Organoid-based personalized medicine: from tumor outcome prediction to autologous transplantation.

Inter-individual variation largely influences disease susceptibility, as well as response to therapy. In a clinical context, the optimal treatment of a disease should consider inter-individual variation and formulate tailored decisions at an individual level. In recent years, emerging organoid technologies promise to capture part of an individual's phenotypic variability and prove helpful in providing clinically relevant molecular insights. Organoids are stem cell-derived three-dimensional models that contain multiple cell types that can self-organize and give rise to complex structures mimicking the organization and functionality of the tissue of origin. Organoids represent thus a more faithful recapitulation of the dynamics of the tissues of interest, compared to conventional monolayer cultures, thus supporting their use in evaluating disease prognosis, or as a tool to predict treatment outcomes. Additionally, the individualized nature of patient-derived organoids enables the use of autologous organoids as a source of transplantable material not limited by histocompatibility. An increasing amount of preclinical evidence has paved the way for clinical trials exploring the applications of organoid-based technologies, some of which are in phase I/II. This review focuses on the recent progress concerning the use of patient-derived organoids in personalized medicine, including (1) diagnostics and disease prognosis, (2) treatment outcome prediction to guide therapeutic advice and (3) organoid transplantation or cell-based therapies. We discuss examples of these potential applications and the challenges associated with their future implementation.

The diagnostic power of salivary electrolytes for Sjögren's disease: a systematic literature review and meta-analysis.

OBJECTIVES: To perform a systematic review and meta-analysis to determine the power of salivary electrolytes for the diagnosis of Sjögren's disease (SjD). METHODS: A literature search was conducted (last search March 2023) using PubMed and Web of Science and completed with a manual search. Articles were screened for reports of human salivary ion concentrations, comparing SjD patients with healthy controls and/or sicca patients. Articles not using the SjD classification criteria or performing the classification as part of the experimental design were excluded. Forest plots were used to present the meta-analyses results for each ion, distinguishing between salivary type (unstimulated and stimulated whole saliva, submandibular/sublingual and parotid saliva). RESULTS: A total of 21 out of 722 articles were eligible for inclusion. For SjD patients a significant increase in salivary ion concentration was observed for sodium, chloride and calcium when comparing to healthy controls. Significant differences between SjD and sicca patients were noted for sodium, chloride, phosphate, calcium, phosphate, nitrite and nitrate. Stimulated whole saliva showed larger variability in results between studies in comparison to other types of saliva (unstimulated whole saliva, submandibular/sublingual saliva and parotid saliva). CONCLUSIONS: Despite differences in saliva type, salivary ion levels could be utilised for the screening for SjD. Making use of chloride in combination with sodium would be most promising for distinguishing SjD patients from healthy controls and adding phosphate to potentially make a distinguishment with sicca patients. Unstimulated whole saliva should be the first choice when testing salivary ion concentrations.

Immunohistological profiling confirms salivary gland-like nature of the tubarial glands and suggests closest resemblance to the palatal salivary glands.

BACKGROUND AND PURPOSE: High label uptake in 68 Ga-PSMA-11 PET/CT recently identified a bilateral nasopharyngeal structure as a salivary gland (SG)-like additional 'area of interest', to be considered in conditions affecting SGs. These structures were termed 'tubarial glands'. We aimed to further characterize their histological and immunohistochemical position compared to established SGs. METHODS: Tubarial gland tissue was compared with parotid, submandibular, sublingual, palatal and labial SGs tissue using immunohistological techniques. RESULTS: Expression of acinar cell-associated aquaporin-5 (AQP5) was detected in tubarial glands, in an apical location associated in control, established SGs with polarized, secretory acinar cells. Keratin14 (KRT14) expression in cells peripheral to AQP5+ clusters also suggested presence of myoepithelial cells. α-amylase, prolactin-induced protein, proline rich protein Haelll subfamily 2, and Muc5B expression suggests mucous acinar cell presence, and presence of muco-serous acinar cells peripheral to putative mucous acinar cells. Expression of adrenergic receptor-ß1 by acinar-like cells of the tubarial gland suggests ability to transduce sympathetic neuronal signaling. In terms of ductal architecture, tubarial glands contained large excretory-like ducts (similar to all other SGs), and squamous ducts, comprised of intermingled KRT14+ and KRT7+ cells. These squamous ducts were also observed in palatal, sublingual and labial SGs. No striated or intercalated ducts were observed, similar to palatal SGs. CONCLUSION: Based on histological and immunohistochemical analyses, the tubarial glands resemble SGs. They most convincingly echo characteristics of the palatal SGs in terms of ductal cells, and both the palatal and labial SGs when considering acinar cells.

