Biopsy Sections Research Articles

A novel method for making cell blocks with higher cellular yield.

Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from elevenand five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.

Association of gastroesophageal reflux disease with gastritis activity in schoolchildren living in a region with a high incidence of gastric and esophageal cancer

Aim. To study the activity of the inflammatory process in the gastric mucosa (GM) in gastroesophageal reflux disease (GERD) in schoolchildren living in a region with a high incidence of stomach and esophageal cancer. Material and methods. The transverse method in the Republic of Tyva collected data on the presence of gastroenterological complaints in 1535 schoolchildren aged 7-17 years. The main diagnostic criterion for GERD was the presence of weekly heartburn in the subjects. Subsequently, esophagogastroduodenoscopy was performed with the taking of biopsy material of the mucosa of the antrum and body of the stomach in 246 children. Morphological assessment of GM was carried out in accordance with the Sydney classification after staining biopsy sections with hematoxylin-eosin. Diagnosis of Helicobacter pylori was carried out after staining according to Giemsa. The studies were approved by the ethics committee and the consent of the examined was obtained. Results. Schoolchildren in Tyva have a high prevalence of GERD (9.5%). Associative relationship of GERD with gastritis activity, both in the body (p=0.7338) and in the antrum (p=0.2036) was not revealed. In the antrum, the activity of gastritis was higher than in the body of the stomach (p=0.0001). In children with GERD, when infected with Helicobacter pylori, there was an increase in activity, both in the antrum (p=0.0001) and in the body of the stomach (p=0.0159). At the same time, in children with GERD, the inflammatory process is associated with infection by a microorganism, regardless of its belonging to the Cag A strain. Conclusion. An association of GERD with highly active Helicobacter pylori associated gastritis has been established, regardless of whether the microorganism belongs to the Cag A strain.

Case Series: Large B-cell Lymphoma with IRF4 Rearrangement Involving the Lung and the Tonsil in Two Different Patients

Abstract Introduction/Objective Large B-cell lymphoma (LBCL) with IRF4 rearrangement is a new entity described in the 2017 WHO classification. It is characterized by a monotonous proliferation of medium to large sized cells with strong expression of IRF4/MUM1 and IRF4 rearrangement. It accounts for only 0.05% of all diffuse LBCLs and affects predominantly children and young adult with median age of 12 years. The reported sites of disease include lymph nodes of head and neck, Waldeyer ring and gastrointestinal tract. Here, we report two cases of Large B-cell Lymphoma with IRF4 rearrangement in two female patients, 73-year old and 71-year old respectively. The first patient presented with a lung nodule, while the second presented with right palatine tonsillitis. Hematoxylin and Eosin staining, immunohistochemistry and Fluorescence In-Situ Hybridization were used to characterize the lesions. H&E stained sections of the lung nodule biopsy from the first patient showed diffuse proliferation of medium- to large-sized cells (Figures A and B). Immunohistochemistry shows tumor cells are positive for CD20 (figure C), CD10, BCL6, and IRF4/MUM1(figure D). FISH study shows that tumor cells carry the IRF4 rearrangement but are negative for MYC, BCL2, and BCL6 translocations. To the best of our knowledge, this is the first case of LBCL with IRF4 rearrangement reported involving the lung in an older patient. H&E stained sections of the right palatine tonsil from the second patient shows effacement of the normal architecture which is replaced by sheets of large lymphoid cells. Immunohistochemistry shows tumor cells positive for CD20, CD10, BCL-6, BCL-2, MUM-1, C-MYC, and Ki-67 (80%). FISH study shows that tumor cells carry the IRF4 rearrangement which is consistent with a diagnosis of LBCL with IRF4 rearrangement. Methods/Case Report NA. Results (if a Case Study enter NA) NA. Conclusion NA.

