Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren's syndrome.

BMP6 is a central cytokine in the induction of Sjögren's syndrome-associated (SS-associated) secretory hypofunction. However, the upstream initiation leading to the production of this cytokine in SS is unknown. In this study, RNA ISH on salivary gland sections taken from patients with SS indicated monocytic lineage cells as a cellular source of BMP6. RNA-Seq data on human salivary glands suggested that TLR4 signaling was an upstream regulator of BMP6, which was confirmed by in vitro cell assays and single-cell transcriptomics of human PBMCs. Further investigation showed that HSP70 was an endogenous natural TLR4 ligand that stimulated BMP6 expression in SS. Release of HSP70 from epithelial cells could be triggered by overexpression of lysosome-associated membrane protein 3 (LAMP3), a protein also associated with SS in several transcriptome studies. In vitro studies supported the idea that HSP70 was released as a result of lysosomal exocytosis initiated by LAMP3 expression, and reverse transcription PCR on RNA from minor salivary glands of patients with SS confirmed a positive correlation between BMP6 and LAMP3 expression. BMP6 expression could be experimentally induced in mice by overexpression of LAMP3, which developed an SS-like phenotype. The newly identified LAMP3/HSP70/BMP6 axis provided an etiological model for SS gland dysfunction and autoimmunity.

Focal lymphocytic sialadenitis and ectopic germinal centers in oral reactive lesions and primary Sjögren's syndrome: a comparative study.

Focal lymphocytic sialadenitis (FLS), an important diagnostic criterion for Sjögren's syndrome (SS) diagnosis, can also be observed when assessing minor salivary gland (mSG) biopsies from healthy asymptomatic individuals (non-SS patients). Fifty cases of primary SS (pSS group) and 31 cases of oral reactive lesions (non-SS non-sicca group) containing also typical FLS features, were assessed by morphological and immunohistochemical (CD10, CD23 and Bcl-6) analysis, aiming at the detection of GCs. All pSS cases showed FLS with focus score (FS) ≥ 1. In the non-SS non-sicca group, 12, 10 and 9 cases showed FLS with FS ≥ 1, FLS with FS < 1 and FLS associated with chronic sclerosing sialadenitis with FS < 1, respectively. The morphological analysis revealed similar frequency of GCs in pSS (20%) and non-SS non-sicca group (19%). The area (p = 0.052) and largest diameter (p = 0.245) of GCs were higher in pSS than non-SS non-sicca group. The FS and number of foci were significantly higher in pSS than non-SS non-sicca group with FS < 1. Immunohistochemistry confirmed all morphological findings (GCs showing CD23 and Bcl-6 positivity, with variable CD10 expression) and additionally in 3 and 1 cases of the pSS and non-SS non-sicca group, respectively. Moreover, another 6 and 2 cases of the pSS and non-SS non-sicca group with FS ≥ 1, respectively, showed positivity only for CD23. FLS can also be observed when assessing oral reactive lesions, which showed similar frequency of GCs with those found in pSS patients. Further studies, including functional analysis of lymphocytic populations and GCs in FLS, are encouraged.

Spongiotic hyperplasia of the oral mucosa: case series and immunohistochemical analysis.

The localized juvenile spongiotic gingival hyperplasia (LJSGH) mainly affects the maxillary vestibular attached gingiva of juvenile patients, without sex predilection. Similar lesions involving extragingival sites have not been reported to date. Here, we report 2 cases of extragingival soft tissue lesions with similar clinicopathological features to those reported in LJSGH and 12 cases of intraoral reactive soft tissue lesions microscopically showing LJSGH-like focal areas. The 2 cases were adult patients, affecting the maxillary alveolar ridge (55-year-old female) and hard palate (78-year-old male), which were diagnosed as "spongiotic hyperplasia of the oral mucosa." The 12 intraoral reactive soft tissue lesions (6 men and 6 women; mean age, 49.5 years) were diagnosed as inflammatory fibrous hyperplasia (n = 6), peripheral ossifying fibroma (n = 3), and pyogenic granuloma (n = 3), each of them presenting LJSGH-like focal areas. By immunohistochemistry, the spongiotic hyperplasia areas showed positivity for CK19, CK14, CK34ßE12, and CAM5.2 (weak/focal), while CK4 was negative. Considering the anatomical locations (extragingival) of these 2 cases, the term "spongiotic hyperplasia of the oral mucosa" is suggested. Moreover, LJSGH-like focal areas can be detected when microscopically assessing common intraoral reactive soft tissue lesions.

