Cytoplasmic Pattern Research Articles

Utility of MUC4 in the diagnosis of secretory carcinoma of salivary glands

Salivary gland tumors are diverse in morphology and both benign and malignant tumors may pose diagnostic challenges especially in small biopsies. Secretory carcinoma (SC) is histologically characterized by microcysts, follicles, solid growth pattern and occasional papillary structures, and absence of zymogen granules. SC is molecularly defined by the presence of novel gene fusion ETV6::NTRK3. Among the positive stains (S100 and mammaglobin), MUC4 is now another promising marker for the diagnosis of SC, that would enable the pathologists to exclude other morphologically close simulators. Aim of this study was to report clinicopathological features and assess utility of MUC4 in the diagnosis of SC. MUC4 was performed on 22 cases of SC. Glass slides were reviewed to record morphological patterns and staining of S100, mammaglobin, DOG1 and MUC4. Age ranged from 9 to 63 years with mean age of 34.41 ± 16.28 years. The male: female ratio was 72.7 %:27.3 %. The majority occurred in major salivary glands. A combination of patterns was seen; microfollicles were the most prevalent (90 %) followed by papillary-cystic and macrofollicles. MUC4 was positive in 19/21 (90 %) cases with almost equal number of 2+ and 3+ staining. MUC4 was negative in all cases of acinic cell carcinoma, polymorphous adenocarcinoma, adenoid cystic carcinoma, salivary duct carcinoma, myopepithelioma and myoeithelial carcinoma, cystadenoma and cribriform adenocarcinoma and all except 3 cases of mucoepidermoid carcinoma tested. Overall sensitivity of MUC4 was 95.4 %, specificity 90 %, p-value being <0.01, positive predictive value 87.5 % and negative predictive value 96.4 %. A characteristic cytoplasmic granular pattern was observed in 76.1 % tumors. S100 and mammaglobin were positive in all the performed cases. DOG1 was positive in 6/11 (28.5 %) tumors. In conclusion, MUC4 is a useful addition to a diagnostic immunohistochemical panel for SC, and to distinguish it from close potential mimickers such as acinic cell carcinoma, especially in practice settings where molecular testing is unavailable.

Utilization of p53 and p16 Immunohistochemistry in the Classification of Human Papillomavirus–Associated, p53 Wild-Type, and p53 Abnormal Oral Epithelial Dysplasia

p53 immunohistochemistry (IHC) has recently been shown to be a clinically useful marker for predicting risk of progression to invasive squamous cell carcinoma in oral epithelial dysplasia (OED). The literature supports the use of p53 IHC as a marker to identify TP53 mutation in in situ and invasive vulvar lesions and as a surrogate marker for high-risk human papillomavirus (HPV) infection, but there is little documentation for similar use in OED. The purpose of this study was to determine whether p53 IHC is a reliable surrogate marker for detecting both TP53 mutation and high-risk HPV infection in OED. We studied 57 cases of OED (11 mild, 18 moderate, and 28 severe), and all were stained for p16 and p53 IHC. High-risk HPV RNA in situ hybridization (ISH) was performed in selected cases (all p16-positive cases and all OED showing abundant apoptotic cells and karyorrhectic cells; N = 27). Targeted next-generation sequencing (NGS) was performed in 33 p16-negative cases and all high-risk HPV RNA ISH–negative cases (N = 36). We identified 21 cases with p53 basal sparing patterns (mid-epithelial and markedly reduced [null-like]), 14 cases with p53 wild-type patterns (scattered basal and patchy basal/parabasal), and 22 cases with p53 abnormal patterns (18 overexpression, 3 null, and 1 novel cytoplasmic pattern). Among cases with p53 basal sparing patterns, 20 were positive for p16 (20/21, 95%), and all were positive for high-risk HPV RNA ISH (21/21, 100%). The 36 sequenced cases had IHC patterns concordant with TP53 mutation status in 92% (33/36) of lesions. This study demonstrates that p53 IHC expression patterns are sensitive and specific for detection of both high-risk HPV infection and TP53 mutation. Coupled with selective p16 IHC testing, this IHC panel can accurately subclassify OED into HPV-associated, p53 wild-type (conventional), and p53 abnormal OED.