Ovarian Cancer Metastasis to Lung Parenchyma without Peritoneal Dissemination: A Rare Case

Ovarian cancer is the most lethal gynecological malignancy worldwide. Despite the advances in treatment for ovary cancer, the 5-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains as high as 30%. Clinical stage is the most important prognostic factor and most patients are at an advanced stage. There is often no clearly identifiable precursor lesion; therefore, the events which lead to metastatic disease are poorly understood. Solitary lung parenchyma metastasis is extremely rare. We report a case of 39-year-old woman who was admitted with a lung mass. She had a history of high-grade serous carcinoma ovary, which was detected and treated 6 years ago with cytoreductive surgery as well ashyperthermic intraperitoneal and adjuvant chemotherapy. During the routine follow-up, positron emission tomography computed tomography scan showed two nodules of soft-tissue density in the lung parenchyma of the left lower lobe with a dimension of 2.8 × 2.5 cm. Intraoperatively, two conglomerated lung nodules seen in lung parenchyma of the left lower lobe. Deeper nodule was in close proximity to the left lower lobe bronchus and left lower lobe pulmonary artery. Video-assisted thoracoscopic surgery was conducted to perform left lower lobectomy. Postoperatively, the patient was stable. Microscopically, the tumor was classified as a high-grade serous carcinoma, and the patient was diagnosed for having an ovarian cancer metastasis. Ovarian cancer rarely metastasizes to the lung parenchyma in a relapse. However, excision of lesion with further chemotherapy showed excellent results. Surgical excision biopsy and intraoperative frozen sectioning, followed by lobectomy or segmentectomy, are both diagnostic and therapeutic.

Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression.

Hypertensive nephropathy (HN) requires a kidney biopsy as diagnostic gold-standard but histological findings are unspecific and specific prognostic markers are missing. We aimed at identifying candidate prognostic markers based on glomerular protein signatures. We studied adult patients (n = 17) with eGFR >30 ml/min/1.73m2 and proteinuria <3 g/d from the Norwegian Kidney Biopsy Registry, including subjects non progressing (NP, n = 9), or progressing (P, n = 8) to end-stage renal disease (ESRD) within an average follow-up of 22 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction. Proteomic analyses were performed using Q-exactive HF mass spectrometer and relative glomerular protein abundances were compared between P and NP patients. Immunohistochemistry (IHC) was used to validate selected data. Amongst 1870 quality filtered proteins, 58 were differentially expressed in P and NP patients' glomeruli, with absolute fold changes (FC) ≥1.5, p ≤ 0.05. Supervised classifier analysis (K nearest neighbor) identified a set of five proteins, including Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in P, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in NP glomeruli, correctly classifying 16/17 kidney biopsy samples. Geneset Enrichment Analysis (GSEA), showed that metabolic pathways were generally enriched in P, and structural cell pathways in NP. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. IHC analysis confirmed overexpression of BBOX1 and Cadherin 16 in glomeruli from P patients. In conclusion, glomerular proteomic profiling can be used to discriminate P from NP HN patients.

Grade of liver siderosis in beta-thalassaemia major patients receiving different amount of blood transfusion

Background and objectives: A progressive accumulation of body iron easily occurs as a result of long-term transfusions in patients with anaemia of genetic disorders such as thalassaemia. Iron deposit in liver biopsy sections was studied in beta-thalassaemia major patients to assess the grade of liver siderosis and to correlate the grade with amount of blood transfused. Materials and methods: Beta-thalassaemia major patients having splenomegaly and selected for splenectomy were enrolled. Liver biopsy was taken from every patient during the splenectomy. Liver tissue was sectioned and stained with Perls’ prussian blue method for the presence of iron deposition. The degree of iron deposition was expressed as grades of siderosis from 0 to 4. Results: A total of 30 beta-thalassemia patients were enrolled in the study. Out of 30 patients, 7 were males (23.3%) and 23 females (76.7%). The mean age of patients was 15.2 ± 1.4 years. The mean serum iron and ferritin levels of the study cases were above the normal range. Blood received by all patients was 51.5 ± 11.6 units (range 31 to 88 units). Out of 30 patients, grade 1, 2, 3 and 4 liver siderosis was present in 1, 3, 9 and 17 patients respectively. Serum ferritin level of patients with grade 4 siderosis was significantly higher (p = 0.03) compared to grade 3 cases. Pearson’s correlation coefficient test revealed significant positive correlation between grades of liver siderosis and amount of blood transfusion received (0.626, p &lt; 0.01). Conclusion: Grade of liver siderosis is associated with increased units of blood transfusion and is a good indicator for transfusional iron overload in beta-thalassaemia major patients. IMC J Med Sci. 2023. 17(1): 004. DOI: https://doi.org/10.55010/imcjms.17.004 *Correspondence: Dr. Souvik Basak, Department of General Surgery, Medical College, Kolkata, West Bengal, India. Email: sb009cmc@gmail.com