Human papillomavirus co-infection and survival in oral and oropharyngeal squamous cell carcinoma: A study in 235 Brazilian patients.

OBJECTIVES: While unknown for oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinoma (OSCC), some studies assessing cervical carcinoma have shown that human papillomavirus (HPV) co-infection can be associated with its prognosis. METHODS: Through in situ hybridization (HPV and Epstein-Barr virus [EBV] probes) and immunohistochemistry (p16INK4a, cyclin D1, p53, and Ki-67 antibodies), 126 OPSCC and 109 OSCC samples were assessed. RESULTS: All patients were EBV-negative. OPSCC (25%) showed a significant association with HPV compared to OSCC (11%). Almost all HPV-associated cases were p16INK4a-positive. Regarding OPSCC and OSCC, 23 and 7 cases were positive for high-risk HPV (HRHPV) only, 6 and 3 cases for low-risk HPV (LRHPV) only, and 3 and 2 cases for HRHPV/LRHPV, respectively. HPV-associated carcinomas showed a significantly higher proliferative index than HPV-unassociated carcinomas. Both carcinomas showed a similar overall survival rate, which was not affected by the HPV status. However, when comparing HPV-associated subgroups, patients with HRHPV/LRHPV-associated carcinomas showed worse survival. CONCLUSION: LRHPV-associated and HRHPV/LRHPV-associated cases can also be detected when assessing OSCC and OPSCC. Further studies, especially in populations with a high prevalence of HPV-associated OPSCC, are necessary to understand the clinicopathological behavior of these neoplasm subgroups.

Prevalence of human papillomavirus 6 and 11 variants in recurrent respiratory papillomatosis.

Human papillomavirus (HPV) types 6 and 11 are the etiological agents of recurrent respiratory papillomatosis (RRP). We examined the prevalence and distribution of HPVs 6 and 11 genetic variants in juvenile onset (JORRP) and adult onset (AORRP) laryngeal papillomas. Cases of JORRP and AORRP were collected, retrospectively. HPV detection and genotyping were accessed by polymerase chain reaction-sequencing in 67 RRP samples. Overall, the most prevalent HPV-6 variants were from B1 (55.8%) and B3 (27.9%) sublineages, whereas among HPV-11 positive samples A2 (62.5%) variants were predominant. A higher prevalence of HPV-6 B1 was observed in JORRP (83.3% B1 and 16.7% B3), compared with AORRP cases (58.3% B1 and 41.7% B3). HPV-11 A2 variants were more prevalent both in JORRP (57.2%) and in AORRP cases (70.0%). Nevertheless, with the exception that HPV-6 B1 were significantly less likely to recur, there was a lack of association between any particular HPVs 6 or 11 variant and clinicopathological features. Our data do not support an association between HPVs 6 and 11 variability and RRP.

First description of ultramutated endometrial cancer caused by germline loss-of-function and somatic exonuclease domain mutations in POLE gene.

Endometrial cancer (EC) harboring heterozygous POLE proofreading inactivating mutations (POLE-exo*) is associated with an increased number of somatic mutations that result in a distinctive anti-tumor immune response. However, the consequences of such POLE mutations in the context of the missing wild-type allele have not yet been described in endometrial tumors. A 72-year-old woman harboring a germline monoallelic frameshift mutation (p.Pro269fsTer26) in POLE was diagnosed with an EC having a somatic heterozygous mutation in the exonuclease domain of POLE (S459F). Targeted gene sequencing revealed an ultramutated phenotype (381 mutations/Mb) in the tumor and a 2-fold excess of mutations on the DNA leading strand. Additionally, we observed a mutational signature similar to the COSMIC signature 10, a higher mutation rate in this tumor than in endometrial tumors with heterozygous POLE-exo*, and an increased number of T lymphocytes. This is the first report of an ultramutated EC harboring a somatic POLE-exo* mutation in association with a germline loss-of-function mutation in this gene. The absence of a wild type POLE allele led to a particularly high mutational burden.

