagnosis of pSS. Histopathological finding of lymphocytic sialoadenitis is highly informative, but represents an invasive diagnostic method. Focal lymphocytic infiltrates of minor labial salivary glands are considered target-organ specific for pSS, but it may also be present in other autoimune diseases as well as in aquired immunodeficiency syndrome (AIDS) (12-14). External factors such as smoking and medications have also been suggested to influence the focal lymphocytic infiltrates (15).
The anti-SSA and/or anti-SSB autoantibodies are considered typical among pSS patients. Depending on different laboratory methods, anti-SSA and/or anti-SSB autoantibodies are detected in about 50% to 70% of patients with pSS (16). It is well known that negative serology results occur in 10% to 50% of patients with pSS and correlate with a milder form of the disease (17). The presence of autoantibodies (anti-SSA and/or anti-SSB) have been included in all classifications and usually precede MSGB on routine pSS diagnosis.
The approach to the definition of glandular involvement in pSS is constantly evolving, but in daily practice, clinicians tend to use less invasive diagnostic methods. Thus having access to less invasive but sensitive confirmatory tests that complement existing immunological and/or histopathological findings would be a great advantage. The objective assessment of oral and ocular dryness could be one of such complementary tests. Current literature suggests good agreement between ocular component and pSS, but the previous results regarding xerostomia are inconclusive and possibly confusing. Evaluation of xerostomia in patients with clinically suspected pSS is a diagnostic challenge. Sialometry results can vary considerably because xerostomia can be a side effect of numerous medications (antidepressants, antihistamines, diuretics, etc.) or previous treatment (e.g., radiotherapy of the head and neck) (18).
Sialometry is widely applied in diagnosing xerostomia. Several methods for collecting saliva have been reported. Salivary flow measurement is simple and quick diagnostic method, needs no special equipment and was shown to have good reproducibility. Sialometry has a sensitivity (56.1%) and specificity (80.7%) that is lower than the sensitivity (84.4%) and specificity (86.2%) of MSGB (19); however, sialometry, unlike MSGB, is completely uninvasive diagnostic method. Unstimulated whole saliva (UWS) flow rate ≤0.1 ml/minute is one of the classification criteria for the diagnosis of pSS (10, 20, 21).
In the study by Liquidato et al., authors concluded that both isolated MSGB and sialometry can be used in screening patients with clinically suspected pSS because those diagnostic methods have shown no difference in sensitivity. If there are limitations in the classification of patients, the combination of a „positive“ histopathological finding and sialometry increases the chance of making a correct diagnosis (22).
The main objective of our study was to retrospectively evaluate correlation between MSGB, serological findings and sialometry in patients with clinically suspected pSS.
SUBJECTS AND METHODS
This is a retrospective study involving patients with subjective complaint of oral and ocular dryness who were examined at the Department of Internal Medicine, Clinical Hospital Centre „Sestre milosrdnice“, Zagreb in a period of three years. Clinical oral examination with appropriate diagnostic tests was performed at the Department of Oral Medicine, School of Dental Medicine, University of Zagreb and classification was made based on revised AECG criteria (10). The symptoms of ocular and/or oral dryness or various extraglandular symptoms were the initial reason for specialist consultation and raised suspicion of pSS.
The study was conducted after approval by the Ethics Committee of the Clinical Hospital Centre „Sestre milosrednice“ regarding the retrospective collection of data. After signing the informed consent, the patients were included in the study. The study was performed in accordance with the World Medical Association, Declaration of Helsinki (23).
The diagnosis of pSS was made by experienced rheumatologists in collaboration with oral medicine specialists and ophthalmologists. Patients were classified into two groups, pSS group and non-pSS group. Primary Sjögren’s syndrome was diagnosed based on revised AECG classification criteria, considering the results of the 6-item screening questionnaire for oral and ocular dryness, ocular test (Schirmer’s test), measurement of timed UWS flow rate, MSGB, presence of autoantibodies (anti-SSA and/or anti-SSB), and expert clinical judgement (10). Non-pSS group included patients who have not been diagnosed with pSS because they were not fulfilling the revised AECG diagnostic criteria. Only patients who underwent MSGB were included in the study after signing informed consent.
