Biopsy Fragments Research Articles

Biofilm and Its Characteristics in Venous Ulcers.

The aim of the study was to analyze the characteristics of the biofilm of venous ulcers in terms of location and formation and to relate the presence of the biofilm to ulcer characteristics including duration, injured area, and necrotic tissue. Descriptive clinical study. We obtained 2 biopsy fragments (tissue samples) from 44 patients with venous ulcers treated at a public outpatient clinic in a university hospital in Belo Horizonte, Brazil. Ulcers were photographed and classified according to the duration. In addition, the wound size and proportion of wound surface covered by necrotic tissue were measured. One fragment from each ulcer underwent microbiological analysis, while the other was analyzed using transmission electron microscopy. Data analysis was limited to fragments from patients with bacteria in the microbiological analysis. Data analysis is based on samples obtained from 21 ulcers in 21 patients who had bacteria in their ulcer based on microbiologic analysis of a tissue sample. Most ulcers were open for 2 to 10years, 57% (n=12) were 16 cm2 or smaller, and the proportion of the wound bed covered by necrotic tissue coverage varied widely. Of the 21/44 patients (48%) with bacteria in their ulcers, only 3 patients had bacterial biofilm present in the transmission electron microscopy, corresponding to 7% of the 44 patients. Pseudomonas aeruginosa was the most frequent bacterium, identified in 10 fragments. The biofilm was not present on the surface but in a layer slightly below it. The detection of biofilms was not directly related to the duration of the ulcer. It was not possible to establish a correlation between the size of the lesion and the presence of these microorganisms due to the small sample size. Our findings indicate that detecting biofilm in venous ulcers is challenging, as it does not uniformly occur throughout the wound bed, can occur at different depths, and is often not present on the wound surface. There is a need to develop studies that can contribute to the detection of biofilm in clinical practice.

Evaluation of the Tissue Immune Microenvironment in the Diabetic Patients with Dental Implant

Long-term hyperglycemia in people with diabetes very frequently leads to damage and/or dysfunction of many tissues and organs of the human body, causing increased clinical morbidity. There is, however, an increased correlation between glycemic control and the onset of these consequences and in particular microvascular and macrovascular complications. The results of some studies have suggested that diabetes exerts an influence on the failure rates of implants compared to non-diabetic patients. One study demonstrates that HbA1c level (higher or lower than the threshold of 8.1) also influences the survival rate of dental implants. Biopsy fragments from 8 consecutive patients who had a titanium dental implant as well as diabetes were included in our study. The control group was represented by 4 patients with dental implants and who did not have diabetes. After the histological processing of the tissues, cell counts of CD4, CD8, CD20 and CD3 lymphocytes were performed by using QuPath. The findings characterize the composition of lymphocyte populations in the oral mucosa surrounding dental implants. In diabetic patients, poor glycemic control has been associated with a decreased CD4/ CD8, contrary to our findings in tissue. The decreased number of CD20 positive lymphocytes could be associated with the dysfunction of humoral immunity generally observed in patients with diabetes. In our study, a 2.5 x higher value of TCD4+ lymphocytes was noted compared to the average of TCD8+ type lymphocytes. These results support the involvement of these types of inflammatory cells in the potimplant bone healing processes. This process starts on the 10th day post-implant. Different studies raise, in these cases, the question of innate immune response vs adaptive immune response. Suppression of processes induced by CD8+ lymphocytes is associated with stimulation of bone formation. Our results support this hypothesis.

A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

Clinical Tools for Optimizing Therapeutic Decision-Making in Prostate Cancer: A Five-Year Retrospective Analysis.