Why do drug treatments fail in Sjögren's disease? Considerations for treatment, trial design and interpretation of clinical efficacy.

INTRODUCTION: Despite ongoing efforts to develop effective therapeutics, no disease-modifying drugs have been officially licensed for the indication of Sjögren's disease (SjD). This is partly due to heterogeneity in disease manifestations, which complicates drug target selection, trial design and interpretation of clinical efficacy in SjD. AREAS COVERED: Here, we summarize developments and comment on challenges in 1) identifying the right target for treatment, 2) selection of the primary study endpoint for trials and definition of clinically relevant response to treatment, 3) inclusion criteria and patient stratification, 4) distinguishing between disease activity and damage and 5) establishing the effect of treatment considering measurement error, natural variation, and placebo or nocebo responses. EXPERT OPINION: Targets that are involved in both the immune cell response and dysregulation of glandular epithelial cells (e.g. B-lymphocytes, type-I interferon) are of particular interest to treat both glandular and extra-glandular manifestations of SjD. The recent development of composite study endpoints (CRESS and STAR) may be a crucial step forward in the search for clinically effective systemic treatment of patients with SjD. Important additional areas for future research are symptom-based and/or molecular pathway-based patient stratification, prevention of irreversible damage, and establishing the effect of treatment.

Indication of Activated Senescence Pathways in the Temporal Arteries of Patients With Giant Cell Arteritis.

OBJECTIVE: Giant cell arteritis (GCA) affects almost exclusively individuals above 50 years old, suggesting a role of aging-related changes such as cellular senescence in its pathobiology. The kinases p21(WAF1/CIP1) and p16/INK4A play key roles in 2 distinct pathways leading to senescence. The proinflammatory molecules interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are key components of the senescence-associated secretory phenotype (SASP), are effective targets of treatment in GCA. Here, we aimed to investigate the presence of p21+ and p16+ cells producing these SASP cytokines in temporal artery biopsies (TABs) of patients with GCA. METHODS: Eight patients with GCA and 14 age-matched, non-GCA individuals who underwent a TAB were included. Immunohistochemical staining of p21, p16, IL-6, and GM-CSF was performed. Multiplex immunofluorescent staining was performed to investigate the colocalization of p21 and p16 with IL-6, GM-CSF, and immune cell markers (CD68, CD3, CD20). RESULTS: We found that expression levels of p16, p21, IL-6, and GM-CSF were elevated in the TABs of patients with GCA. Both p16- and p21-expressing cells were mainly found near the internal lamina elastica, especially among giant cells and macrophages, although p21 and p16 expression could be found in all 3 layers of the vessels. Expression of p16 and p21 was occasionally found in T cells but not B cells. The p16+ and p21+ cells expressing GM-CSF/IL-6 were detected throughout the TABs. CONCLUSION: Our data suggest the presence of activated senescence pathways at the site of vascular inflammation in GCA and support further research into the role of senescence in the pathophysiology of GCA.

The lacrimal gland in Sjögren's syndrome: can we unravel its mystery using ultrasound?