Exclusion criteria were: non signing informed consent; patients with radiation-induced xerostomia, sarcoidosis-associated xerostomia, current use of anticholinergic drugs; patients with history of secondary SS, hepatitis C virus (HCV) infection, human immuno-deficiency virus (HIV) infection, pre-existing lymphoma, graft-versus-host disease (GvHD); patients without clinical, laboratory and histopathological findings.
The results of clinical, laboratory and histopathological findings were stored in medical files. The data collected included age, gender, family history, body mass index (BMI), subjective symptoms of xerostomia and xerophthalmia, ocular tests, cerebrovascular risk factors (hypertension, diabetes mellitus (DM), smoking, hyperlipidemia), as well as history of other comorbidities. The time (in months) from the initial onset of symptoms to the diagnosis of pSS was also recorded as well as information of previous or current use of immunosuppressants before the MSGB.
Clinical oral examination included measurement of UWS flow rate according to a sialometry protocol (20, 24). Patients were asked to refrain from eating, drinking, smoking and brushing their teeth for at least two hours prior to saliva sampling. Saliva was collected in graduated tubes for five minutes using the spitting method. The UWS flow rate ≤0.1 ml/minute indicated reduced secretion of salivary glands (10, 20, 21).
Minor salivary gland biopsy was performed from the inner surface of the lower lip mucosa under local anesthesia at the Department of Oral Medicine, School of Dental Medicine, University of Zagreb. Histopathological analysis of MSGB was performed at the Department of Pathology and Cytology, University Hospital Centre Zagreb. A “positive” biopsy finding is a classification criteria that highly indicates pSS and refers to the presence of focal lymphocytic sialoadenitis with a focus score ≥1 at 4 mm2 of glandular tissue (1 focus is a cluster of 50 CD4 + cells) (25).
We also used EULAR Sjögren’s Syndrome Patient Reported Index (ESSPRI) to measure perception of patients’ symptoms (26). It is a very simple questionnaire to assess disease symptoms and activity on a 10-point scale for pain, fatigue and dryness. The scores from three questions are added together, resulting in a total score that varies from 3 (very mild symptoms) to 30 (maximum symptoms). With good construct validity, it is used as an outcome measure in clinical trials (27).
Statistical analysis was performed using MedCalc version 11.4 software. The normal distribution of continuous variables was assessed by the Kolomogrov-Smirnov test. The continuous variables are expressed as mean ± standard deviation (SD) or median (interquartile range) as appropriate. Categorical variables are expressed as a number (%). Student’s t-test was used to compare mean values between groups where the distribution was normal. Mann-Whitney non-parametric test was used when the distribution of values was not normal. The significance level (P value) for all analyses was defined as P <.05. The correlation between clinical, serological and histological findings was analysed using Spearman correlation coefficient.
RESULTS
A total of 56 MSGB were performed at the Department of Oral Medicine, School of Dental Medicine, University of Zagreb. Of the initial number of patients, 36 patients (34 female and 2 male) were included in the study because they met the inclusion criteria. Twenty patients were excluded due to lack of clinical or/and laboratory findings or presence of other exclusion criteria.
The mean age of the patients was 53.4 years (ranging from 23 to 77 years) and 34 patients were female (94.4%). According to the revised AECG criteria, 36 patients were divided into two groups, a pSS group (n=28; male/female: 2/26) and a non-pSS group (n=8; male/female: 0/8). Patients in the non-pSS group had subjective oral and ocular dryness but did not meet the diagnostic criteria for pSS diagnosis because they had negative serology and/or histopathological findings that were not consistent with the revised AECG diagnostic criteria. Eighteen patients (64.3%) did not meet serological criteria for the diagnosis of pSS but met the histopathologic criteria. The demographic, clinical and laboratory characteristics of pSS and non-pSS patients are showed in Table 1.
