Salivary Gland Biopsy—Diagnostic Tool
In experienced hands, minor salivary gland biopsy is a well-tolerated procedure associated with a low complication rate . While some patients are willing to have three or even four biopsies removed for the purposes of follow up and research, there is an ethical limit on the number of consecutive lip biopsies.
Salivary gland involvement has a central role when assessing the development and pathogenesis of SS. Notably, SS histopathology is strongly associated with autoantibodies but only correlates weakly with xerostomia in SS patients. The diagnostic role of salivary gland histology still remains widely accepted and a central part of both AECG and the new proposed classification criteria endorsed by the ACR in 2012 .
The autoantibodies Ro/SSA and La/SSB are valuable diagnostic tools, they appear early in the disease, persist, and correlate with focus scores. Histological focus scoring has been employed to describe salivary gland involvement in SS, where a positive biopsy with mononuclear cell infiltrates comprising of ≥50 mononuclear cells per 4 mm2 results in a positive score value of 1–12 according to numbers of foci seen.
Few studies have assessed other histopathological features in the salivary gland environment and their possible associations with diagnosis and stage of disease. The destruction of salivary gland tissue in SS is commonly accompanied by the development of adipose tissue and fibrosis where adipocytes can occupy a large part of the gland. A recent study  highlights the possibility that there could be a relationship between the disease activity and adipose tissue replacement in the gland, and that fat replacement could be a helpful support in the diagnostic evaluation of the glandular tissue. Furthermore, a possible active role of adipocytes in the immune reactions in the glandular environment has been suggested [40, 41]. During the last years, novel diagnostic tools have been investigated. These would include salivary gland ultrasound imaging where parenchymal inhomogeneity appears to be the method with most promising results . Even if its role in the early stages of disease is debated, it is worthy of note that, when used in association with traditional tests, ultrasound improves the diagnostic sensitivity of the AECG [43–46].
Proteomic biomarker profiles of unstimulated whole saliva from SS patients have been investigated for potential as a tool for patient subclassification . Further studies will be necessary to determine the utility of such an approach. To facilitate the evaluation of treatment efficacy in clinical trials and to select subgroup of patients for personalized treatment, the availability of new prognostic markers is needed.
The classic glandular lesion is composed of a lymphoid infiltrate of T and B lymphocytes, whose distribution may vary according to disease severity. Macrophages, plasma cells, NK cells, and dendritic cells are also present in varying degree [48, 49]. A great effort has been made to in deeply characterizing the role of different T cell subsets in pSS. In pSS patients, the specific T helper subset, Th 17 cells, mainly defined by secretion of cytokine IL-17, has been found in elevated numbers both in the periphery and also present in the salivary gland tissue [50, 51]. The follicular T helper cells are another subset derived from naïve lymphocytes under the stimulus of IL-12 secreted by dendritic cells, and these cells are involved in the crosstalk between T and B cells. It has been indicated that these cells participate in the pathogenesis of pSS by promoting B-cell maturation .
B cell hyperactivity represents a key hallmark in the pathogenesis of SS and hypergammaglobulinemia, autoantibody production and alterations of B cell subpopulations are distinctive features of pSS patients. Patients with pSS present up to 16 times increased risk for developing non-Hodgkins lymphoma (NHL)  and mucosa associated lymphoid tissue (MALT) lymphoma in the salivary glands and gastrointestinal tract compared to healthy individuals. Lymphoid organization in the form of germinal center (GC)-like structures has been identified in the salivary glands of a subgroup of SS patients . Notably, the identification of germinal center-like structures has been suggested to be a possible predictor of the development of lymphoma since, based on results from a study from Theander et al., the majority of the GC+ patients developed lymphoma later on . This novel finding may allow identification of high-risk patients for repeated lymphoma screening and selection of candidates for advanced B-cell directed biological treatment.
Negative status for anti-Ro/SSA and/or anti-La/SSB is suggested to be a protective factor for evolution toward lymphoma in these patients . However, the development of lymphomas in pSS is not confined only to serologically positive patients for anti-Ro and anti-La. Accordingly, the rheumatologists are encouraged to include the minor salivary gland biopsy in the routine work-up . CD4+ T lymphocytopenia is an additional strong risk factor for developing lymphoma . Lymphomas often develop in salivary glands of SS patients where the disease is active. Nocturne and Mariette recently launched a “2014 proposed scenario for the pathophysiology of pSS-associated lymphoma” . They envisioned that immune complexes with antibodies against specific antigens such as SSA/Ro and SSB/La or others, continuously stimulate autoimmune B cells containing rheumatoid factor activity. Furthermore, that defects in control of NF-kB activation accentuate B cell over-activation and promote survival of B cells and oncogenic mutations.
The HLA carries the major genetic influence on susceptibility to autoimmune diseases, as it is an important key in antigen presentation and immune response. A meta-analysis found DRB1*03:01, DQA1*05:01, DQB1*02.01, and DRB1*03 to be risk factors for pSS while DQA1*02:01, DQA1*03:01, and DQB1*05:01 alleles were protective . DRB1*03-DQB1*02 was the significant risk haplotype associated with pSS on a worldwide level in that study. This is in line with our previous study where we also found components of the DRB1*03-DQB1*02-DQA1*0501 haplotype as the strongest contributors to the formation of an anti-Ro/La response in a study of pSS Caucasions .