According to a recent survey of patients with the autoimmune disease primary Sjögren's syndrome (pSS), dry eye symptoms are present in 95-98% of pSS patients. As one of the most disabling symptoms mentioned by pSS patients, dry eyes have demonstrable effects on quality of their life, leading to eye dryness, itching, and pain, with some patients describing as a recurrent sensation of sand or gravel in the eyes. The symptoms are matched only in prevalence by dry mouth and chronic fatigue. In contrast to the prevalence of dry eye symptoms in pSS and their burden on pSS patients, our comprehension of dry eye disease development is minimal; specifically how function of the tear-fluid producing gland the lacrimal gland (LG), manifests. The comparison becomes stronger again when we consider what we know about dysfunction of the salivary gland (SG) in pSS, for example the appreciation of the transcriptome of 'innately activated' B cells invading the SG, their complicity in formation of lymphoepithelial lesions, and the ability of the SG epithelium to actively contribute to the inflammatory milieu. The exploration of ultrasound imaging as an additional modality to garner information about SG dysfunction in pSS has opened many doors for non-invasive, repeatable imaging in pSS. Here we summarise SG histology and ultrasound phenotype briefly and then juxtapose this with available studies examining LG pathology and ultrasound, and our understanding of LG dysfunction in pSS.

Lymphoepithelial lesions in the salivary glands of primary Sjögren's syndrome patients: the perfect storm?

In patients with primary Sjögren's syndrome (pSS), inflamed salivary gland (SG) tissue may contain lymphoepithelial lesions (LELs). LELs are histopathological phenomena whereby B cells are present in hyperplastic ductal epithelium of the SG. Despite the potential role of LELs in pSS pathogenesis, studies on their formation, detection, and prevalence in benign lesions (not complicated with lymphoma) are scarce. Recent evidence however shows that LELs are present in approximately half of the patients with pSS, both in minor and major SGs. Migration of a small number of B cells into the epithelium appears to be a critical initial step in LEL formation. These intra-epithelial B cells are proliferative, exhibit an innate-like phenotype, and may be linked to MALT lymphoma development. Alongside intra-epithelial B cells, the hyperplastic epithelial partner in LELs also engages in the local immune reaction. Epithelial cells are a source of cytokines and chemokines, with CXCL10 in particular playing a potential role in LEL formation. Importantly, LELs also have a negative impact on the maintenance of SG homeostasis by SG progenitor cell (SGPC) populations, likely due to dysregulation of SGPC lineage commitment or induction of plasticity. In conclusion, LEL formation mirrors a perfect storm of B and epithelial cell interaction culminating in increased risk of B cell derailment and SGPC dysregulation in pSS patients. We therefore argue that attenuation of LEL formation is an important treatment goal to preserve SG function and prevent B cell derailment in pSS.

Role of quiescent cells in the homeostatic maintenance of the adult submandibular salivary gland.

Stem/progenitor cells are required for maintenance of salivary gland (SG) function and serve as untapped reservoirs to create functional cells. Despite recent advancements in the identification of stem/progenitor pools, in the submandibular gland (SMG), a knowledge gap remains. Furthermore, the contribution to adult SMG homeostasis of stem/progenitor cells originating from embryonic development is unclear. Here, we employ an H2B-GFP embryonic and adult pulse-and-chase system to characterize potential SMG stem/progenitor cells (SGSCs) based on quiescence at different stages. Phenotypical profiling of quiescent cells in the SMG revealed that label-retaining cells (LRCs) of embryonic or adult origin co-localized with CK8+ ductal or vimentin + mesenchymal, but not with CK5+ or CK14 + stem/progenitor cells. These SMG LRCs failed to self-renew in vitro while non-label retaining cells displayed differentiation and long-term expansion potential as organoids. Collectively, our data suggest that an active cycling population of cells is responsible for SMG homeostasis with organoid forming potential.

Parotid salivary sodium levels of Sjögren's syndrome patients suggest B-cell mediated epithelial sodium channel disruption.

