Biopsy Material Research Articles

Proteinuria and proximal tubular epithelial cells: correlation between immunofluorescence, histology, and degree of proteinuria

Proteins are filtered from the blood through the glomerular filtration barrier. Filtered proteins are reabsorbed by proximal tubular epithelial cells (PTECs), which have been shown to possess the ability to regulate protein reabsorption. Histologically, these reabsorbed proteins are seen as tubular protein reabsorption droplets (TPRDs). Experimental studies indicate that PTECs play an important role in regulating proteinuria but the correlations between TPRD and the degree of proteinuria in human kidney biopsies have not been investigated in detail. Consecutive native kidney biopsies with non-proliferative glomerular disease performed at the OSUWMC for a 1-year period were analyzed. Cases with acute glomerular diseases and inadequate biopsies were excluded. The staining intensity and the percentage of TPRDs, as well as other morphologic parameters, were assessed. A total of 109 kidney biopsies were included in the study. A reverse correlation was identified between the percentage of albumin TPRDs and proteinuria (p = 0.047). There were positive correlations between proteinuria and the staining intensity for IgG TPRDs (p = 0.05) and the degree of acute tubular necrosis (ATN) (p = 0.015). In patients with no ATN, positive correlations between proteinuria and albumin and IgG TPRDs were seen, whereas in patients with ATN, these correlations were lost. A positive correlation was seen between proteinuria and chronic kidney injury. A strong correlation was noted between the degree of proteinuria and podocyte foot process effacement. Our data indicate that PTECs regulate proteinuria by absorbing proteins from the urine filtrate. Therefore, based on the human renal biopsy material, our study confirms that well-functioning renal PTECs play an important role in the regulation of proteinuria.

Blood cytokines in children with erosive gastritis

Aim: To evaluate the cytokine profile of blood serum in children with erosive gastritis depending on the activity of the inflammatory process, bacterial invasion of H. pylori and family predisposition to peptic ulcer disease. Gastroscopy was performed with the collection of biopsy material from the gastric mucosa in 168 children aged 7-17 years with gastroenterological complaints. Subsequently, a morphological examination of biopsy specimens confirmed the diagnosis of gastritis in all examined patients and determined H. pylori invasion. The content of cytokines in the blood serum (IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, IL-1β, IFNα, TNFα) was determined using the enzyme immunoassay method. When analyzing cytokine levels in schoolchildren infected with H. pylori, there were no differences in cytokine concentrations (p 0.05). While in uninfected children in the presence of erosive changes, a decrease in IL-2 content was noted (p = 0.026). In individuals with a family history of peptic ulcer disease with erosive gastritis, an increase in the content of IL-8 was observed (p = 0.006), which is known to play an important role in maintaining innate immunity. Whereas, in the absence of a family predisposition, schoolchildren with erosive gastritis showed a decrease in IL-2 (p = 0.027), which is similar to the level of IL-2 in schoolchildren with erosive gastritis without H. pylori infection. IL-2 is considered an activator of the antitumor response and this property is being actively studied in patients with gastric cancer. In the context of these data, it can be assumed that in individuals with erosive gastritis, even without a family predisposition and H. pylori infection, inhibition of IL-2 synthesis is observed. What causes this influence is an open question. Thus, the variety of components of the cytokine profile involved in the regulation of the inflammatory process and the influencing negative factors create difficulties in assessing and, even more so, predicting the role and significance of changes in the content of a particular cytokine in the blood serum in children with erosive gastritis.

Blood lactate in children with starged inguinal hernia

Target. To study the level of blood lactate in children with strangulated inguinal hernias (SIH). Material and methods. 46 boys with UPG under the age of 3 years were examined. The examination included anamnesis, examination, laboratory data, including blood lactate, ultrasound, Doppler sonography, examination of biopsy and surgical material. Results. Blood lactate in children of group 1 (up to 12 hours from the moment of strangulation) was 1.8±0.3 mmol/l, in children of group 2 (from 12 to 24 hours) - 2.5±0.2 mmol/l, in children of group 3 (after 24 hours) - 2.8±0.3 mmol/l. All children were operated on. In 6 children, bowel resection was performed; in 6, testicular infarction was detected. Discussion. A significant excess of normal blood lactate levels occurred in children with strangulated inguinal hernias who were admitted later than 12 hours from the moment of strangulation. Conclusion. The study of blood lactate in children with strangulated inguinal hernias can be used in the diagnosis of intestinal and testicular ischemia.