The effective staging of prostate cancer is essential for optimizing treatment and predicting outcomes. This study assessed the correlation between detailed preoperative diagnostic scores and postoperative outcomes to evaluate the accuracy of cancer restaging and its impact on treatment decisions and prognosis after prostatectomy. This retrospective study analyzed 133 prostate cancer patients who underwent prostatectomies at "Pius Brinzeu" Clinical Emergency Hospital in Timisoara over five years. Preoperative Gleason scores increased significantly across risk categories, from an average of 6.21 in low-risk patients to 7.57 in high-risk patients. This trend continued postoperatively, with scores rising from 7.04 to 8.33, respectively. The average increase in Gleason scores from preoperative to postoperative assessments was most pronounced in high-risk patients, at 0.76. Significant changes in clinical staging included increases in NCCN risk, where high-risk patients showed a 30% increase, and ISUP grade, with a 26.7% increase in the high-risk category. Notably, nodal status changes were also significant in high-risk patients, showing a 23.3% increase. The incidence of MRI-detected adenopathy was notably higher in the high-risk group (50%). Furthermore, there were significant correlations between the preoperative CAPRA score and postoperative ISUP grade (r = 0.261) and the preoperative PIRADS score and postoperative ISUP grade (r = 0.306). Similar observations were made between the preoperative and postoperative Gleason scores (r = 0.286) and the number of positive fragments (r = 0.227) with the postoperative ISUP grading. Furthermore, the preoperative CAPRA score was significantly correlated (r = 0.261) with the postoperative ISUP grading. Preoperative MRI findings, which included assessments of adenopathy and seminal vesicle invasion, were also significantly correlated (r = 0.218) with the postoperative pathological findings. Additionally, a significant correlation was found between the preoperative PIRADS score and postoperative ISUP grade (r = 0.306). In forecasting the aggressiveness and staging of prostate cancer following surgery, preoperative PSA levels showed an AUC of 0.631; the preoperative Gleason score had an AUC adjusted to 0.582, and the number of positive biopsy fragments indicated an AUC of 0.566. These results highlight the necessity of accurate and comprehensive preoperative assessments to better predict disease progression and refine treatment strategies.

Improving adequacy rates of bone marrow aspirate and core biopsies through training and a bedside X-ray device.

e23228 Background: Bone marrow (BM) evaluation is crucial to the diagnosis of hematolymphoid disorders. WHO requires an adequate number of well-spread-out spicules in aspirates and defines an adequate BM core biopsy as ≥1.5 cm with at least 10 inter-trabecular areas (ITA). Gross examination of the core biopsy at the bedside can be misleading due to attached clot, thick periosteum, and cortical bone, or tangential biopsies, which histologically results in biopsy without adequate ITAs. Suboptimal BM biopsies hinder appropriate diagnosis, delay patient care, and may require the patient to undergo additional BM biopsies. Bedside image analysis via a cabinet x-ray system can be utilized as a point-of-care tool to assess the amount of evaluable marrow permitting re-biopsy at the time of the procedure. Methods: The study was conducted over 5 months at Westchester Medical Center. We retrospectively estimated BM adequacy rates by analyzing the aspirate quality and measuring the BM core biopsy length. Subsequently, a training demonstration to the proceduralist (fellows in training) by an experienced attending was performed and the adequacy rates were reassessed. BM biopsies were analyzed with a cabinet x-ray system, Faxitron, soon after biopsy, then subjected to routine histology and processing. High-resolution images of the core biopsy were obtained. The length of the core biopsy and the number of ITAs were calculated from the x-ray images. Measurement of the gross biopsy length and microscopic assessment of evaluable marrow were compared to the measurements obtained by x-ray imaging. Results: Our adequacy rates which met aspirate quality were 5% (1/20) pre-intervention and 65% (13/20) post-intervention. The aspirate smear quality improved after the demonstration technique to 65%. Only 13/53 core biopsies exceeded the WHO standard of 1.5 cm by microscopy, while 34/53 were grossly estimated to be > 1.5 cm. There was general concordance between the number of ITAs and the length of core biopsies by microscopy. In approximately 20% of cases, the x-ray images highlighted suboptimal core biopsy ( < 10 ITA/cortical bone/cartilage), which weren’t apparent by gross examination alone at the bedside. Utilizing x-ray image measurements, core biopsy fragment length was shown to be in concordance with microscopic estimation of length and number of ITAs. X-ray imaging readily identified clot sections (non-radiopaque), sclerotic cortical bone (absence of trabeculae), and periosteum (soft tissue shadow) in all 10 sub-1 cm biopsies. Conclusions: We conclude that a standard instruction technique of slide preparation can improve the quality of bone marrow aspirate. Core biopsy length and number of ITAs can easily be estimated using bedside point-of-care x-ray analysis to ensure adequate BM evaluations. We propose training and the routine use of bedside x-ray machine to improve the adequacy rates of BM biopsies.