Patients with primary Sjögren's syndrome (SS) suffer widely from lack of saliva production. Here we investigate potential mechanisms underpinning changes in SS patient saliva composition. Sodium concentration was significantly higher in all saliva samples collected: unstimulated submandibular/sublingual (SmSl) saliva (p<0.0001), stimulated SmSl saliva (p=0.002) and stimulated parotid (PG) (p<0.0001) saliva, compared to non-SS sicca controls. Chloride, phosphate and potassium ion concentrations, α-amylase activity and total protein content correlations were less consistently changed between SS and non-SS saliva types. Stimulated PG salivary sodium levels correlated with the degree of CD45+ lymphocytic cell infiltrate in the parotid glands (r=0.69, p<0.001), and even more strongly so with infiltrating CD20+ B cells (r=0.73, p<0.0001). CD3+ T cells were only moderately correlated with salivary sodium (r=0.23, p=0.015). In non-SS control or focus score (FS) negative SS PG tissue, the epithelial sodium channel (ENaC), responsible for sodium transport out of saliva, was localised to the apical membrane of luminal striated duct cells. In PG tissue from FS+ SS patients, apical ENaC expression appeared absent. We hypothesise that B cell-related proinflammatory cytokines in SS salivary glands may dysregulate sodium transport channels in SS.

The Transcriptome of Paired Major and Minor Salivary Gland Tissue in Patients With Primary Sjögren's Syndrome.

Background: While all salivary glands (SGs) can be involved in primary Sjögren's syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients. Methods: Paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology. Results: With principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<0.05, log2FC<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R2 = 0.86, p-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients. Conclusion: Transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.

A Distinct Macrophage Subset Mediating Tissue Destruction and Neovascularization in Giant Cell Arteritis: Implication of the YKL-40/Interleukin-13 Receptor α2 Axis.

OBJECTIVE: Macrophages mediate inflammation, angiogenesis, and tissue destruction in giant cell arteritis (GCA). Serum levels of the macrophage-associated protein YKL-40 (chitinase 3-like protein 1), previously linked to angiogenesis and tissue remodeling, remain elevated in GCA despite glucocorticoid treatment. This study was undertaken to investigate the contribution of YKL-40 to vasculopathy in GCA. METHODS: Immunohistochemistry was performed on GCA temporal artery biopsy specimens (n = 12) and aortas (n = 10) for detection of YKL-40, its receptor interleukin-13 receptor α2 (IL-13Rα2), macrophage markers PU.1 and CD206, and the tissue-destructive protein matrix metalloproteinase 9 (MMP-9). Ten noninflamed temporal artery biopsy specimens served as controls. In vitro experiments with granulocyte-macrophage colony-stimulating factor (GM-CSF)- or macrophage colony-stimulating factor (M-CSF)-skewed monocyte-derived macrophages were conducted to study the dynamics of YKL-40 production. Next, small interfering RNA-mediated knockdown of YKL-40 in GM-CSF-skewed macrophages was performed to study its effect on MMP-9 production. Finally, the angiogenic potential of YKL-40 was investigated by tube formation experiments using human microvascular endothelial cells (HMVECs). RESULTS: YKL-40 was abundantly expressed by a CD206+MMP-9+ macrophage subset in inflamed temporal arteries and aortas. GM-CSF-skewed macrophages from GCA patients, but not healthy controls, released significantly higher levels of YKL-40 compared to M-CSF-skewed macrophages (P = 0.039). In inflamed temporal arteries, IL-13Rα2 was expressed by macrophages and endothelial cells. Functionally, knockdown of YKL-40 led to a 10-50% reduction in MMP-9 production by macrophages, whereas exposure of HMVECS to YKL-40 led to significantly increased tube formation. CONCLUSION: In GCA, a GM-CSF-skewed, CD206+MMP-9+ macrophage subset expresses high levels of YKL-40 which may stimulate tissue destruction and angiogenesis through IL-13Rα2 signaling. Targeting YKL-40 or GM-CSF may inhibit macrophages that are currently insufficiently suppressed by glucocorticoids.