The results showed that the two groups shared most of the features similarly. There are statistically significant differences between these two groups in BMI and obesity. Proportion of obese patients in the pSS group was higher than in the non-pSS group (71.4% vs 12.5%, p=0.005). There was a higher proportion of smokers in the pSS group (17.9% vs 0%, p=0.566), although it was not statistically signifficant. The time from onset of symptoms to diagnosis of pSS ranged from 1 to 84 months, with a mean of 36 months. Two (5.6%) of a total of 36 patients underwent immunosuppressive therapy prior to MSGB.
The perception of symptoms was similar among the groups according to the ESSPRI assessment (Table 2). It is very interesting to notice that the perception of pain measured by the ESSPRI questionnaire was statistically significant lower (p=0.041) in the pSS group than in the non-pSS group, but we must take into account the small number of patients included in interpreting the results. Studies with a larger number of patients are needed to make relevant conclusions.
Serological testing was performed in all 36 patients. At least one autoantibody (anti-SSA or anti-SSB) was positive in 14 patients (38.9%) (Table 1). The frequency of anti-SSA autoantibodies in the pSS group was statistically significant higher than in the non-pSS group (42.9% vs 0%, p=0.033), while the frequency of anti-SSB autoantibodies was similar in both groups (35.7% vs 25.0%, p=0.69).
Mann–Whitney test showed that UWS flow rate was statistically significant lower in pSS group (mean UWS flow rate 0.148 +/– 0.147 vs 0.463 +/– 0.259 ml/minute, p=0.003). Unstimulated whole salivary flow rate is an objective diagnostic test to assess the involvement of the salivary glands. It was performed in all patients (n=36). The results of 22 (61.1%) patients showed hyposalivation according to the revised AECG diagnostic criteria for diagnosing pSS (abnormal UWS flow rate ≤ 0.1 ml/ minute) (Table 1). In the pSS group, 75% of patients had low UWS flow rate (vs 12.5% in non-pSS group). The results of UWS flow rates are presented in Figure 1. The difference is statistically significant (Mann–Whitney test p=0.003).
The correlation between histopathological and serological findings, salivary gland involvement and clinical features were assessed by Spearman rank correlation. As expected, pSS diagnosis strongly correlated with MSGB (rho=0.93, p<0.001), BMI (rho=0.41, p=0.013), the presence of anti-SSA autoantibody (rho=0.38, p=0.023) and negatively correlated with UWS flow rate (rho= –0.51, p=0.002) and ESSPRI pain score (rho= –0.35, p=0.037). Also, positive MSGB results were in negative correlation with UWS flow rate (rho= –0.41, p=0.012) but in a stronger correlation with patients below the diagnostic flow rate cutoff (≤0.1 ml/minute, rho=0.46, p=0.005). The detection of either autoantibody (anti-SSA or anti-SSB) was not in correlation with other parameters but both were correlated well with diagnosis (rho= –0.48, p=0.003 and rho= –0.42, p=0.011, for A and B, respectively).
It is interesting to note that diagnostic criteria did not correlate statistically significant with ESSPRI scores or most demographic or clinical parameters. A good negative correlation was observed only for anti-SSA and anti-SSB autoantibodies and the age of the patient at the time of diagnosis (rho= –0.48, p=0.003 and rho= –0.42, p=0.011 for A and B, respectively). ESSPRI pain and fatigue scores correlated between themselves (rho= 0.73, p<0.0001) but neither correlated with ESSPRI dryness score. Interestingly the dryness score correlated well with patient BMI (rho= 0.57, p=0.0003). Additionally, exercising was negatively correlated with both ESSPRI fatigue (rho –0.37, p=0.026) and ESSPRI total scores (rho= –0.41, p=0.014) indicating that it can alleviate some of the subjective symptoms and improve overall self-perception.