Machine Learning-Assisted Classification of Paraffin-Embedded Brain Tumors with Raman Spectroscopy.

Raman spectroscopy (RS) has demonstrated its utility in neurooncological diagnostics, spanning from intraoperative tumor detection to the analysis of tissue samples peri- and postoperatively. In this study, we employed Raman spectroscopy (RS) to monitor alterations in the molecular vibrational characteristics of a broad range of formalin-fixed, paraffin-embedded (FFPE) intracranial neoplasms (including primary brain tumors and meningiomas, as well as brain metastases) and considered specific challenges when employing RS on FFPE tissue during the routine neuropathological workflow. We spectroscopically measured 82 intracranial neoplasms on CaF2 slides (in total, 679 individual measurements) and set up a machine learning framework to classify spectral characteristics by splitting our data into training cohorts and external validation cohorts. The effectiveness of our machine learning algorithms was assessed by using common performance metrics such as AUROC and AUPR values. With our trained random forest algorithms, we distinguished among various types of gliomas and identified the primary origin in cases of brain metastases. Moreover, we spectroscopically diagnosed tumor types by using biopsy fragments of pure necrotic tissue, a task unattainable through conventional light microscopy. In order to address misclassifications and enhance the assessment of our models, we sought out significant Raman bands suitable for tumor identification. Through the validation phase, we affirmed a considerable complexity within the spectroscopic data, potentially arising not only from the biological tissue subjected to a rigorous chemical procedure but also from residual components of the fixation and paraffin-embedding process. The present study demonstrates not only the potential applications but also the constraints of RS as a diagnostic tool in neuropathology, considering the challenges associated with conducting vibrational spectroscopic analysis on formalin-fixed, paraffin-embedded (FFPE) tissue.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 μm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

Retrospective analysis of discordant results between histology and other clinical diagnostic tests on helicobacter pylori infection.

A reliable test is essential for diagnosing Helicobacter pylori (H. pylori) infection, and crucial for managing H. pylori-related diseases. Serving as an excellent method for detecting H. pylori infection, histologic examination is a test that clinicians heavily rely on, especially when complemented with immunohistochemistry (IHC). Additionally, other diagnostic tests for H. pylori, such as the rapid urease test (CLO test) and stool antigen test (SA), are also highly sensitive and specific. Typically, the results of histology and other tests align with each other. However, on rare occasions, discrepancy between histopathology and other H. pylori diagnostic tests occurs. To investigate the discordance between histology and other H. pylori tests, the underlying causes, and the impact on clinical management. Pathology reports of gastric biopsies were retrieved spanning August 2013 and July 2018. Reports were included in the study only if there were other H. pylori tests within seven days of the biopsy. These additional tests include CLO test, SA, and H. pylori culture. Concordance between histopathology and other tests was determined based on the consistency of results. In instances where histology results were negative while other tests were positive, the slides were retrieved for re-assessment, and the clinical chart was reviewed. Of 1396 pathology reports were identified, each accompanied by one additional H. pylori test. The concordance rates in detecting H. pylori infection between biopsy and other tests did not exhibit significant differences based on the number of biopsy fragments. 117 discrepant cases were identified. Only 20 cases (9 with CLO test and 11 with SA) had negative biopsy but positive results in other tests. Four cases initially stained with Warthin-Starry turned out to be positive for H. pylori with subsequent IHC staining. Among the remaining 16 true discrepant cases, 10 patients were on proton pump inhibitors before the biopsy and/or other tests. Most patients underwent treatment, except for two who were untreated, and two patients who were lost to follow-up. There are rare discrepant cases with negative biopsy but positive in SA or CLO test. Various factors may contribute to this inconsistency. Most patients in such cases had undergone treatment.

Caveats in Interpretation of Molecular Diagnostics in Heart Allografts.