Molecular analyses of ADAMTS-1, -4, -5, and IL-17 a cytokine relationship in patients with ulcerative colitis

BackgroundUlcerative colitis (UC) is a chronic inflammatory bowel disease that develops due to the impaired immune response in genetically susceptible individuals, and its etiopathogenesis is not fully elucidated. IL-17 A is a cytokine that is produced by a type of immune cell called Th17 cells and is involved in the immune response and inflammation. On the other hand, ADAMTS-1, -4, and − 5 are enzymes that are involved in the breakdown of extracellular matrix proteins, including proteoglycans, which are important components of the intestinal wall. This study aimed to evaluate the relationship between interleukin 17 (IL-17 A) cytokine, which plays a role in the pathogenesis of ulcerative colitis, and the inflammation-controlled a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1, -4, and − 5 protein members.MethodsBowel tissue samples and blood serum from 51 patients with UC and 51 healthy controls were included in this study. mRNA expression levels of the ADAMTS-1, -4, -5, and IL-17 A were analyzed by RT-qPCR, and immunohistochemical analyses were performed to evaluate ADAMTS-1, -4, -5, and IL-17 A proteins in tissue samples. In addition, ELISA analysis determined serum levels of the ADAMTS-1, -4, -5, and IL-17 A.ResultsRT-qPCR results reveal that the expression of ADAMTS-1, -4, -5, and IL-17 A genes in the UC tissue samples were significantly high according to the control tissue samples. Also, ADAMTS-1, -4, -5, and IL-17 A proteins revealed enhanced expression pattern UC groups according to the control. Also, ADAMTS-1, -4, -5, and IL-17 A protein showed cytoplasmic localization patterns in both control and UC groups. The serum levels of ADAMTS-1,-5, and IL-17 A were significantly higher in UC samples than in the control group.ConclusionsWe observed a positive correlation between the ADAMTS-1, -5 and IL17A cytokine expression in UC samples. These results provide a new understanding of controlling crucial ADAMTS family protein members by IL-17 A cytokines with UC.

Rare haematological complication of a rheumatological disease

Abstract A 16-year-old female presented with complaints of low-grade fever for 4 months associated with generalised weakness, loss of appetite, significant loss of weight and loss of hair. On examination, pallor was evident. Rest of the examination was normal. Initial investigations revealed haemoglobin of 7 g/dL, white blood cell count of 3000 cells/mm3 and platelet count of 25,000/mm3. Bone marrow biopsy revealed 30% cellularity, suggestive of hypoplastic marrow. Further investigations revealed anti-nuclear antibody (4+ homogeneous pattern and 2+ cytoplasmic pattern); anti-dsDNA antibody was positive, low complements. A diagnosis of hypoplastic anaemia secondary to systemic lupus erythematosus was made.

Evaluation of Transforming Growth Factor Beta-1 in the vein wall of males with varicocele.

This study aims to investigate potential differences in the presence of Transforming Growth Factor-Beta 1 (TGF-β1) between the vein walls of patients with varicocele and those of healthy individuals. The study comprised a total of 40 participants, divided into two groups. The control group (Group 1) consisted of 20 patients who underwent coronary bypass surgery, while the varicocele group (Group 2) included 20 patients scheduled for varicocelectomy. The cytoplasmic and nuclear staining patterns of TGF-β1 immunohistochemistry were assessed in tissue samples under light microscopy, identifying any differences in TGF-β1 presence between varicocele patient vein walls and normal (saphenous) veins. The varicocele group demonstrated lower nuclear and cytoplasmic TGF-β1 staining rates compared to the control group. After controlling for the independent factor of age, significantly lower nuclear and cytoplasmic staining was still observed in the varicocele group. This study is the first of its kind to compare TGF-β1 staining in the vein walls of varicocele patients and healthy individuals. Previous studies focusing on varicose veins reported elevated TGF-β1 expression. Contrarily, our study observed lower TGF-β1 expression in varicocele patient veins, marking a unique contribution to the field.

