Diagnosis and Differential Diagnosis

Diagnostic Tests

Salivary Gland Biopsy

Minor salivary gland biopsy (MSGB) (Fig. 10.2) displays a crucial role for diagnosis, prognosis, and risk stratification [64, 65]. According to American/European Consensus SS classification criteria of 2016, diagnosis cannot be established without a positive MSGB or positive anti-SSA/Ro antibodies [37]. Moreover, intense lymphocytic infiltration has been identified as an independent histopathological risk factor for NHL development [66], which is the leading cause of excess mortality in SS patients [67–69].

The presence of a lymphocytic infiltrate of ≥50 lymphocytes per 4 mm² of glandular parenchyma, usually located in the periductal area, is considered a positive focus score. The average number of these lymphocytic aggregates per 4 mm² of salivary gland tissue is the focus score (Fig. 10.3) [64]. Despite the fact that only focus score appears in the American/European consensus criteria, Tarpley score (measure of glandular architecture derangement) is also commonly used [65, 70]. Another important histopathological feature is the presence of germinal center (GC)-like structures (Fig. 10.4). The latter have been associated with higher focus score, higher frequency of extraglandular manifestations, hypergammaglobulinemia, increased RF levels, and higher prevalence of positive anti-SSA/Ro and/or anti-SSB/La autoantibodies. The presence of GC-like structures has also been suggested to confer increased risk for lymphoma development. However, the contradicting results on GC-like structures significance from various studies – possibly due to poor definition on one hand and under-detection in H&E staining on the other – underline the need for uniform criteria and further research [65, 70–73].

The advantages of MSGB include easy accessibility, avoidance of skin incisions, and local anesthesia. Parotid biopsy is reserved only to rule out lymphoma in case of persistent parotid enlargement [74, 75]. MSGB sensitivity and specificity are considered to be higher than 75% and 90%, respectively [64, 76, 77].

Ocular Tests

Mainly aqueous deficiency but also meibomian gland dysfunction and neuropathic pain contribute to increased tear evaporation rate, reduced tear film stability, and ocular discomfort in SS patients [82].

Keratoconjunctivitis sicca (KCS) symptoms can be quantified using patient questionnaires, like the Ocular Surface Disease Index (OSDI). The main available objective tests are Schirmer’s test (Fig. 10.5), ocular surface dye staining (Fig. 10.6), and tear breakup time (TBUT) [82–84]. Among those, only the first two are included in the latest classification criteria [37].

Disease Activity Indexes

There are two indexes, introduced by European League Against Rheumatism (EULAR), which are used to assess SS activity from the patient’s and the clinician’s point of view. The first one is the EULAR Sjögren’s Syndrome Patient Report Index (ESSPRI), which consists of three visual-analogue scales measuring severity of sicca symptoms, fatigue, and pain [85]. The second one is the EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI), which assesses the activity in 12 different domains representing organ systems. Since its introduction, ESSDAI has become the gold standard in terms of disease activity assessment because it serves as point of reference among physicians; it is widely used in randomized controlled trials as outcome measure and has been correlated with biomarkers and lymphoma development risk [42, 43].

Clinical Manifestations and Disease Management

SS can manifest with a plethora of clinical signs and symptoms, frequently vague and definitely not pathognomonic, often resulting in diagnostic delays [86]. Oral and ocular dryness, sometimes accompanied by xeroderma, upper respiratory desiccation, and vaginal dryness with subsequent dyspareunia are among the most common complaints [10]. Extraglandular manifestations (Fig. 10.7) occur in at least one third of individuals and can schematically be divided into three groups: nonspecific, periepithelial, and immune complexes mediated [6, 87–94]. The disease often runs a benign, indolent course with the exception of severe systemic complications causing excess morbidity and most importantly lymphoma, which is one of the main causes of mortality in SS [42, 67].

Lymphomagenesis and Lymphoproliferation

SS is unique among autoimmune diseases as for malignant transformation risk. Those patients seem to have a 10–44-fold greater risk of developing lymphoma compared to general population, whereas systemic lupus erythematosus patients and rheumatoid arthritis patients only have a seven-fold and four-fold greater risk, respectively [67, 121]. In other words, 2.7–9.8% of SS patients are diagnosed with non-Hodgkin lymphoma (NHL) and that risk increases by 2.2% per year of age [78, 122].

Mucosa-associated lymphoid tissue (MALT) lymphomas represent 60% of cases [67]. Most common sites are the salivary glands, especially the parotid and submandibular glands, but other mucosal sites of MALT lymphoma development include the orbits, nasopharynx, stomach, thyroid, and lung [78]. Other subtypes of lymphoma found in these patients are the diffuse large B-cell lymphoma (DLBCL) and the nodal marginal zone lymphoma (NMZL), which – together with MALT – account for more than 90% of total SS-associated lymphoma cases [67].

Multiple research studies have been focusing their efforts on correlating clinical, serological, and histopathological features with risk of lymphomagenesis [66, 72, 123–126], and an attempt has also been made to formulate a predictive score for SS-related NHL development based on data collected at the time of diagnosis [123] (Fig. 10.8). However, the molecular etiopathogenetic aspects of malignant transformation still remain largely elusive and ill-defined.