Histologic separation of injury, T cell-mediated rejection, or antibody-mediated rejection in allograft heart biopsies is difficult. A critical review of publications was performed to evaluate the caveats of using molecular diagnostics (MDX) to distinguish between these entities. Typically, only 1 to 2 fragments of unknown histologic appearance are evaluated. Archetype and molecular classifier analyses use gene lists derived from histologic labels and associated reproducibility issues influence the accuracy of the derived MDX classes. Archetypes A1, A2, and A3 archetypes created by bioinformatics were renamed no rejection, T cell-mediated rejection, and antibody-mediated rejection despite as little as 40% concordance with histologic diagnoses and overlapping archetype scores. Additional archetypes S4 and minor injury were created using arbitrary cutoffs based on visual examination of principal component analysis plots. Therapeutic implications of the numerous discrepancies with histology remain unexplored. Many MDX-derived observations are ambiguous and open to alternate logical explanations. Better molecular methods and more rigorous validation studies are needed to advance the field. Ideally, these methods should analyze all available biopsy fragments to minimize sampling issues. It is also desirable to incorporate spatial transcriptomics into the workflow, so that gene expression data can be directly compared with the underlying histology lesions.

Evaluating testicular tissue for future autotransplantation: focus on cancer cell contamination and presence of spermatogonia in tissue cryobanked for boys diagnosed with a hematological malignancy.

What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy? Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts. Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients. This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021. Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available. The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed. The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range. ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored. The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest. N/A.

Neointimal myofibroblasts contribute to maintaining Th1/Tc1 and Th17/Tc17 inflammation in giant cell arteritis

Vascular smooth muscle cells (VSMCs) have been shown to play a role in the pathogenesis of giant cell arteritis (GCA) through their capacity to produce chemokines recruiting T cells and monocytes in the arterial wall and their ability to migrate and proliferate in the neointima where they acquire a myofibroblast (MF) phenotype, leading to vascular stenosis. This study aimed to investigate if MFs could also impact T-cell polarization.Confocal microscopy was used to analyze fresh fragments of temporal artery biopsies (TABs). Healthy TAB sections were cultured to obtain MFs, which were then treated or not with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) and analyzed by immunofluorescence and RT-PCR. After peripheral blood mononuclear cells and MFs were co-cultured for seven days, T-cell polarization was analyzed by flow cytometry.In the neointima of GCA arteries, we observed a phenotypic heterogeneity among VSMCs that was consistent with a MF phenotype (α-SMA+CD90+desmin+MYH11+) with a high level of STAT1 phosphorylation. Co-culture experiments showed that MFs sustain Th1/Tc1 and Th17/Tc17 polarizations. The increased Th1 and Tc1 polarization was further enhanced following the stimulation of MFs with IFN-γ and TNF-α, which induced STAT1 phosphorylation in MFs. These findings correlated with increases in the production of IL-1β, IL-6, IL-12 and IL-23 by MFs.Our study showed that MFs play an additional role in the pathogenesis of GCA through their ability to maintain Th17/Tc17 and Th1/Tc1 polarizations, the latter being further enhanced in case of stimulation of MF with IFN-γ and TNF-α.

The Value of Tumor Infiltrating Lymphocytes (TIL) for Predicting the Response to Neoadjuvant Chemotherapy (NAC) in Breast Cancer according to the Molecular Subtypes.

The antitumor host immune response is an important factor in breast cancer, but its role is not fully established. The role of tumor infiltrating lymphocytes (TIL) as an immunological biomarker in breast cancer has been significantly explored in recent years. The number of patients treated with neoadjuvant chemotherapy (NAC) has increased and the identification of a biomarker to predict the probability of pCR (pathological complete response) is a high priority. We evaluated 334 cases of BC treated with NAC followed by surgical resection from 2020-2022 at the Ist Clinic of Oncological Surgery, Oncological Institute "Prof Dr I Chiricuta" Cluj Napoca. Of the above, 122 cases were available for histological evaluation both in pre-NAC biopsy and post-NAC resection tissue. Evaluation of biopsy fragments and resection parts were performed using hematoxylin eosin (H&E). The TIL evaluation took place according to the recommendations of the International TIL Working Group (ITILWG). There was a strong association between elevated levels of pre-NAC TIL. At the same time, there is a statistically significant correlation between stromal TIL and tumor grade, the number of lymph node metastases, the molecular subtype and the number of mitoses (p < 0.005). Intratumoral TIL showed a significant correlation with tumor size, distant metastasis, molecular subtype, number of mitosis, stage and lymph node metastasis (p < 0.005). We also demonstrated that high pre-NAC STIL represents a strong predictive marker for pCR. This study reveals the role of TIL as a predictive biomarker in breast cancer not only for the well-established TNBC (triple negative breast cancer) and HER2+ (Her2 overexpressed) subtypes but also in Luminal A and B molecular subtypes. In this scenario, the evaluation of sTIL as a novel predictive and therapy-predicting factor should become a routinely performed analysis that could guide clinicians when choosing the most appropriate therapy.