Epithelial-immune cell interplay in primary Sjögren syndrome salivary gland pathogenesis.

In primary Sjögren syndrome (pSS), the function of the salivary glands is often considerably reduced. Multiple innate immune pathways are likely dysregulated in the salivary gland epithelium in pSS, including the nuclear factor-κB pathway, the inflammasome and interferon signalling. The ductal cells of the salivary gland in pSS are characteristically surrounded by a CD4+ T cell-rich and B cell-rich infiltrate, implying a degree of communication between epithelial cells and immune cells. B cell infiltrates within the ducts can initiate the development of lymphoepithelial lesions, including basal ductal cell hyperplasia. Vice versa, the epithelium provides chronic activation signals to the glandular B cell fraction. This continuous stimulation might ultimately drive the development of mucosa-associated lymphoid tissue lymphoma. This Review discusses changes in the cells of the salivary gland epithelium in pSS (including acinar, ductal and progenitor cells), and the proposed interplay of these cells with environmental stimuli and the immune system. Current therapeutic options are insufficient to address both lymphocytic infiltration and salivary gland dysfunction. Successful rescue of salivary gland function in pSS will probably demand a multimodal therapeutic approach and an appreciation of the complicity of the salivary gland epithelium in the development of pSS.

Mouse parotid salivary gland organoids for the in vitro study of stem cell radiation response.

OBJECTIVE: Hyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro. MATERIALS AND METHODS: Single cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response. RESULTS: Single cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland. CONCLUSIONS: Murine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.

Checkpoint inhibition-induced sicca: a type II interferonopathy?

The advent of immune checkpoint inhibitor (ICI) therapy for treatment of cancers is unfortunately coupled with a broad panoply of side effects, related to non-specific activation of the immune system. One such side effect is the development of sicca complaints. This culminates in a proportion of patients who, according to the ACR-EULAR 2016 criteria, can be classified as suffering from the autoimmune disease primary Sjögren's syndrome (pSS). Although salivary gland (SG) loss of function is often seen after ICI therapy, the similarities with 'classical' pSS patients would appear to end there. Despite the presence of focal lymphocytic sialadenitis typical for SS in salivary gland biopsies from patients receiving ICI therapy, the nature of the immune infiltration (foci) following ICI use (T-cell dominated) is starkly different to that in pSS (B-cell dominated). The SG parenchyma post-ICI use does not present with germinal centres, lymphoepithelial lesions or IgG plasma cells, which are frequently found in the SG in pSS. Here we review the functional deterioration of SGs following ICI use, the SG parenchyma phenotype associated with this, and ultrasound abnormalities. We conclude by suggesting that ICI-induced SG dysfunction may represent a new interferonopathy, driven by IFNγ, and that this 'pSS' patient cohort may require a different management than classical pSS patients.

Novel approaches for rescuing function of the salivary gland epithelium in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by dysfunction and inflammatory lymphocytic infiltration of exocrine glands, namely the salivary and lacrimal glands. pSS patients often suffer from sicca (dry mouth) complaints, including dental caries, and difficulties in eating, sleeping and speaking. A large body of literature points to a central role for salivary gland (SG) epithelial cells in the development of this pathology. Here we summarise recent studies concerning the role of SG epithelial cells in pSS, which strongly indicate their intrinsic activation and early involvement during the disease process. Based on that, we propose possible future interventions targeting SG epithelial cells, to treat SG dysfunction pSS.

Lack of Conventional Acinar Cells in Parotid Salivary Gland of Patient Taking an Anti-PD-L1 Immune Checkpoint Inhibitor.