Expression of CDX2, CK20 and CK7 in the epithelium of the gastric mucosa in schoolchildren with CagA H. pylori-associated gastritis

Aim. To study the association of protein expression CDX2, CK20, CK7 in the epithelium of the gastric mucosa in schoolchildren with CagA H. pylori-associated gastritis. Material and methods. Gastroscopy was performed with the collection of biopsy material from the mucous membrane of the antrum and body of the stomach in 89 schoolchildren (7-17 years old) with gastroenterological complaints. The morphological method confirmed the diagnosis of gastritis and determined the presence of H. pylori. Gastric biopsies were examined immunohistochemically to identify the proteins CDX2, CK20, CK7 in the epithelium. Blood was also collected to determine the CagA strain of H. pylori using enzyme immunoassay. Results. In schoolchildren infected with CagA H. pylori, higher expression of CK7 was found in both parts of the stomach (in the body (p = 0.044), in the antrum (p = 0.014)) compared to uninfected children. Changes in the expression of CDX2 and CK20 in children infected with H. pylori CagA have not been established. CDX2 protein was detected more often among schoolchildren aged 12-17 years with H. pylori infection than in children without H. pylori (p = 0.062) and in children with CagA H. pylori (p = 0.017). In the group of younger children with CagA H. pylori, there were higher levels of CK7 expression in the epithelium of the antrum of the stomach, compared with uninfected children (p = 0.033). They also had higher CK7 in the body than in CagA H. pylori-associated gastritis in children of the older age group (p = 0.001). Boys with CagA H. pylori had the highest expression of CK7 in the gastric body, which was higher than in uninfected boys (p = 0.021). Conclusion. Thus, in children, the features of the association of CDX2, CK20, CK7 with CagA H. pylori-associated gastritis have been established, including depending on the age and gender of the child.

Key Anabolic Markers in Human M. Soleus after 21-Day Head-Down Tilt Bedrest

Prolonged bed rest can have a significant negative effect on skeletal muscle, leading to muscle wasting and reduced strength. This process can occur in as little as 10 days in healthy individuals, with the loss of muscle mass and strength being particularly pronounced during the first week of immobilization. Head-down tilt bed rest (HDT) is a method used to simulate the physiological changes that occur in weightlessness during spaceflight. This technique involves lying in bed with the head tilted downward. This paper is dedicated to the analysis of key anabolic markers of the soleus muscle during 21 days of HDT BR. The HDT BR experiment was conducted at the Institute of Biomedical Problems, Russian Academy of Sciences. Six healthy male volunteers, aged 25–35 years, were subjected to 21 days of strict bed rest with a tilt angle of –6°. A needle biopsy of the m.soleus was performed using the Bergström method before the start of HDT BR and on day 21 of HDT BR. The biopsy material was immediately frozen in liquid nitrogen for further Western blot and PCR analysis. Examination of mTORC1 substrates showed a significant decrease in p70 and 4EBP1 phosphorylation after HDT BR. We also observed a significant decrease in the phosphorylation of another ribosomal kinase, p90RSK, a significant increase in eEF2 phosphorylation and an increase in eEF2k mRNA expression. In addition, the phosphorylation of AMPK and its substrate ACC decreased after HDT BR. The data obtained in this work support the hypothesis that a decrease in protein synthesis, together with an increase in proteolysis, contributes to the development of human m. soleus atrophy after 21 days of HDT BR.

Report of 3 Cases Diagnosed with Basal Cell Carcinoma and Systemic Mastocytosis with Favorable Prognosis