Several non-HLA regions have been implicated in pSS. Gene expression microarray studies on labial salivary glands and peripheral blood showed dysregulation of type I interferon-inducible genes [59, 60]. Two GWAS studies have recently been performed on pSS, one on European descents, and one on Chinese descents [61••, 62••]. Among SS-associated non-HLA genes discovered by GWAS, we find STAT4 and IRF5 encoding transcription factors, BLK coding for B cell kinase, as well as genes encoding the IL-12A cytokine, and interestingly, genes involved in NF-kB signaling and the CXCR5 chemokine production . CXCR5 is receptor of CXCL13, which directs B cells to lymphoid follicles . Mice deficient of Cxcl13 or its receptor Cxcr5 fail to form these structures [64, 65]. In addition to the interferon signature appearing from the microarray gene expression profiling of minor salivary glands of primary SS patients and healthy controls, particularly two other interesting observations were made by Hjelmervik et al. . Firstly, CXCL13 which directs B cells chemotaxis was differently expressed in patients and controls. CXCL13 were among the genes that were expressed in 9 of 10 patients with pSS, whereas it was only expressed in 1 of 10 healthy individuals . This is supported by a recent study by Kramer et al. who found CXCL13 to be elevated in serum and saliva of SS patient and in mice models . Secondly, we found lymphotoxin-β (LTB) to be among the most highly expressed genes in inflamed salivary glands of pSS patients. Lymphotoxin-β receptor (LTβR) signaling is crucial for the formation of lymphoid tissue, and LTβ can activate NF-kB pathways that promote inflammation [67–69]. When we in a later experiment blocked the Ltβr in animal models of SS, there was an increase in salivary secretion and a reduction of inflammation in the glands . We experienced an amelioration of SS after neutralization of Ltβr signaling. A congenic mouse model was also developed that differed genotypically from the control mice only in two non-MHC loci, which were sufficient for the congenic mice to develop sialadenitis spontaneously . Cxcl13 and Ltβ were among the genes that were found differentially expressed in salivary glands of the NOD congenic mice compared to control mice. Furthermore, Ltβ blockade also reduced Cxcl13 in lacrimal glands of a NOD model of SS improving the corneal integrity [72, 73]. Recently, early BAFF receptor blockade was shown to mitigate murine SS [74•]. Concomitant targeting of CXCL13 and BAFF receptors prevented salivary hypofunction [74•]. CXCL13 has been proposed as a biomarker for SS and a possible therapeutic target .
Epigenetic factors such as altered patterns of DNA methylation have been implicated in models of autoimmune disease. Recent studies have reported epigenetic alterations such as changes in DNA methylation, histone modification, micro-RNA expression, and have found defective DNA methylation to be associated with SSB gene expression and lymphocyte infiltration in pSS [75–77]. Just recently, a genome-wide DNA methylation study on human labial salivary glands of SS was presented . Several genes and pathways previously thought to be involved in disease-related processes as well as a number of new candidates were discovered. Interestingly, a correlation was recognized between DNA methylation and a set of genes previously found highly differentially expressed in pSS and healthy salivary glands [59, 78]. Furthermore, genome-wide DNA methylation profiles showed prominent hypomethylation of interferon-regulated genes in whole blood and CD19+ cells in pSS [79•].
As mentioned, increased expression of type I interferon-regulated genes have been demonstrated in autoimmune diseases . An interferon signature was demonstrated in sera and minor salivary glands of pSS [59, 81], and in CD14 monocytes of pSS patients it was found associated with disease activity and higher B cell activation factor (BAFF) gene expression . CXCL10 is one of the most strongly upregulated type I interferon-regulated genes and was found upregulated in salivary glands of pSS patients [59, 80]. A causal relationship between type I interferon production and development of autoimmune disease has been suggested, and as a biomarker of active disease, the interferon signature is an interesting target for research aiming at new treatment possibilities . The contribution of type I and type II interferon signatures to SS pathogenesis and lymphomagenesis was recently investigated, and interferon γ/interferon α mRNA ratio was proposed as a novel biomarker for prediction of in situ lymphoma development in SS .
Type I interferons are involved in innate immune response against viral infection and help to regulate the activity of the immune system. A link between the LTβR and interferon pathways and mouse models has pointed to the pathogenic role of the lymphotoxin and interferon I pathways in human autoimmune diseases . The lymphotoxin pathway has a role in orchestrating the development of homeostasis of lymph nodes through regulation of homeostatic chemokines, and the LTβR signaling is essential in differentiating stromal cells and macrophages in lymphoid organs to produce interferon I in response to virus infection. A possible potential of the lymphotoxin network as a tool for treatment of autoimmune diseases has been suggested .
Virus has been suggested as one of the environmental factors that could trigger SS and be involved in SS pathogenesis, and the observation that infection can precipitate autoimmune abnormality is not new . A number of candidates have been suspected to trigger such a disease, such as human herpes virus 6 (HHV6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis C virus (HCV), human T lymphotropic virus type 1 (HTLV-I), and human immunodeficiency viruses (HIV) . However, the connection is not clear, and more studies are justified.
Autoimmune diseases aggregate in families. In a population-based family study of 105 Taiwan patients with SS who had an affected first-degree relative, Kuo et al.  found a relative risk (RR) for SS in siblings of patients with SS to be 18.99, 11.31 in offspring, and 12.46 in parents. In first-degree relatives of SS patients the RR were 6.25 for having systemic lupus erythematosus, 3.38 for multiple sclerosis and 2.95 for rheumatoid arthritis .