Correlation between HER2/neu protein overexpression on Immunohistochemistry and Fluorescent in Situ Hybridization (FISH) in breast carcinoma: Problems in developing countries.

To correlate the results of HER2/neu protein overexpression on immunohistochemistry (IHC) and gene amplification on fluorescence in situ hybridization (FISH) and to document the problems faced in performing FISH procedure. This was an observational retrospective study covering five years from January 1st, 2015 - December 31st, 2019 at Histopathology Department of Shifa International Hospital (SIH), Islamabad. All cases of breast cancer that underwent florescence in situ hybridization (FISH) were retrieved. Correlation between HER2/neu overexpression on IHC and its amplification on FISH was analyzed. Problems in application of FISH were recorded. Out of 451 cases submitted for HER2/neu testing by FISH, 68 cases (15%) were rejected. Gene amplification was seen in 139 (36.29%) cases. Total cases with HER2/neu IHC score of 2+ were 330 cases and out of which gene amplification was seen in 98 cases (29.69%) whereas 93.1% (41/44) 3+ IHC cases were amplified. Poor fixation, inadequate amount of tumor with crushing artefacts and dye application to the biopsy fragments were causes of sample rejection. Her2/neu amplification was seen in most Her2/neu 3+ cases and approximately one-third of Her2neu 2+ cases. Proper fixation, adequate biopsy material with standardized processing is required to yield useful results on FISH.

Visceral stromal sarcoma in a dog: Case report

Visceral stromal sarcoma is a tumor that may cause no symptoms and, when it does, symptoms are unspecific. The tumor is usually an incidental finding, as it is detected while treating unrelated conditions. In this study, we aimed to highlight the importance of complementary exams in diagnosing stromal sarcoma in dogs. A nine-year-old Lhasa Apso dog was admitted into a veterinary hospital with a distended abdomen and decreased defecation. Ultrasound and hematological exams were requested. The hematology test revealed thrombocytosis, neutrophilia, and monocytosis. The ultrasound exam showed the spleen with increased dimensions. Presence of a neoformation occupying the entire left and right mesogastric abdominal topography was observed, apparently originating in the splenic tail, with irregular and coarse shape, mixed echogenicity, vascularized in some portions, and with cavitary areas and irregular walls in others, with anechoic content and echogenic dots in suspension measuring across the vascularized and cavitary portions. Associated with the formation, the mesentery and peritoneum were visible with a slight increase in echogenicity and coarse echotexture. These findings suggested that initial treatment should be surgical, which confirmed that the tumor did not originate in the spleen but in the mesentery. The tumor was removed, and an end-to-end anastomosis was performed at the site the tumor was adhered to. The tumor was taken for a biopsy and multiple nodular mass fragments were found, together measuring 10.0 × 9.0 × 2.5 cm. The inner surface of fragments of the fragments was observed to be whitish-beige, with yellowish areas, smooth and irregular. Biopsy confirmed a diagnosis of visceral stromal sarcoma and the patient was referred to an oncologist. Surgery was the most appropriate treatment in this case due to the size of the tumor, which could compromise the function of other systems. The complementary exam was critical in determining the diagnosis and deciding on the best course of action. In veterinary clinics where the complementary exams are not available, this case could have gone unnoticed.