An experimental challenge model for Leishmania donovani in beagle dogs, showing a similar pattern of parasite burden in the peripheral blood and liver.

Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-022-07681-7.

S2350 Mind the Steroids: Herpes Esophagitis in an Immunocompetent Patient After Steroid Therapy

Introduction: Infective esophagitis (IE) is rare, but well known to complicate the immunosuppressed state; particularly the HIV infected, transplant recipient, cancer patient and those treated with chronic corticosteroids or immunomodulators. Common infections include candida, cytomegalovirus, and herpes simplex virus (HSV). Steroids have been used for symptomatic relief of an acute sore throat. Short courses are considered benign with low risk of infectious complications. We report an immunocompetent patient found to have herpes esophagitis after a short course of corticosteroids. Case Description/Methods: A 41-year-old African American male with history of hypertension presented with worsening sore throat and odynophagia restricting oral intake. Prior to presentation, he had a 10-day history of sore throat. He was diagnosed with streptococcal pharyngitis and was on antibiotics and steroids 7 days ago. Given persistent symptoms he underwent laryngoscopy which demonstrated bilateral tonsillar exudates and normal pharyngeal mucosa, prompting admission and initiation of IV antibiotics and dexamethasone. He had modest improvement of pain, however 8 days later expressed worsening burning retrosternal chest pain and progressed oral intolerance. He underwent upper endoscopy revealing multiple circumferential erosions and Los Angeles grade D esophagitis of the lower third of the esophagus (Figure A). Biopsies demonstrated viral intranuclear inclusions that stained positive for HSV1/HSV2 (Figure B, C). His symptoms abated while on IV acyclovir allowing transition to oral valacyclovir. Discussion: Corticosteroids are well known for their anti-inflammatory effect but immunosuppression with short courses may be underestimated. Infectious sequalae such as herpetic esophagitis may complicate steroid use even when therapy duration is short. This patient was found to have progressive odynophagia while on corticosteroids administered for analgesia and had significant improvement after antiviral therapy. It is easy to overlook IE as a cause of odynophagia in the immunocompetent, however, with history of steroid use, a diagnosis of IE, especially HSV esophagitis, should be considered in the differential. Therefore, one should have a low threshold for early endoscopy when evaluating these patients.Figure 1.: A: Endoscopic photograph displaying multiple circumferential erosions and Los Angeles grade D esophagitis of the lower third of the esophagus. B: (x200): Photomicrograph of hematoxylin and eosin-stained section of the esophageal biopsy, showing multiple multinucleated epithelial cells. Cowdry type A is observed. C: (x200): IHC study using antibodies against HSV-1. Numerous intranuclear viral inclusions are positive with HSV-1 within the infected cells.

Prognostic Implications of a Morphometric Evaluation for Chronic Changes on All Diagnostic Native Kidney Biopsies.