Immunohistochemistry analysis of checkpoint kinase 2 in oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor gene that acts on the cellular response to DNA damage. However, the role of Chk2 in OSCC prognosis is not yet fully understood. The objective of this study was to evaluate Chk2 immunoexpression in OSCC and to elucidate the association between its expression and clinicopathological parameters of prognostic importance, including overall survival, disease-free survival, and metastasis-free survival. Methods: Chk2 expression was analyzed in 101 samples from patients with OSCC using immunohistochemistry. We stratified the patients into high expression (> 66% of cells positive for Chk2) and low expression (< 66%) groups. Results: Chk2 showed high expression in 57.43% of OSCC. In our study, the expression of Chk2 did not correlate with any of the prognostic parameters evaluated. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to Chk2 expression. Conclusion: Despite the great importance of Chk2 in the development of different types of cancer, our findings do not favor Chk2 as a prognostic marker in oral squamous cell carcinoma.

LAMP3 induces apoptosis and autoantigen release in Sjögren's syndrome patients.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease characterized by dysfunction of secretory epithelia with only palliative therapy. Patients present with a constellation of symptoms, and the diversity of symptomatic presentation has made it difficult to understand the underlying disease mechanisms. In this study, aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjögren's syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjögren's syndrome cases. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and α-fodrin protein, common autoantigens in Sjögren's syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease.ClinicalTrials.gov Identifier: NCT00001196, NCT00001390, NCT02327884.

Lynch syndrome identification in a Brazilian cohort of endometrial cancer screened by a universal approach.

OBJECTIVE: To report the frequency of Lynch syndrome (LS) in a cohort of patients from Southeast Brazil bearing endometrial cancer (EC), using a tumor screening universal approach. METHODS: A total of 242 endometrial carcinomas were screened by immunohistochemistry (IHC) and microsatellite instability (MSI) for detection of DNA mismatch repair deficiency (dMMR). MLH1 methylation was assessed to identify sporadic cases. Patients with dMMR tumors were recruited for germline variant analysis by next-generation sequencing of the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. RESULTS: Ninety-three out of 242 tumors (38.5%) were classified as dMMR based on MSI and IHC results. Of these, 54 cases were selected for germline analysis, and 37/54 (68.5%) were available for sequencing. Ten patients (10/37, 27%) harbored germline pathogenic or likely pathogenic variants, most of them in the MSH6 gene (4/10, 40%). Seven variants of uncertain significance were found. Eight novel germline variants were identified. The LS prevalence in our cohort was of at least 4.1%. LS patients presented lower mean age at cancer diagnosis compared with patients diagnosed with sporadic EC. Individuals with dMMR tumors, without germline pathogenic variants detected in LS-genes ("Lynch-like" syndrome), had an intermediate mean age at cancer diagnosis between LS and sporadic cases. CONCLUSION: This is the first report of the LS prevalence in EC screened by a universal approach in Brazil. Our findings contribute to a better understanding of the mutational landscape of this syndrome in Brazil, which is relevant for improved identification, genetic counseling, prevention and control of cancer in LS.

Gingival ossifying myopericytoma in a pediatric patient: Immunohistochemical analysis and literature review.

Myopericytoma is a rare mesenchymal tumor characterized by a hemangiopericytoma-like vascular pattern with perivascular myoid differentiation. To date, only 11 cases of oral myopericytoma have been reported. To the best of our knowledge, myopericytoma with gingival involvement and associated with calcifications has not been reported, expanding their clinicopathological spectrum. Herein, we report a 12-year-old girl female patient who presented a gingival nodule diagnosed as ossifying myopericytoma, which should be considered in the differential diagnosis when assessing oral soft tissue lesions, especially in pediatric patients.

Oral hairy leukoplakia in a child using a corticosteroid nasal spray.

We report a case of atypical oral hairy leukoplakia (OHL) in a 9-year-old immunocompetent girl treated with fluticasone propionate nasal spray for allergic rhinitis. The OHL in childhood is uncommon and should be included in a differential diagnosis of white lesions in the oral mucosa.

EBV-negative lymphoepithelial-like carcinoma of the lower lip.

Lymphoepithelial-like carcinoma (LEC) is a rare malignant neoplasm, which can be associated with Epstein-Barr virus (EBV) infection. Histologically, LEC is an undifferentiated carcinoma with an intermixed reactive lymphoplasmacytic infiltrate. LEC appears to be an uncommon tumor type of lip carcinoma. An 82-year-old white woman presented a lesion on her lower lip that developed over the last year. The lesion was characterized by ulceration with flat edges, hardened base, painful, and absence of regional lymphadenopathy. Microscopical analysis evidenced an intense inflammatory infiltrate, composed of lymphoplasmacytic cells, associated with scarce pleomorphic epithelial cells. Immunohistochemistry highlighted the LEC cells with strong expression of pan-CK AE1/AE3, EMA, p63, and p53. CD138 was also faintly positive. Ki-67 was >85%. In situ hybridization analysis did not show evidence of EBV. A diagnostic of EBV-negative LEC was made. We present an uncommon type of lip carcinoma, which can represent a diagnostic challenge for clinicians and pathologists.