S3492 A Rare Case of Antimitochondrial Antibody-Negative Primary Biliary Cholangitis With a Positive Cytoplasmic Pattern of Antinuclear Antibody

S3492 A Rare Case of Antimitochondrial Antibody-Negative Primary Biliary Cholangitis With a Positive Cytoplasmic Pattern of Antinuclear Antibody

B-131 The Prevalence and Clinical Relevance of the DFS Immunofluorescent Staining Pattern in a Large ANA-positive Cohort

Abstract Background The dense fine-speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine antinuclear antibody (ANA) screening. Although various researchers have studied the DFS pattern in recent years, it presents a clinical puzzle since its significance remains elusive. Anti-DFS-70 antibodies occur in healthy individuals with different medical conditions. Unlike the other ANA patterns, they are not associated with systemic autoimmune disease. The present study examined the association of a DFS ANA pattern with the medical conditions of patients who presented to a tertiary care medical center. Methods Retrospective chart review of all laboratory values (January 2020 through June 2022) with ANA- and DFS-positive patterns in patients with ANA tests ordered by various clinicians and compared to the patient diagnosis reported. UMass Human IRB approves this study. Results Of 8885 ANA samples analyzed, 67.8% (6022) were reported negative. Of total positive ANA (2863) samples, 78.6% (2257) had nuclear patterns, 9.1% (260) had mitotic patterns, and 12.1% (346) had cytoplasmic patterns. Of the positive ANA samples, 14.6% (417) had DFS patterns. For samples with DFS pattern, titers ranged from 1:40 to 1:1280; 62.5% were between1:40, and 1:80. Of samples positive for ANA DFS patterns, 76% (317) were from female patients, and 90% (375) were from patients &amp;gt;21 years of age. Among patients with DFS patterns, the most commonly reported diseases were neurological, followed by respiratory, digestive, circulatory, and musculoskeletal (Table 1). Conclusion Clinicians should use a positive ANA with a DFS pattern to indicate pathological phenomena other than autoimmune diseases. Only 3.1% of the patients with DFS patterns have a systemic rheumatic disorder. Clinicians should pay close attention to ANA Patterns, especially DFS staining when using ANA testing.

Expression pattern, prognostic value and potential microRNA silencing of FZD8 in breast cancer.

Breast cancer (BC) is one of the most widespread types of cancer affecting females, and therefore, early diagnosis is critical. BC is a complex heterogeneous disease affected by several key pathways. Among these, WNT proteins and their frizzled receptors (FZD) have been demonstrated to be crucial in regulating a number of cellular and molecular events in BC tumorigenesis. The role of the WNT receptor, FZD8, in BC has received minimal attention; for that reason, the present study examined the prognostic value of its protein expression pattern in a BC cohort. FZD8 cytoplasmic expression pattern analysis revealed that ~38% of the primary samples presented with a high expression profile, whereas ~63% of the samples had a low expression profile. Overall, ~46% of the malignant tissues in the lymph node-positive samples exhibited an increased FZD8 cytoplasmic expression, whereas 54% exhibited low expression levels. An increased expression of FZD8 was associated with several clinicopathological characteristics of the patients, including a low survival rate, tumor vascular invasion, tumor size and grade, and molecular subtypes. Affymetrix microarray triple-negative BC datasets were analyzed and compared with healthy breast tissues in order to predict the potential interfering microRNAs (miRNAs) in the WNT/FZD8 signaling pathway. A total of 29 miRNAs with the potential to interact with the WNT/FZD8 signaling pathway were identified, eight of which exhibited a significant prediction score. The target genes for each predicted miRNA were identified. On the whole, the findings of the present study suggest that FZD8 is a potential prognostic marker for BC, shedding some light onto the silencing mechanisms involved in the complex BC signaling.