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. ObjectiveThe goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. MethodsFifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. ResultsBlood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were A-B- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). ConclusionsAlthough C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

P158 Defining distinct fibroblast populations in systemic sclerosis skin using functional characterization and single-cell RNA sequencing

Abstract Background/Aims Fibroblasts isolated by traditional explant culture from skin biopsies in systemic sclerosis (SSc) have a profibrotic and “migratory” phenotype. Single cell RNAseq (scRNAseq) studies suggest other fibroblast subpopulations may be important in SSc pathogenesis. We have isolated a second fibroblast population termed “resident” from whole skin in SSc. We used bulk and scRNAseq to further characterise these populations. Methods Forearm skin from dcSSc (n = 3) and HC (n = 3) was biopsied. “Migratory” fibroblasts were isolated by standard explant culture. The remaining biopsy fragments underwent collagenase digestion to yield a second “resident” fibroblast population. Functional characterization included 3-D collagen gel contraction, migratory scratch-wound assays and western blot comparing expression of pro-collagen I, CTGF and αSMA. Significant difference in gene expression was defined by fold change≥1.5, and adjusted p-value&amp;lt;0.05. scRNAseq was performed on 12 SSc whole skin biopsies, and 3 HC, using the 10X Genomics platform. Results Both SSc fibroblast populations showed a fibrotic phenotype with overexpression of COL1, CTGF and αSMA compared to HC, but with reduced expression αSMA in resident compared to migratory SSc fibroblasts (p &amp;lt; 0.05). RNAseq confirmed 1483 genes being significantly differentially expressed between the SSc fibroblast populations (Table 1). All four populations clustered separately on hierarchical clustering. 10 fibroblast clusters were identified using scRNAseq of whole skin. Of these, cluster 0 and 4 showed greater expression of COL10A1, and STC2 in keeping with migratory fibroblasts. Increased expression of CCL2, and EGR1 were seen in clusters 3 and 6, representing the resident fibroblast population. Gene set enrichment analysis (GSEA) confirmed concordance between KEGG pathway upregulation of the migratory and resident fibroblast clusters and bulk RNAseq analysis. Resident fibroblasts showed upregulation of complement and coagulation genes, and JAK/STAT signalling pathway. There was upregulation of glycolysis gluconeogenesis, and PPAR signalling in migratory fibroblasts. There was agreement in GSEA findings between relevant scRNAseq clusters and bulkRNAseq for resident and migratory subpopulations. Conclusion We have isolated for the first time two distinct profibrotic fibroblast populations from SSc skin with altered expression of key genes associated with SSc pathogenesis. Future work will define their role in SSc pathogenesis, potentially facilitating a more targeted treatment approach. Disclosure K.E. Clark: None. S. Xu: None. V.H. Ong: None. C.D. Buckley: None. C. Denton: None.

Diagnostic challenges in children with celiac disease. A five-year experience with new guidelines in a General Pediatrics Department

Background. The clinical characteristics of patients with celiac disease cover a wide range of signs and symptoms. Our study aimed to describe the main clinical features and investigations performed on our celiac patients diagnosed in the past five years. Material and methods. We collected data from medical records of patients aged 0 to 18 years diagnosed with celiac disease. We included in the analysis patients newly diagnosed with celiac disease, in our department, between January 2017 and August 2022. Results. In this period, 30 children were diagnosed with celiac disease in our general pediatrics department. The mean age at diagnosis ± standard deviation was 6.3±4.2 years (median age=4.3 years). The period between onset of symptoms ranged between 3 months to 5 years (median=6 months). Twenty children (66%) had features of classical celiac disease. Thirteen/30 patients (43.3%) were found to have iron deficiency anemia at diagnosis. In 23/30 (76%) of the children, the values of the IgA-TGA were more than 10 times the upper limit of the normal values. Nineteen children were diagnosed between 2017 and 2019. Fifteen of these 19 patients (79%) were diagnosed based on positive levels of antibodies and duodenal biopsy samples showing Marsh 2 or 3 features, and 4/19 (21%) based on positive IgA-TGA and AEM and a genetic test showing HLA DQ2 and/or DQ8. Conclusions. Classical celiac disease remains the predominant phenotype in our patients. Adherence to new guidelines allowed in the past 2 years a diagnosis without histological examination of biopsy fragments in most of the patients.