Role of epstein-barr virus in the severity of recurrent respiratory papillomatosis.

OBJECTIVE: The objective was to investigate the prevalence of the Epstein-Barr virus (EBV) and its association with human papilloma virus (HPV) detection, clinicopathological features, and the severity of recurrent respiratory papillomatosis (RRP). METHODS: Cases of juvenile recurrent respiratory papillomatosis (JRRP) (n = 36) and adult recurrent respiratory papillomatosis (ARRP) (n = 44) were collected retrospectively and subdivided into low- and high-risk severity groups based on the Derkay score. We performed HPV detection and genotyping using a reverse hybridization protocol and investigated the presence of EBV by polymerase chain reaction (PCR) and in situ hybridization. CD21 levels were accessed by immunohistochemistry. RESULTS: All samples were HPV-positive, including 49 cases of HPV 6, 26 cases of HPV 11, four cases of HPV 6 and 11 coinfections, and one case of HPV 16. EBV-DNA was detected in nine samples by PCR, although none of the cases were positive by means of in situ hybridization. CD21 immunoexpression was not statistically associated with any of the variables analyzed. HPV 6 detection was significantly higher in ARRP cases (P = 0.03), whereas HPV 11 was more prevalent in JRRP cases (P = 0.02) and was even more prevalent in JRRP cases of greater severity (Derkay laryngoscopic scale ≥20) (P = 0.04). CONCLUSION: The presence of EBV does not seem to play an important role in the progression/severity of RRP. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E611-E618, 2020.

Relationship between inflammation and the severity of Recurrent Respiratory Papillomatosis.

OBJECTIVE: To characterize inflammatory cells in Recurrent Respiratory Papillomatosis (RRP) and to correlate it with severity using the Derkay laryngoscopic scale. MATERIALS AND METHODS: The data and biopsies from 36 patients with Juvenile (JRRP) and 56 patients with Adult (ARRP) were collected and analyzed under light microscopy. The patients were separated into groups according to the Derkay index: ≥20 for the most severe and < 20 for the less severe cases. Immunohistochemical analysis using CD3, CD4, CD8, CD15, CD20, CD68, FoxP3 and MUM-1 antibodies was performed, and the inflammatory cells were quantified. All the clinicopathological characteristics and the results of the immunohistochemical analysis were compared among the groups proposed using the Chi-Square test and correlated through the Spearman correlation test. RESULTS: The ARRP showed significantly higher quantities of CD3+, CD8+ and MUM1+ cells (p < .05) than the JRRP samples. The presence of CD15+ cells showed positive correlation with the Derkay index (p < .05), while the MUM-1+ cells showed an inverse correlation (p = .01). CONCLUSION: There are differences between the inflammatory cells population in the juvenile and adult groups and it can be related to disease severity.

FOXP3+ and CD25+ cells are reduced in patients with stage IV, grade C periodontitis: A comparative clinical study.

BACKGROUND AND OBJECTIVE: Some studies suggest that regulatory T cells (Tregs) have suppressive effects on inflammatory osteolysis. The aim of this study was to evaluate Treg immunomarkers in periodontitis-affected tissues from patients with periodontitis and clinically healthy gingiva (control). MATERIAL AND METHODS: The presence and distribution of positive cells for CD4, CD25 and FOXP3 (Treg immunomarkers) in periodontitis-affected tissues (epithelium and lamina propria) of 30 patients (ten per group) with a diagnosis of stage IV, grade C periodontitis (IV-C), stage III, grade B periodontitis (III-B) and the control were evaluated. A two-way ANOVA followed by Fisher's LSD test was used to demonstrate differences between the groups and immunomarkers; Student's t test was used to demonstrate differences between the epithelium and the lamina propria. RESULTS: Both IV-C and III-B periodontitis presented a significantly high proportion of immune-stained cells for all immunomarkers when compared to the control group. Notably, CD25+ and FOXP3+ cells were detected in a significantly higher number in III-B than IV-C periodontitis (P < .05). CONCLUSION: Our results suggest the participation of Tregs on the osteoimmunological mechanisms in IV-C and III-B periodontitis patients, notably contributing to strategies for alveolar bone regeneration in clinical treatment decisions.