Semiquantitative visual inspection for glomerulosclerosis, interstitial fibrosis, and arteriosclerosis is often used to assess chronic changes in native kidney biopsies. Morphometric evaluation of these and other chronic changes may improve the prognostic assessment. We studied a historical cohort of patients who underwent a native kidney biopsy between 1993 and 2015 and were followed through 2021 for ESKD and for progressive CKD (defined as experiencing 50% eGFR decline, temporary dialysis, or ESKD). Pathologist scores for the percentages of globally sclerosed glomeruli (GSG), interstitial fibrosis and tubular atrophy (IFTA), and arteriosclerosis (luminal stenosis) were available. We scanned biopsy sections into high-resolution images to trace microstructures. Morphometry measures were percentage of GSG; percentage of glomerulosclerosis (percentage of GSG, ischemic-appearing glomeruli, or segmentally sclerosed glomeruli); percentage of IFTA; IFTA foci density; percentage of artery luminal stenosis; arteriolar hyalinosis counts; and measures of nephron size. Models assessed risk of ESKD or progressive CKD with biopsy measures adjusted for age, hypertension, diabetes, body mass index, eGFR, and proteinuria. Of 353 patients (followed for a median 7.5 years), 75 developed ESKD and 139 experienced progressive CKD events. Visually estimated scores by pathologists versus morphometry measures for percentages of GSG, IFTA, and luminal stenosis did not substantively differ in predicting outcomes. However, adding percentage of glomerulosclerosis, IFTA foci density, and arteriolar hyalinosis improved outcome prediction. A 10-point score using percentage of glomerulosclerosis, percentage of IFTA, IFTA foci density, and any arteriolar hyalinosis outperformed a 10-point score based on percentages of GSG, IFTA, and luminal stenosis >50% in discriminating risk of ESKD or progressive CKD. Morphometric characterization of glomerulosclerosis, IFTA, and arteriolar hyalinosis on kidney biopsy improves prediction of long-term kidney outcomes.

Clinical feature and sural biopsy study in nitrous oxide-induced peripheral neuropathy.

ObjectiveThe objective was to analyze the clinical characteristics and pathological characteristics of sural biopsy in nitrous oxide (N2O) -induced peripheral neuropathy.MethodsWe recruited 18 patients with N2O abuse-induced neurological disorders and reported their demographic data, clinical manifestations, laboratory examinations, and nerve conduction studies. Seven patients underwent sural nerve biopsy pathologic examination.ResultsAll 18 patients had polyneuropathy, the nerve conduction results showed significant reductions in motor and sensory amplitudes, slowing of conduction velocities, and prolongation of latencies in most tested nerves compared to the controls. Toluidine blue staining of semi-thin sections of sural nerve biopsy showed decreased myelinated nerve fiber density, increased thin myelinated nerve fiber density, and axonal regeneration. Electron microscopy showed axonal degeneration and nerve regeneration.ConclusionThe main manifestations of peripheral nerve damage caused by the abuse of N2O are lower limb weakness and distal sensory disorder. The nerve conduction study results demonstrated that mixed axonal and demyelinating neuropathy was the most common type of neuropathy. Sural biopsy showed the main pathological change was chronic axonal degeneration.

Human IRP1 Translocates to the Nucleus in a Cell-Specific and Iron-Dependent Manner.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with mutually exclusive RNA-binding or enzymatic activities that depend on the presence of a 4Fe-4S cluster. While IRP1 is a well-established cytosolic protein, work in a Drosophila model suggested that it may also exhibit nuclear localization. Herein, we addressed whether mammalian IRP1 can likewise translocate to the nucleus. We utilized primary cells and tissues from wild type and Irp1−/− mice, as well as human cell lines and tissue biopsy sections. IRP1 subcellular localization was analyzed by Western blotting, immunofluorescence and immunohistochemistry. We did not detect presence of nuclear IRP1 in wild type mouse embryonic fibroblasts (MEFs), primary hepatocytes or whole mouse liver. However, we observed IRP1-positive nuclei in human liver but not ovary sections. Biochemical fractionation studies revealed presence of IRP1 in the nucleus of human Huh7 and HepG2 hepatoma cells, but not HeLa cervical cancer cells. Importantly, nuclear IRP1 was only evident in iron-replete cells and disappeared following pharmacological iron chelation. These data provide the first experimental evidence for nuclear IRP1 expression in mammals, which appears to be species- and cell-specific. Furthermore, they suggest that the nuclear translocation of IRP1 is mediated by an iron-dependent mechanism.