Background: Salivary glands (SGs) can be damaged by immune checkpoint inhibitor (ICI) therapy. In patients with ICI-induced SG dysfunction, 60% progress to fulfill classification criteria for primary Sjögren's syndrome (pSS), owing to immune foci in SGs and/or anti-SSA autoantibody positivity. We report the SG tissue analysis of a patient with SG dysfunction after treatment with a programmed death ligand-1 (PD-L1) inhibitor, compared to that of a dry mouth ("sicca") control and pSS patient. Case presentation: The patient received the PD-L1 inhibitor durvalumab (10 mg/kg, every 2 weeks by intravenous infusion) as adjuvant treatment for stage 3 non-small cell lung carcinoma, following concurrent chemo radiotherapy. At 43 weeks after 21 cycles of Durvalumab, the patient was not capable of producing unstimulated or stimulated parotid gland saliva, and a biopsy was taken. Immunohistochemical analysis showed no classical AQP5+ CK7- acinar cell clusters (CK7 marks intercalated ducts, IDs). In contrast, the parenchyma was dominated by hybrid epithelial "structures" with ID-like morphology, containing a mixture of AQP5+CK7-, AQP5-CK7+, and AQP5+CK7+ cells (30 structures/mm2). These structures were present at lower frequencies in sicca control (2/mm2) and pSS (10/mm2) tissue. Hybrid structures contained proliferating (Ki67+) cells and senescent (p16+) cells. Striated ducts showed no abnormal morphology post PD-L1 treatment, in contrast to pSS tissue. PD-L1 expression was detected in the SG parenchyma following anti-PD-L1 therapy. The SG post-PD-L1 therapy further demonstrated focal lymphocytic sialadentitis, harboring disperse, and focal CD4+ T cell-rich infiltrates. CD8+ T cells were also present. In this patient, these CD4+ and CD8+ T cells were observed in-between and inside hybrid structures. CD20+ B-cells were infrequently detected following PD-L1 blockade, in contrast to their preponderance in pSS SG tissue. Conclusion: This patient lacked conventional SG acinar cells following anti-PD-L1 therapy and demonstrated presence of hybrid intercalated duct-like structures. Understanding which mechanisms and dynamics underpinning this aberrant parenchyma may be crucial to understand how SG dysfunction post ICI therapy, and potentially other affected organs. Furthermore, although the patient treated with anti-PD-L1 antibody examined here fulfills the criteria for pSS and demonstrated focal lymphocytic sialadentitis, the further histopathological characteristics do not resemble pSS.

Progenitor cell niche senescence reflects pathology of the parotid salivary gland in primary Sjögren's syndrome.

OBJECTIVE: Salivary gland (SG) progenitor cells (SGPCs) maintain SG homeostasis. We have previously shown that in primary Sjögren's syndrome (pSS), SGPCs are likely to be senescent, and may underpin SG dysfunction. This study assessed the extent of senescence of cells in a SGPC niche in pSS patients' SGs, and its correlation with functional and clinical parameters. METHODS: The expression of p16 and p21 as markers of senescence in both total SG epithelium and a SGPC niche (basal striated duct cells, BSD) was examined in SGs of pSS (n = 35), incomplete pSS (n = 13) (patients with some signs of pSS, but not fulfilling all classification criteria) and non-SS sicca control (n = 21) patients. This was correlated with functional and clinical parameters. RESULTS: pSS patient SGs contained significantly more p16+ cells both in the epithelium in general (P <0.01) and in the BSD layer (P <0.001), than non-SS SGs. Significant correlations were found in pSS patients between p16+ BSD cells and secretion of unstimulated whole saliva, stimulated whole saliva, stimulated parotid saliva, CD45+ infiltrate, ultrasound total score and ACR-EULAR classification score, but not with EULAR Sjögren's syndrome disease activity index (ESSDAI) and EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) scores. Correlations with total epithelium p16+ cells were weaker. Incomplete pSS patients also had increased numbers of p16+ epithelial and BSD cells. Based on protein and mRNA expression, p21+ appears not to play a significant role in the SG in pSS. CONCLUSION: These findings suggest SGPC senescence may be an early feature of primary Sjögren's syndrome and may contribute to defective SG function in pSS but not to systemic disease activity.