Abstract Introduction/Objective The co-existence of basal cell carcinoma (BCC) and systemic mastocytosis is an exceptionally rare clinical occurrence, and the prognosis is not well-documented. Understanding the clinical and histopathological features of this uncommon association is relevant for accurate diagnosis, management, and prognostication. Methods/Case Report We conducted a retrospective analysis of three cases of BCC with concurrent systemic mastocytosis that were identified in the pathology database of the University of Pennsylvania from 2010 to 2023. Patient demographics, clinical and histopathologic characteristics, and treatment outcomes were reviewed. Histopathologic assessment, immunohistochemistry, and C-KIT D816V mutation analysis (with an allele-specific PCR assay) were performed on biopsy material. Next-Generation Sequencing (NGS) was used to identify other hematologic neoplasm-associated mutations. Results (if a Case Study enter NA) All three cases were female with a median age of 55.3 years (50, 69, 47). In all three cases, atypical CD117+ tryptase+ CD25+ mast cells with mild to moderate pleomorphism were found in the dermis adjacent to the BCC cells. All three cases had bone marrow involvement. Serum tryptase levels were mildly elevated in all 3 patients (maximum 65.5 μg/L). All three cases harbor a C-KIT D816V mutation and a history of systemic glucocorticosteroids before the diagnosis of BCC. One of the cases was treated with phototherapy. NGS detected the following somatic mutations: Case #2 DNMT3A p.R882H c.2645G>A; Case #3 JAK2 V617F. Case #1 had multiple relapses of BCC, but a subsequent marrow biopsy did not identify any involvement by mastocytosis. Case #2 had mast cell leukemia, which typically has a poor prognosis; however, this patient has survived for several years. Case #3 had essential thrombocythemia and systemic mastocytosis with an associated hematological neoplasm, and her symptoms were stable. Treatment modalities for the three patients ranged from surgical excision to anti-mastocytosis therapy, with favorable outcomes (Table 1). Conclusion The coexistence of BCC with mastocytosis poses diagnostic challenges due to its infrequency and varied clinical presentations. Previous literature reports on treatments of mastocytosis such as phototherapy and steroids, which have been identified as risk factors for the development of basal cell carcinoma. However, recognizing this unique combination in clinical practice may aid in providing appropriate treatment strategies, ultimately leading to favorable patient outcomes.

Optimization of Turnaround Time and Insufficient Quantity Frequency for Molecular Testing on FFPE Material

Abstract Introduction/Objective With clinical decisions relying progressively more on molecular data extracted from formalin- fixed, paraffin-embedded (FFPE) tissue, it is pivotal to optimize the use of tissue for diagnostic and prognostic purposes. This optimization can be reached by identifying actionable instances that can decrease the turnaround time (TAT) and the rate of “quantity not sufficient” (QNS) results Methods/Case Report From January 2023 to December 2023, the total TAT from the time a molecular study is ordered to its delivery to the molecular lab at LL200A (Tomsich Laboratories), as well as the TAT from the time between the ordering pathologist circling the region of interest to its delivery to LL200A were broken down by subspecialty and site. In addition, the QNS results of Caris genomic profiling were evaluated considering subspecialty and site, amount of material present in the initial sample, and number of levels cut for other ancillary testing from the same material. Results (if a Case Study enter NA) 83% of subspecialties took longer than 48 hours to have material ordered for molecular studies delivered to LL200A. The median TAT for all specialties was 58 hours (average: 71.6 hours). When examining TAT from circling to LL200A, the median time was 19 hours (average: 28.4 hours). Regarding the QNS rate of the Caris assay, most QNS cases were gastrointestinal tract specimens (27%) or specimens obtained from either CT-guided core needle biopsies (30%) or endoscopic biopsies (32%). The longest dimension of QNS cases ranged from 7.5 cm to 0.1 cm (median: 1.3cm)—the average number of slides ordered before Caris ranged from 1 to 52 (median: 12). Conclusion The initial results pointed towards the time from circling to LL200A as one actionable item that could improve the TAT of molecular tests. Possible solutions include enacting a dedicated tumor circling service. In addition, to decrease the rate of Caris QNS results, we propose working together with proceduralists to increase biopsy material yield and to work with pathologists to preserve limited specimens for molecular studies.

Diagnostic Approach to Mesenchymal and Spindle Cell Tumors of the Breast

Mesenchymal and spindle cell tumors of the breast represent a broad and heterogeneous group of lesions that may be sampled on core needle biopsy or surgical excision. Mesenchymal lesions unique to the breast are those that derive from the specialized breast myofibroblast, such as mammary myofibroblastoma and pseudoangiomatous stromal hyperplasia. However, any mesenchymal lesion arising in extramammary soft tissue may also arise in the breast, including fibroblastic, peripheral nerve sheath, adipocytic, and vascular lesions. The spindle cell lesions pose the greatest diagnostic challenge, due to the significant radiographic, morphologic, and immunophenotypic overlap within the category of mesenchymal lesions and more broadly with other nonmesenchymal breast lesions. The distinction is particularly challenging on the limited material of breast core needle biopsies, and caution should be taken before definitively classifying a breast spindle cell lesion on core needle biopsy to avoid unnecessary treatment if misdiagnosed. Consideration of a wide differential diagnosis, adequate sampling of a resection specimen, use of a targeted immunopanel, and selective use of molecular assays are essential steps for accurate classification of mesenchymal lesions in the breast. This review covers the clinical, histologic, and immunophenotypic features of mesenchymal tumors of the breast, with a special emphasis on the differential diagnoses unique to the breast and challenges encountered on breast core needle biopsy.