Borrelia Infected Neoplastic Glial Cells in Five Patients With Glioblastoma Multiformae

Glioblastoma multiformae (GBM) is a primary malignancy of brain astrocytes. Transcranial stereotactic biopsy specimens from five deceased and anonymous patients with GBM were retrieved from archival storage two years after the death of the patients. All patients were residents of the Long Island, New York metropolitan region where borrelia burgdorferi and borrelia miyamotoi infections are hyperendemic. Fluorescence in situ hybridization (FISH) using two DNA probes specific for genes of burgdorferi borrelia and an additional FISH method study with two DNA probes specific for miyamotoi borrelia each and individually produced high signal borrelia DNA hybridizations inside malignant cells in brain biopsies from all five decedents. Bright signal bacterial DNA hybridizations inside live borrelia spirochetes were found at all levels in serial sections of tumor biopsy fragments in low magnification imaging and outright invasion of the nuclei of individual tumor cells in high resolution imaging. Herein is the first report of a cluster of GBM patients with universal borrelia microbe invasion of neoplastic glial cells.

Prognostic value of xenograft engraftment in patients with metastatic high-risk neuroblastoma.

Successful engraftment of human cancer biopsies in immunodeficient mice correlates with the poor prognosis of patients. This was reported 30 years ago for children with neuroblastoma, but the standard of care treatment evolved significantly during the last 15 years, leading to improved survival of these patients. Here, we evaluated the association of patient-derived xenograft (PDX) engraftment and prognosis in patients receiving up-to-date treatments for cancers classified as metastatic (stage M) high-risk neuroblastoma (HR-NB) by the International Neuroblastoma Risk Group Staging System (INRGSS). We obtained biopsies from patients with stage M HR-NB. We inoculated biopsy fragments subcutaneously in mice. We studied the association of PDX engraftment with event-free survival (EFS) and overall survival (OS) of patients. Since 2009, we established 17 PDX from 97 samples of 66 patients with stage M HR-NB, with a follow-up of at least two years. Factors associated with higher probability of engraftment were the death as outcome (p=.0006) and the amplification of the gene MYCN in tumors (p=.0271). Patients whose biopsies established a PDX had significantly shorter EFS and OS (p=.0039 and .0002, respectively) than patients whose samples did not engraft. The association of PDX engraftment and OS was significant in patients without MYCN amplification (p=.0041), but not in patients with MYCN amplification (p=.2707). Positive PDX engraftment is a factor related to poor prognosis and fatal outcome in patients with stage M HR-NB treated with up-to-date therapies.

Cytoplasmic poly(A)-binding protein 1 as a biomarker to assist early diagnosis and prognosis of esophageal squamous cell carcinoma in endoscopic biopsy fragments.

Esophageal squamous cell carcinoma (ESCC) has high mortality worldwide, but its early diagnosis and prognosis are very difficult. Cytoplasmic poly(A)-binding protein 1 (PABPC1) plays an important role in regulating most cellular processes, resulting in a close relationship to tumor genesis and malignant development. Therefore, this work aimed to evaluate the clinical value of PABPC1 as a biomarker for the early diagnosis and prognosis of ESCC in endoscopic patients. A total of 185 patients with lesions found by endoscopy were involved in this study, including 116 finally diagnosed with ESCCs and 69 with nonmalignant lesions. Biopsy fragments and surgical specimens were collected to assess PABPC1 expression by immunohistochemistry, and the association between the expression and survival was analyzed and compared in both samples. The average ratio of positive tumor cells to total tumor cells in the biopsy fragments was lower than that in surgical specimens, leading to a cutoff value of only 10% for the former in ROC analysis (AOC = 0.808, P < 0.001). However, PABPC1 high expression (PABPC1-HE) in both biopsy fragments and surgical specimens was associated with poor survival. When PABPC1 expression was used as a biomarker to diagnose ESCC in biopsy fragments, sensitivity, specificity, positive predictive value, and negative predictive value reached 44.8, 100.0, 100.0, and 51.9%, respectively. Among the 116 ESCC patients, 32 received postoperative concurrent chemoradiotherapy. Postoperative treatment increased the overall survival (OS) but not disease-free survival in lymph node-positive patients (P = 0.007 and 0.957, respectively). Nevertheless, PABPC1-HE predicted shorter OS regardless of the postoperative treatment in both endoscopic biopsy samples and surgical specimens. PABPC1 expression can be used as a biomarker to detect ESCC from endoscopic lesions. At the same time, PABPC1-HE is a predictor of poor survival regardless of postoperative chemoradiotherapy in endoscopic biopsy samples of ESCC.