Current belief on etiopathogenesis of lymphoma focuses on four interrelated axes: chronic inflammation, B-cell activation, defective immunosurveillance, and epigenetic alterations [4, 104]. Focus score of at least 3 in MSGBs of SS patients has been identified as an independent and important predicting factor for NHL development, and the use of this threshold has a positive predictive value of 16% and a negative predictive value of 98% for that kind of malignant transformation [66]. Furthermore, the activation of P2X7 receptor-NLRP3 inflammasome complex (with subsequent increase of pro-inflammatory cytokines IL-18 and IL-1β serum levels, among others) correlates with lymphocytic infiltration severity, ESSDAI scores, and lymphoproliferation risk. The hypothesis of inflammasome activation secondary to increased accumulation of proinflammatory nucleic acid shreds, ineffectively degraded and cleared, has recently been supported [127, 128].

Another factor promoting chronic inflammation seems to be IFNγ, as the mRNA levels in MSGBs have been associated with a higher degree of lymphocytic infiltration, shown to be a predisposing factor for lymphomagenesis [129]. Moreover, a functional variant of TNFAIP3 gene (rs2230926), encoding the A20 protein, leads to unopposed NF-κB pathway activation and is therefore involved in inflammatory process perpetuation, B-cell survival, and more aggressive disease phenotype with earlier disease onset and increased lymphoproliferation risk [130].

B-cell activating factor (BAFF), produced by various immune cells but also by the salivary epithelial cells and regulated by both type I and II IFNs, is of utmost importance for the maturation, proliferation, and survival of B cells. BAFF levels are found increased in SS patients’ serum with a history of lymphoma, and high levels persist for years after treatment and remission [4, 131, 132]. Another equally important observation is the association of specific BAFF polymorphisms with SS-related lymphomagenesis and in particular the significantly different prevalence of the protective AA genotype of the rs12583006 polymorphism, as well as of the protective haplotypes TACAC and TACC and of the risk haplotype TTTC in SS patients prone to NHL development (high risk determined by the presence of adverse predictors), compared to low-risk individuals [133]. Additionally, a specific mutation (His159Tyr) of BAFF receptor (BAFF-R) is of interest, as it has been identified in more than two thirds of SS-associated MALT lymphoma. This mutation, leading to NF-kB pathway activation, was linked to earlier development of lymphoma or adverse immunological features (hypergammaglobulinemia and positive RF) [78, 134].

Finally, Fms-like tyrosine kinase 3 ligand (Flt-3 l) is a protein that acts as a cytokine and a growth factor, activating Flt-3 (or CD135) on the surface of hematopoietic progenitor cells, and therefore considered to be a mediator of B-cell survival. Higher levels of Flt-3l are strongly associated with history of lymphoma, detectable years before malignant transformation, and not affected by treatment [135].

The vicious circle of autoreactive B-cell chronic stimulation and immunocomplexes formation might finally lead to the favorable, monoclonal expansion of rheumatoid factor (RF)-reactive B cells and to the lymphomatous transformation, under defective immunosurveillance [4]. As a matter of fact, IFNα mRNA levels in MSGBs seem to strongly correlate with the expression of pro-apoptotic molecules (tumor suppressor gene p53 and auto-antigen Ro52, with the latter negatively regulating the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene) [132]. Moreover, decreased prevalence of a specific TREX1 variant (rs11797 AA genotype) in SS-related non-MALT cases was observed. This variant was shown to associate with higher mRNA IFNα levels in SS salivary gland tissues [136].

Further oncogenic mechanisms, likely to contribute to malignant turn in SS, are the over-expression of Bcl-2 due to a translocation involving chromosomes 14 and 18, leading to inhibition of apoptosis and increased B-cell survival. Apart from IFNα effect on p53 levels, specific mutations of this tumor suppressor gene were described 20 years ago in MSGBs from SS-associated NHL cases [4, 132, 137].

Epigenetic changes, involving methylating enzymes and transcription of noncoding micro-RNAs, are also implicated in lymphomagenesis [138]. MiR200b miRNAs, which regulate the expression of oncogenes and tumor suppressor genes, have been found to be downregulated in MSGBs with advanced lymphocytic infiltration and MALT lymphoma [139]. Especially miR200b-5p strongly discriminates SS patients who already have or will develop NHL from the rest of them or from sicca controls [140]. As for methylating enzymes, DNA methyltransferase (DNMT)3B and methyl-CpG-binding protein 2 (MeCP2) have been found decreased in SS-lymphoma patients [141], while methylene-tetrahydrofolate reductase (MTHFR) gene variants, leading to defective methylation and impaired stability of DNA, have lately been suggested as susceptibility factors for non-MALT lymphoma [142].

It becomes clear that the multifactorial process of lymphomagenesis is rather complicated, and the finely tuned balance between opposing forces can become deranged and lead to adverse outcomes. An example of that is the IFNγ/IFNα mRNA ratio in MSGBs, which has emerged as a histopathological biomarker for the prediction of in situ lymphoma development [132].

SS-related hematological malignancies are correlated with an eightfold higher mortality risk compared to general population [69], and one in five deaths of SS is attributable to lymphoma [68]. Follow-up every 6 months is recommended for high-risk patients. Overall, NHL 5-year survival is estimated at approximately 92%, but higher disease activity is linked to higher possibility of relapse and death [143–145]. Wait and watch strategy is suitable for MALT lymphomas localized in the salivary glands, while rituximab and chemotherapy are employed in case of disseminated or aggressive disease [106, 146, 147, 149].

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