STRBC1 and cyTRBC1 Expression Distinguishes Indolent T-Lymphoblastic Proliferations From T-Lymphoblastic Leukemia/Lymphoma.

Indolent T-lymphoblastic proliferation (iT-LBP) consists of a proliferation of non-neoplastic TdT + T cells in extrathymic tissues, requiring no treatment. However, due to overlapping clinical and histologic features, distinguishing iT-LBP from T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) can be challenging. Recently, flow cytometry-based evaluation of TRBC1 has been used to detect of T-cell clonality in TCRαβ + mature T-cell lymphomas and aid in the differential diagnosis between T-ALL and normal thymocytes. We present a case of iT-LBP with high-grade serous ovarian carcinoma (HGSOC). To investigate the potential utility of TRBC1 expression in distinguishing iT-LBP from T-ALL/LBL, we assessed both surface (s) and cytoplasmic (cy) TRBC1 expression patterns on blast cells from the patient with iT-LBP and HGSOC as well as 11 patients diagnosed with T-ALL/LBL. The results revealed that sTRBC1 and cyTRBC1 exhibited polytypic expression patterns in patient with iT-LBP and HGSOC, while cyTRBC1 showed monotypic expression in those with T-ALL/LBL. This suggests that evaluation of sTRBC1 and cyTRBC1 expression can serve as a simple, rapid, and effective approach to differentiate between iT-LBP and T-ALL/LBL.

Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer.

This study aimed to evaluate AT1-R expression in normal and cancerous human kidneys, how these expressions are modified, and AT1-R functionality. AT-1R mRNA expression, determined by real-time PCR, was detected in all samples. AT-1R mRNA increased in well-differentiated cancer (G1, p < 0.01) and decreased 2.9-fold in undifferentiated cancer (G4, p < 0.001) compared with normal kidney tissues. Immunocytochemistry analysis showed that the AT-1R was expressed in the normal tubular epithelium. The glomerulus was also immunoreactive, and as expected, the smooth muscle cells of the vessel walls also expressed the receptor. A total of 35 out of 42 tumors were AT-1R positive, with the cell tumors showing varying numbers of immunoreactive cells, which were stained in a diffuse cytoplasmic and membranous pattern. Computer-assisted counting of the stained tumor cells showed that the number of AT-1R-positive cells increased in the well-differentiated cancers. The functionality of AT-1R was assessed in primary cultures of kidney epithelial cells obtained from three G3 kidney cancer tissues and corresponding histologically proven non-malignant tissue adjacent to the tumor. Indeed, Ang II stimulated, in a dose-dependent manner, the 24 h proliferation of normal kidney cells and cancer cells in the primary culture and phosphorylated extracellular regulated kinases 1 and 2. In conclusion, Ang II may be involved in the growth or function of neoplastic kidney tissue.

Laboratory risk factors for coexistent primary biliary cholangitis in patients with Sjögren’s syndrome: a retrospective study

BackgroundLimited research exists on the laboratory characteristics of coexistent primary biliary cholangitis (PBC) and Sjögren’s syndrome (SS). This study aimed to investigate the laboratory risk factors for the coexistence of PBC in patients with SS.MethodsEighty-two patients with coexistent SS and PBC (median age 52.50 years) and 82 age- and sex-matched SS controls were retrospectively enrolled between July 2015 and July 2021. The clinical and laboratory characteristics of the two groups were compared. Laboratory risk factors for the coexistence of PBC in patients with SS were analyzed using logistic regression analysis.ResultsBoth groups had a similar prevalence of hypertension, diabetes, thyroid disease, and interstitial lung disease. Compared with the SS group, patients in the SS + PBC group had higher levels of liver enzymes, immunoglobulins M (IgM), G2, and G3 (P < 0.05). The percentage of patients with an antinuclear antibody (ANA) titre > 1:10000 in the SS + PBC group was 56.1%, higher than that in the SS group (19.5%, P < 0.05). Additionally, cytoplasmic, centromeric, and nuclear membranous patterns of ANA and positive anti-centromere antibody (ACA) were observed more frequently in the SS + PBC group (P < 0.05). Logistic regression analysis showed that elevated IgM levels, high ANA titre, cytoplasmic pattern, and ACA were independent risk factors for PBC coexistence in SS.ConclusionsIn addition to established risk factors, elevated IgM levels, positive ACA, and high ANA titre with cytoplasmic pattern provide clues to clinicians for the early screening and diagnosis of PBC in patients with SS.