Clinical and metabolic features of diabetic encephalopathy

BACKGROUND: Actual mindset, concerning the pathogenesis of type 2 diabetes mellitus and its complications, is described in the glucocentric, lipocentric and lipokine theories. At the same time, it is known, that acute cerebrovascular disorder in type 2 diabetes mellitus can also develop with normoglycemia. The mechanism of association between obesity and type 2 diabetes mellitus in metabolic syndrome has not yet been studied, and 40% of diabetic patients are not obese. In the physiological state hyperglycemia is prevented by pancreatic regulatory oligopeptides rather than insulin, and protein glycation occurs before the development of diabetes symptoms. That facts indicate the need to continue investigation of the pathogenesis in general and diabetic encephalopathy in particular from the standpoint of metabology in order to substantiate the peptidergic theory of the diabetes mellitus and dyscirculatory encephalopathy pathogenesis.&#x0D; AIM: To consider little-known clinical, morphological and metabolic manifestations of diabetic encephalopathy from the standpoint of improving the disease diagnosis.&#x0D; MATHERIALS AND METHODS: Among 162 elderly and senile patients in the pre-stroke and stroke stages of diabetic encephalopathy, modern methods of radiological, laboratory, histological, psychometric and clinical analysis have been used: the relationship between low total cholesterol and disease progression has been determined according to Schulte and MMSE psychometric tests with the same hyperglycemia in different age groups sick; cases of amyloid angioencephalopathy on magnetic resonance imaging in the ultrasensitive weighted imaging mode and amyloidosis have been identified.&#x0D; Staining post-mortem ultrathin tissue biopsy sections with Congo red allowed to find amyloid deposition in 12% of autopsy cases in the absence of diagnosing amyloidosis during life; preparations, in which Congo red staining was positive for the presence of amyloid, were taken for inspection in polarizing light and typing of amyloid. Staining of histological preparations with hematoxylin-eosin and Van Gieson revealed a morphological characteristic of diabetic encephalopathy different from that of atherogenic and hypertensive dyscirculatory encephalopathy; using immunological studies, a statistically significant increased content of induced interleukin-1 production, a trigger for the serum amyloid analogue in the liver synthesis, was determined, and an experimental method for beta-amyloid peptide adhesion by monocytes and the density of amyloid expression on the surface of monocytes in patients with diabetic encephalopathy at senile age at 23 times higher than the density of the peptide in the elderly and in the patients with dyscirculatory encephalopathy of the same old age.&#x0D; RESULTS: It has been established that a fairly frequent and early manifestation of metabolic disorders in diabetic encephalopathy is a violation of protein metabolism with its final conformation into toxic amyloid tissue components. These observations indicate the multifactorial nature of diabetic encephalopathy. Literature data on the parametabolism of the serotonin mediator and tryptophan in the pathogenesis of diabetic encephalopathy are presented.&#x0D; CONCLUSIONS: Modern immunological, morphological, and histochemical capabilities make it possible to diagnose the conformation of the protein matrix and develop a protein-centric hypothesis of diabetic encephalopathy.

Direct proteomic and high-resolution microscopy biopsy analysis identifies distinct ventricular fates in severe aortic stenosis