DISCUSSION
The objective of our study was to retrospectively evaluate correlation between MSGB, serological findings and sialometry in patients with clinically suspected pSS. From our experience, in early stage pSS can be misdiagnosed as burning mouth symptoms. Several reports frequently show a delay in the diagnosis of pSS that can range from 3 to 11 years (18.). Already in early stage of disease patients have reduced UWS flow rate, sometimes even without salivary gland ultrasonography abnormalities (28). Using UWS flow rate may improve the timely diagnosis of the disease and should be part of the classification criteria for diagnosing pSS.
The fact that multiple classification criteria for pSS are available could introduce some confusion in clinical trials and research, resulting in different cohorts of patients and in non-comparable results (29, 30). It is interesting to mention that in the classification criteria of the American College of Rheumatology (ACR) (31) the oral component is completely left out. Many medical experts in this field did not agree with such classification criteria (29). In order to develop international consensus on classification criteria, the ACR-EULAR (European League Against Rheumatism) criteria were introduced, endorsed by both ACR and EULAR (21, 32).
The MSGB has been standardized as one of major parameter used in a composite of criteria to provide reliable sensitivity and specificity for the classification of pSS (10, 21, 31). It is an invasive diagnostic method and there is no algorithm or any recommendation when to indicate MSGB. Unfortunately, there are no clear guidelines when MSGB is indicated in the treatment of patients with pSS. There is no data in the available literature on how rheumatologists use MSGB results in daily clinical work. Patients with clinically suspected pSS are preselected for having difficulty with their oral mucosa, thus, any operative intervention in the oral area should be undertaken only with good reason. Given the diversity of classification criteria used throughout the years, the lack of experience with MSGB, inconsistent interpretation of results and patient benefit, it is hard to assess in which cases it would be convenient to have MSGB in defining diagnosis of pSS. From our experience, in select clinical situations, the diagnosis of pSS can be made based on noninvasive tests i.e., it can be confirmed by positive serology findings (anti-SSA and/or anti-SSB). Clearly, dryness of the exocrine glands and serologic markers are not a complete replacement for tissue diagnosis, which does provide additional data that may be helpful in certain clinical situations.
Over the past years, salivary gland ultrasound has gained more attention. It was proven to be effective for the detection of typical structural abnormalities in pSS (33, 34). Parotid gland ultrasound can be directly compared to parotid and submandibular glands histopathology (35). Salivary gland ultrasound has a good sensitivity and high specificity, it appeared to mirror dysfunction of the salivary glands, even at the early stages of the disease (36). It is non-invasive, relatively inexpensive, easily accessible and quick (37). Despite the potential of salivary gland ultrasound in diagnosis and classification of pSS, the value of salivary gland ultrasound to assess disease activity and disease progression needs to be established (38).
There are many methods of measuring salivary gland function, although not all of them are practical and adaptable for clinical trials (28, 39). As a measure of salivary gland function, the UWS flow rate has been adopted as one validated measure for the revised AECG and ACR-EULAR criteria for pSS (10, 21). It provides great sensitivity and specificity in differentiating patients with pSS from non-pSS patients. It is simple to administer and non-invasive, needs no special equipment, has a good reproducibility and reflects the physiologic state of the gland. The results of our study showed statistically significant correlation between “positive” biopsy score and clinically reduced (≤0.1 ml/minute) saliva secretion (rho= 0.461, p=0.0047). Our results illustrate the correlation between MSGB, which shows morphological changes, and UWS flow rate, representing the functional aspect. Therefore, UWS flow rate may serve as a noninvasive surrogate biomarker of inflammation and fibrosis as well as an indicator of treatment success in patients with pSS. Unstimulated whole salivary flow rate reflects the basal saliva flow. It should be included in all pSS trials to document glandular parenchymal damage and its possible deterioration over time in pSS. Considering the statistical correlation between a “positive” biopsy score and the serological findings, it was noted that there was not statistically significant correlation between these two parameters (p=0.10). Our results reaffirm that “positive” histopathology finding is a requirement for the diagnosis of pSS in the absence of anti-SSA autoantibodies (22, 40, 41). We highly recommend that physicians should consider performing MSGB in patients with glandular dysfunctions and negative serology. Evidence-based algorithms need to be developed in screening patients who have dryness of the exocrine glands to prevent delaying the diagnosis of pSS, and to avoid unnecessary MSGB in patients who already have oral complications. In patients diagnosed with pSS, objective diagnostic tests for ocular and oral dysfunction should be performed regularly to monitor salivary gland function over time. Future studies aimed at developing effective screening and treatment algorithms are needed to improve the quality of life of these patients. In that respect, continuing research in understanding the underlying etiopathogenesis and possibly incorporating genetic and genomic alterations, might prove beneficial in constructing improved criteria for this complex disorder.