O-159 Impact of open and closed methods of vitrification on oocyte mitochondria function and RNAseq transcriptome profile: a preclinical screening study

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR&amp;gt;0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068

Expression of Connexin 43 in Granular Cell Tumors of the Skin, Tongue and Esophagus.

Granular cell tumors (GCT) are rare neoplasms of Schwann cell origin occurring in the skin and in other organs. The etiopathogenesis of GCT is yet poorly understood. Connexin 43 (Cx43) is the most broadly expressed gap junction protein in humans, the tumoral role of which has been investigated in several types of tumors. Its role in GCT of the skin, oral cavity and gastrointestinal tract is as yet unknown. Herein, we present a study on the immunohistochemical expression of Cx43 in GCT of the skin (n = 15), tongue (n = 4) and esophagus (n = 3). Immunolabeling was scored positive (weak (+), moderate (++) or strong (+++)). Cx43 was expressed by all cases of GCT of the skin, tongue and esophagus (22/22), showing moderate to strong staining. All tissue sections of GCT were characterized by a diffuse, cytoplasmic staining pattern of the tumor cells. None of those showed membranous or nuclear staining. Our results suggest that Cx43 probably plays an important role in the development of this rare tumor entity.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

PD-L1 Expression in Ampullary Adenocarcinoma.