The incidence of aortic valve stenosis (AS), the most common reason for aortic valve replacement (AVR), increases with population ageing. While untreated AS is associated with high mortality, different hemodynamic subtypes range from normal left-ventricular function to severe heart failure. However, the molecular nature underlying four different AS subclasses, suggesting vastly different myocardial fates, is unknown. Here, we used direct proteomic analysis of small left-ventricular biopsies to identify unique protein expression profiles and subtype-specific AS mechanisms. Left-ventricular endomyocardial biopsies were harvested from patients during transcatheter AVR, and inclusion criteria were based on echocardiographic diagnosis of severe AS and guideline-defined AS-subtype classification: 1) normal ejection fraction (EF)/high-gradient; 2) low EF/high-gradient; 3) low EF/low-gradient; and 4) paradoxical low-flow/low-gradient AS. Samples from non-failing donor hearts served as control. We analyzed 25 individual left-ventricular biopsies by data-independent acquisition mass spectrometry (DIA-MS), and 26 biopsies by histomorphology and cardiomyocytes by STimulated Emission Depletion (STED) superresolution microscopy. Notably, DIA-MS reliably detected 2273 proteins throughout each individual left-ventricular biopsy, of which 160 proteins showed significant abundance changes between AS-subtype and non-failing samples including the cardiac ryanodine receptor (RyR2). Hierarchical clustering segregated unique proteotypes that identified three hemodynamic AS-subtypes. Additionally, distinct proteotypes were linked with AS-subtype specific differences in cardiomyocyte hypertrophy. Furthermore, superresolution microscopy of immunolabeled biopsy sections showed subcellular RyR2-cluster fragmentation and disruption of the functionally important association with transverse tubules, which occurred specifically in patients with systolic dysfunction and may hence contribute to depressed left-ventricular function in AS.

Abstract A40: Evaluation of the tumor microenvironment in mycosis fungoides using the PanCancer IO 360™ assay and nCounter® platform

Abstract BACKGROUND: Alterations in the tumor microenvironment (TME) can result in a “T-cell inflamed” TME, which is characterized by activation of the PD1/PD-L1 pathway, immunosuppression, and production of anergic, “exhausted” CD8(+)-T-cells. Similar alterations in the TME of Mycosis Fungoides (MF) have been linked to disease progression. However, the relationship between progression and the TME in MF remains incompletely understood. We aimed to further study the TME in MF using the PanCancer IO 360™ 750-gene expression panel and nCounter® platform (Nanostring; Seattle, WA). DESIGN: Clinical and laboratory data from the electronic medical record were reviewed in combination with microscopic review of biopsies from 27 patients to confirm diagnosis of MF and assign samples as “low-grade” (patch/plaque; n=8) or “high-grade” (tumor/large cell transformation, n=19). Patients were also classified as having “limited stage” (TNMB1 &amp;lt;1B, n=8) or “advanced stage” (TNMB1 &amp;gt;1B, n=19) disease. All patients received prior, heterogenous treatment, but no patients received prior anti-PD1 therapy. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE), punch biopsy sections and analyzed per manufacturer’s instructions. Gene expression scores (log2) and a “tissue inflammation score” (TIS) were calculated for each sample. A TIS of &amp;gt;5 has been associated with the presence of a “T-cell inflamed” microenvironment. Samples scores were compared by histologic group and disease stage as described above. RESULTS: Twenty-nine of 30 samples had sufficient RNA integrity for analysis. TIS was &amp;gt;5 in 26/30 samples (median, 7.6; range, 4.3-9.3). No significant differences in TIS were identified between low-grade/limited-stage and high-grade/advanced-stage samples. High-grade samples had significantly increased expression of PD1 and higher amounts of exhausted CD8(+)-cells than low-grade samples (p= 0.015, 0.027). Low-grade samples showed significantly higher expression of ARG1, B7-H3, and genes associated with tissue hypoxia. Expression of genes associated with tumor proliferation was higher in high-grade lesions (p= 0.0019). Limited stage samples showed higher levels of endothelial cells, mast cells, and T-regulatory cells (p=0.033, 0.024, 0.004,) than advanced stage samples. Proliferation, PD1, and exhausted CD8 scores trended higher in advanced stage disease but did not reach statistical significance. CONCLUSION: Our findings indicate that the IO 360™ panel provides comprehensive analysis of the TME in MF. Most samples showed a TIS of &amp;gt;5, suggesting a T-cell inflamed TME in MF. Increased PD1 and exhausted CD8(+) cells were found in high-grade lesions, supporting a role for this pathway in progression. Increased expression of factors associated with angiogenesis (endothelial cells, ARG1) and immunosuppression (B7-H3, tissue hypoxia, mast cells, T-regulatory cells) suggest these mechanisms may play a role in early stages of disease. These findings highlight the complex and dynamic nature of the TME during MF progression, and the need for further study. Citation Format: Stanton Miller, Akshat Patel, Kiran A Kumar, Farrukh Awan, Heather W Goff, Travis Vandergriff, Marisa Juntilla, Franklin Fuda, Weina Chen, Jesse M Jaso. Evaluation of the tumor microenvironment in mycosis fungoides using the PanCancer IO 360™ assay and nCounter® platform [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A40.