We acknowledge some limitations of our study. The study was conducted in a single center with a retrospective nature. The study was performed at the Clinical Hospital Center „Sestre milosrdnice“, which belongs to tertiary health care. Patients with various diseases were included in the study and we performed MSGB and UWS flow rate in all patients. Being a single-centre study, the number of patients included was limited and most importantly, the control group was represented by patients clinically suspected for pSS but diagnosed with non-pSS, which is a heterogeneous entity. However, patients with xerostomia (non-pSS patients) had all undergone MSGB, which to date, remains a cornerstone for pSS diagnosis. It is not possible to exactly determine how many patients with clinically suspected pSS were not biopsied and therefore not included, a factor which could therefore have had an influence on the results. The limited number of cases and the long time interval for the indication of the MSGB and UWS flow rate in some cases under pSS investigation may have modulated the results. Eight of our patients were receiving immunosuppressive therapy, which can downregulate the production of autoantibodies and also altered some histological parameters. Because of the retrospective nature of the study we were not able to evaluate the temporality of the autoantibody presentation. Thus, prospective carrying out of other serological tests (rheumatoid factor, C3, C4, cryoglobulins) would have improved the validity of the clinical judgment and possibly modified the sensitivity of the classification criteria. The methods adopted for ocular staining varied among the participating institutions, forcing us to modify certain items in some criteria.
In conclusion, in our study according to the revised AECG classification criteria only „positive“ histopathological findings, reduced saliva secretion and existence of anti-SSA autoantibody confirmed to have a significant diagnostic value in detecting pSS patients. Also, UWS flow rate appears to be a useful aid in diagnosis of pSS. Unstimulated whole salivary flow rate ≤0.1 ml/minute is highly predictive of “positive” MSGB and can be used as a supplemental method to MSGB in diagnosing the oral component of pSS. As a non-invasive, easy to perform, inexpensive method it might be good monitoring tool to follow the progress of the disease.
Funding: Research funding was provided by the Croatian Ministry of Science, Education and Sport, project Nos. 108-1081874-2416 (Professor Jasenka Markeljević).
Conflict of interest statement: The authors declare no conflict of interest.
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Table 1. Demographic, clinical and laboratory characteristics of pSS group according to the revised AECG criteria
Legend / Legenda: pSS – primary Sjögren’s syndrome / primarni Sjögrenov sindrom; AECG: American-European Consensus Group
* Fischer’s exact test and t-test were used, significant differences indicated in bold / Upotrebljavani su Fisherov test i t-test, značajne razlike označene su podebljanim slovima
Table 2. Results of ESSPRI measurements in the pSS and non-pSS groups.
Legend / Legenda: pSS – primary Sjögren’s syndrome / primarni Sjögrenov sindrom
ESSPRI: European League Against Rheumatism Sjögren’s syndrome patient report index
* Mann-Whitney test, significant p-values highlighted in bold / Mann-Whitneyjev test, značajne p-vrijednosti istaknute su podebljanim slovima
Figure 1. Unstimulated whole saliva flow rate in pSS and non-pSS patients