Ampullary adenocarcinoma is a rare neoplasm often treated by the complex Whipple's procedure. Several histological factors predict poor prognosis including pancreatobiliary morphology, presence of lymphovascular, perineural invasion and local or distant metastasis. Systemic therapy with gemcitabine, 5-fluorouracil regimens are given with variable benefits. Immunotherapy checkpoint inhibitors have shown beneficial anti-tumor effects in several carcinomas, the most remarkable being in non-small cell lung cancer. Administration of these novel drugs is based on immunohistochemical expression (which may or may not be indicative of response to therapy) along with meticulous decision making by the multidisciplinary team. Immunohistochemistry (IHC) is an effective means of immune marker demonstration and has been used in various tumor types for predictive and prognostic purposes. PD-L1 IHC (clone E1L3N) was applied in 101 cases of ampullary adenocarcinoma. Tumor infiltrating lymphocytes were also evaluated. The immunoreactivity was assessed and categorized into following staining thresholds: <1%, <5%, <10% and ≥10% for tumor cells (membranous and/or cytoplasmic staining pattern), and 5% and 10% cut-offs for immune cells. We found that at a 10% cut-off, 73.3% (74/101) patients were men (P = .006) older than 50 years of age (P < .001) presenting with a tumor measuring <3 cm (P = .001). It was significantly associated with intestinal differentiation (P = .004) and grade 1 tumors (P = .001). Twelve patients presented with recurrence as well (P = .03). In the context of ampullary adenocarcinoma, this study highlights the positivity observed with the PD-L1 IHC clone E1L3N at different thresholds, with the particularly stronger associations being evident at a 10% cut-off.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: ALK-positive anaplastic large cell lymphomas (ALCL) are characterized by rearrangements involving the ALK gene on chromosome 2. The translocation t(2;5) (p23;q35) resulting in the NPM1::ALK gene fusion is detected in the majority of ALCL. In 10%–20% of ALCL, ALK is fused to various other partner genes. Compared to NPM1::ALK, variant ALK-fusion proteins are not expressed in the nucleus. The frequency, distribution, and possible prognostic significance of the different variant ALK partner genes have not been analyzed in population-based studies so far. We investigated all patients with ALK-positive ALCL with exclusive cytoplasmic ALK-expression diagnosed in a uniformly treated cohort of ALCL patients in the NHL-BFM study group. Patients and Methods: Between 2000 and 2017, 312 children and adolescents were diagnosed with an ALK-positive ALCL in Austria, Germany, Switzerland and the Czech Republic, and included in the ALCL 99 trial or the NHL-BFM registry 2012. Reference pathology demonstrated exclusive cytoplasmic ALK-staining in 49 tumors. Formalin-fixed-paraffin-embedded or frozen tumors of 43/49 patients were available for molecular analyses. Tumor-DNA was analyzed by an ALK-specific genomic capture high throughput sequencing assay. ALK-partner gene-specific RT-PCR assays were used to identify the respective partner genes when tumor cDNA was available. Results: 41 of 43 available tumors with exclusive cytoplasmic ALK-staining could be analyzed. 13 were NPM1::ALK positive and 28 tumors had a variant ALK partner. TPM3::ALK (n = 9, 32%) and ATIC::ALK (n = 8, 29%) were the most common variant partner genes. XPOI could be identified as a novel ALK fusion partner. The patients´ characteristics of ALCL expressing Variant::ALK were comparable to those with NPM1::ALK fusions. The five-year event-free survival (EFS) and overall survival were comparable between the 284 NPM1::ALK and the group of 28 different Variant::ALK-expressing patients. However, none of the 9 patients with TPM3::ALK positive ALCL relapsed, compared to 5 relapses among 8 patients with ATIC::ALK positive ALCL. Conclusions: In our population-based cohort of ALK-positive ALCL-patients, less than 10% carried variant ALK-fusion partners, 60% of which were TPM3 or ATIC. In patients with exclusive cytoplasmic ALK staining patterns, NPM1::ALK positivity should be excluded by molecular analysis. The tendency of a different relapse risk between ATIC::ALK and TPM3::ALK -positive ALCL-patients suggests that the fusion partner might confer tumor aggressiveness or drug resistance. The research was funded by: KNACK DEN KREBS Fördergemeinschaft Kinderkrebs-Zentrum Hamburg e.V. and HAMBURGER STIFTUNG ZUR FÖRDERUNG DER KREBSBEKÄMPFUNG Keyword: diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Grouper RIP2 inhibits Singapore grouper iridovirus infection by modulating ASC-caspase-1 interaction.

Receptor interacting protein 2 (RIP2), serves as a vital sensor of cell stress, is able to respond to cell survival or inflammation, and is involved in antiviral pathways. However, studies on the property of RIP2 in viral infections in fish have not been reported. In this paper, we cloned and characterized RIP2 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP2) and further discussed the relevance of EcRIP2 to EcASC, comparing the influences of EcRIP2 and EcASC on the modulation of inflammatory factors and the NF-κB activation to reveal the mechanism of EcRIP2 in fish DNA virus infection. Encoded a 602 amino acid protein, EcRIP2 contained two structural domains: S-TKc and CARD. Subcellular localization signified that EcRIP2 existed in cytoplasmic filaments and dot aggregation patterns. After SGIV infection, the EcRIP2 filaments aggregated into larger clusters near the nucleus. The infection of SGIV could notably up-regulate the transcription level of the EcRIP2 gene compared with lipopolysaccharide (LPS) and red grouper nerve necrosis virus (RGNNV). Overexpression of EcRIP2 impeded SGIV replication. The elevated expression levels of inflammatory cytokines induced by SGIV were remarkably hindered by EcRIP2 treatment in a concentration-dependent manner. In contrast, EcASC treatment could up-regulate SGIV-induced cytokine expression in the presence of EcCaspase-1. Enhancing amounts of EcRIP2 could overcome the down regulatory effect of EcASC on NF-κB. Nevertheless, increasing doses of EcASC failed to restrain the NF-κB activation in the existence of EcRIP2. Subsequently, it was validated by a co-immunoprecipitation assay that EcRIP2 dose-dependently competed with EcASC binding to EcCaspase-1. With increasing time to SGIV infection, EcCaspase-1 gradually combined with more EcRIP2 than EcASC. Collectively, this paper highlighted that EcRIP2 may impede SGIV-induced hyperinflammation by competing with EcASC for binding EcCaspase-1, thereby suppressing viral replication of SGIV. Our work supplies novel viewpoints into the modulatory mechanism of RIP2-associated pathway and offers a novel view of RIP2-mediated fish diseases.