Comprehensive Analysis of Fibroblast Activation Protein Expression in Interstitial Lung Diseases.

Rationale: Sustained activation of lung fibroblasts and the resulting oversynthesis of the extracellular matrix are detrimental events for patients with interstitial lung diseases (ILDs). Lung biopsy is a primary evaluation technique for the fibrotic status of ILDs, and is also a major risk factor for triggering acute deterioration. Fibroblast activation protein (FAP) is a long-known surface biomarker of activated fibroblasts, but its expression pattern and diagnostic implications in ILDs are poorly defined. Objectives: The present study aims to comprehensively investigate whether the expression intensity of FAP could be used as a potential readout to estimate or measure the amounts of activated fibroblasts in ILD lungs quantitatively. Methods: FAP expression in human primary lung fibroblasts as well as in clinical lung specimens was first tested using multiple experimental methods, including real-time quantitative PCR (qPCR), Western blot, immunofluorescence staining, deep learning measurement of whole slide immunohistochemistry, as well as single-cell sequencing. In addition, FAP-targeted positron emission tomography/computed tomography imaging PET/CT was applied to various types of patients with ILD, and the correlation between the uptake of FAP tracer and pulmonary function parameters was analyzed. Measurements and Main Results: Here, it was revealed, for the first time, FAP expression was upregulated significantly in the early phase of lung fibroblast activation event in response to a low dose of profibrotic cytokine. Single-cell sequencing data further indicate that nearly all FAP-positive cells in ILD lungs were collagen-producing fibroblasts. Immunohistochemical analysis validated that FAP expression level was closely correlated with the abundance of fibroblastic foci on human lung biopsy sections from patients with ILDs. We found that the total standard uptake value (SUV) of FAP inhibitor (FAPI) PET (SUVtotal) was significantly related to lung function decline in patients with ILD. Conclusions: Our results strongly support that invitro and invivo detection of FAP can assess the profibrotic activity of ILDs, which may aid in early diagnosis and the selection of an appropriate therapeutic window.

IMPACTED TOOTH ASSOCIATED WITH COMPOUND ODONTOMA

Compound odontomas are benign tumors composed of odontogenic tissue that has an orderly pattern. This report describes the case of a 13-year-old boy who was referred for evaluation due to the lack of dental eruption. Intraoral examination revealed the absence of a permanent left central incisor and swelling in the region. The radiographic examination revealed the presence of a lesion in the left premaxilla and the impacted left permanent central incisor. Considering the clinical and radiological examinations, a preliminary diagnosis of compound odontoma was made. On histologic examination, sections of excisional biopsy led to the diagnosis of compound odontoma. The treatment plan included removal of the tumor and extraction of the impacted tooth. To minimize side effects, especially when odontoma is localized in an area with a great impact on facial esthetics, early diagnosis and proper treatment are very important.

LB1030 Automated image analysis methods to detect mast cells in skin

Mast cells are a key cell in the innate immune system. These cells are tissue resident and are considered to act as sentinels for the detection of a multitude of different ligands. Activation of mast cells via stimulation of IgE receptors underlies allergic reactions to food, pollen, pet dander, and many other allergens. Stimulation of mast cells results in their activation leading to degranulation – a process in which the cell releases extracellular mediators like histamine, proteases, and cytokines. These mediators can then go on to activate other cell types nearby or can recruit more immune cells to the tissue. Some chronic pruritic diseases are thought to involve mast cells in the skin and many studies have demonstrated an increase in total mast cell number in skin from atopic dermatitis (AD) or allergic contact dermatitis (CAD). Previous methods relied on staining of skin biopsy sections with a chromogenic stain (like toulidine blue), then manual counting of the mast cells in small sections of the biopsy. More recently, the use of fluorescently-tagged avidin has been used to detect mast cells in skin biopsies, which represents a more specific detection method. A challenge still remains in the differentiation between quiescent (resting) and active (or degranulated) mast cells. Here we describe a method for the automated detection and quantification of quiescent and resting mast cells based on cell morphology, using commercially available image software analysis tools. Our method was applied to human skin biopsies that were treated to induce degranulation and to skin biopsies collected from AD patients. In contrast to earlier results, we do not observe an increase in total mast cell number nor in the number of active mast cells in the skin of AD patients. This method allows for the unbiased detection of mast cells in human skin with out the need to develop de novo proprietary algorithms.

Clinicopathological Correlation in Dermatopathology

Introduction and aim: Getting an accurate diagnosis in dermatopathology requires a combination of clinical and pathological information. Our aim was to assess the importance of the correlation between clinical and histopathological diagnosis in various cutaneous diseases. Materials and Methods: One-hundred cases of variable skin diseases were studied clinically by dermatologist and histopathologically by general pathologists. Cliniopathological correlation was done by direct communication between the dermatologist and the pathologist. Results: Concordance between pathological and clinical diagnoses was seen in 84%, a new pathological diagnosis was issued in 3% and non-diagnostic report in 13 %. After clinicopathological correlation; out of 84%, we accept only 65% as final diagnosis. In addition, the correlation was helpful and provide a diagnosis in another 20% of cases. One new pathological diagnosis was accepted whereas in the remaining cases (14%) further workup were required. Conclusions: Accurate diagnosis of cutaneous disorders depends on clinical skills as well as careful histopathological assessment and best results can be obtained if the patient and biopsy sections are viewed together. Electron microscopy, immunofluorescence assay, immunohistochemistry techniques and molecular pathology are important adjuncts for definite diagnosis of many skin diseases.

Prognostic and Predictive Relevance of Tumor-Infiltrating Lymphocytes in Squamous Cell Head-Neck Cancer Patients Treated with Radical Radiotherapy/Chemo-Radiotherapy.

Microenvironmental conditions control the entrance and thriving of cytotoxic lymphocytes in tumors, allowing or preventing immune-mediated cancer cell death. We investigated the role of tumor-infiltrating lymphocyte (TIL) density in the outcome of radiotherapy in a series of squamous cell head–neck tumors (HNSCC). Moreover, we assessed the link between markers of hypoxia and TIL density. One-hundred twenty-one patients with HNSCC treated prospectively with radical radiotherapy/chemo-radiotherapy were analyzed. The assessment of TIL density was performed on hematoxylin and eosin biopsy sections before radiotherapy. TIL density ranged from 0.8 to 150 lymphocytes per ×40 optical field (median 27.5). Using the median value, patients were grouped into two categories of low and high TIL density. Early T-stage tumors had a significantly higher TIL density (p < 0.003), but we found no association with N-stage. Overexpression of HIF1α, HIF2α, and CA9 was significantly linked with poor infiltration by TILs (p < 0.03). A significant association of high TIL density with better disease-specific overall survival and improved locoregional relapse-free survival was noted (p = 0.008 and 0.02, respectively), which was also confirmed in multivariate analysis. It is concluded that HNSCC phenotypes that allow for the intratumoral accumulation of lymphocytes have a better outcome following radical radiotherapy/chemo-radiotherapy. Intratumoral-activated HIF- and CA9-related pathways characterize immunologically cold tumors and may be used as targets for therapeutic interventions.

Abstract SY25-02: Double-edged “soil”: Stromal microenvironment in breast cancer development

Abstract SY25-02: Double-edged “soil”: Stromal microenvironment in breast cancer development