Double-stranded RNA immunohistochemistry as a screening tool for viral encephalitis.

Viral infections of the central nervous system can be challenging to diagnose because of the wide range of causative agents and nonspecific histologic features. We sought to determine whether detection of double-stranded RNA (dsRNA), produced during active RNA and DNA viral infections, could be used to select cases for metagenomic next-generation sequencing (mNGS) from formalin-fixed, paraffin-embedded brain tissue. Eight commercially available anti-dsRNA antibodies were optimized for immunohistochemistry (IHC) and the top antibody tested in a series of cases with confirmed viral infections (n = 34) and cases with inflammatory brain lesions of unclear etiology (n = 62). Among known positives, anti-dsRNA IHC produced a strong cytoplasmic or nuclear staining pattern for Powassan virus, West Nile virus, rabies virus, JC polyoma virus, and adenovirus while failing to detect Eastern equine encephalitis virus, Jamestown Canyon virus, or any herpesvirus. All the unknown cases were negative by anti-dsRNA IHC, while mNGS detected rare viral reads (0.3-1.3 reads per million total reads) in 2 cases (3%), with only 1 having potential clinical significance. Anti-dsRNA IHC can effectively identify a subset of clinically relevant viral infections but not all. The absence of staining should not exclude cases from mNGS if sufficient clinical and histologic suspicion exists.

Latest Findings on NOD-Like Receptor Family Pyrin Domain Containing Protein 3 Inflammasome and Bone and Articular Diseases

Inflammasomes are important components of the innate immune system. They are assembled by cytoplasmic pattern recognition receptors and play a critical role in the pathogenesis and progression of various inflammatory diseases through regulating the release and activation of inflammatory cytokines and inducing cell prytosis. NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome has been widely studied and has been shown to be closely associated with cardiovascular diseases and metabolic disorders. Bone and joint diseases, such as osteoarthritis and rheumatoid arthritis show high prevalence worldwide and can cause bone and cartilage damage, pain, and dysfunction, adversely affecting the patients' quality of life. The reported findings of some studies indicate that the pathogenesis of various bone and articular diseases is associated with NLRP3 inflammasome. Small molecule antagonists targeting NLRP3 inflammasome have shown considerable therapeutic potentials, but their clinical application still needs further exploration. Herein, we reviewed the composition and function of NLRP3 inflammasome and its association with bone and articular diseases.

Antinuclear Antibody Positivity in Patients With Hair Loss After COVID-19 Infection.

Hair loss is one of the most common disorders after coronavirus disease 2019 (COVID-19) infection. This study aimed to investigate the relationship between COVID-19-related hair loss and antinuclear antibody (ANA) positivity and patterns. ANA positivity and patterns were analyzed in 30 female COVID-19 patients with hair loss complaints and compared in terms of the presence of autoimmunity between patients with and without COVID-19 exhibiting hair loss. ANA positivity and cytoplasmic patterns were detected in 40% of the patients with COVID-19 infection and hair loss. Trichodynia and diffuse hair loss were observed in 63.3% and 53.3%, respectively. In patients with COVID-19-related hair loss, diffuse hair loss and ANA positivity may be related to the high antibody levels triggered by COVID-19 infection.