Unveiling STRs instability in a colorectal cancer FFPE sample: a case report.

In forensic genetics, sometimes formalin-fixed paraffin-embedded (FFPE) biopsy material taken during life is the only biological sample available for individual identification or paternity testing. In most cases, this biological tissue is characterized by the presence of tumor cells characterized by instability and loss of heterozygosity of microsatellites (MSI/LOH) compared to the DNA present in cells of normal tissue.In this case report, two FFPE samples from the same male subject were available for genetic investigation: one sample with colorectal cancer tissue and the other with normal tissue (no cancerous histopathological features). The comparison of the genetic profiles obtained from DNA extracted from the two tissues showed in the tumor tissue the presence of three genomic instability phenomena affecting FGA, CSF1P0, D21S2055 loci, located on three distinct autosomal chromosomes, and one duplication phenomenon affecting the DYS438. Therefore, due to the MSI/LOH phenomena, the genetic profile acquired from the tumor tissue was distorted and thus generated a fictitious genetic profile, not corresponding to the subject's real one (normal tissue free of tumor cells).

Internal Audit of an Oral Pathology Laboratory: Perspectives on Finances and Operational Management.

Background Internal audits are essential tools for enhancing the operational efficiency, quality, and effectiveness of healthcare departments. Audits enable the departments and laboratories to meet the changing needs of the healthcare environment by giving a detailed picture of the department's operations and highlighting areas for possible growth and development. Aims and objectives This study focuses on the biopsies received in the Oral Pathology Department at Saveetha Dental College, aiming to evaluate biopsy trends, financial performance, and resource utilization over one year. Materials and methods The oral pathology department audit covered the period from 1st April 2023 to 31st March 2024. The institutional human ethical committee and scientific review board approved the retrospective audit. It involved a comprehensive analysis of biopsy data, financial records, and material usage. Data on different biopsy types (excisional, incisional, frozen sections), immunohistochemistry, cytology, and special stains were collected and analyzed across four quarters. Financial performance was assessed by comparing total income and expenses, while resource utilization was examined through the use of histopathological blocks and other consumables. Statistical analysis (chi-square) was performed using IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). A P-value less than 0.05 was considered statistically significant. Results We received 1100 cases during the study period. Excisional biopsies were the most common, with 474 (43.09%) cases, followed by incisional biopsies with a total of 432 (39.27%). Out of total cases of 1100, the second quarter (July-September 2023) had the highest case volume of 305 (27.72%), while the third quarter (October-December 2023) recorded the lowest of 250 (22.72%) cases. A financial audit revealed an annual deficit of ₹1,03,321 primarily due to higher expenses towards laboratory reagents. The overall expense incurred per case was ₹448.5. Tissue blocks cost ₹85.23 per case (19.00%) of the average cost per case. The chi-square test analysis was insignificant among the different types of biopsies and the reagent consumption across the four quarters. Conclusion The audit identified critical areas for improvement in both clinical workload and financial management. High volumes of biopsies, but net financial deficits highlight the need for better cost management and resource utilization strategies to maintain sustainability without compromising diagnostic quality.

Calibrating tumor growth and invasion parameters with spectral spatial analysis of cancer biopsy tissues

The reaction-diffusion equation is widely used in mathematical models of cancer. The calibration of model parameters based on limited clinical data is critical to using reaction-diffusion equation simulations for reliable predictions on a per-patient basis. Here, we focus on cell-level data as routinely available from tissue biopsies used for clinical cancer diagnosis. We analyze the spatial architecture in biopsy tissues stained with multiplex immunofluorescence. We derive a two-point correlation function and the corresponding spatial power spectral distribution. We show that this data-deduced power spectral distribution can fit the power spectrum of the solution of reaction-diffusion equations that can then identify patient-specific tumor growth and invasion rates. This approach allows the measurement of patient-specific critical tumor dynamical properties from routinely available biopsy material at a single snapshot in time.

Odontogenic Myxomas Harbor Recurrent Copy Number Alterations and a Distinct Methylation Signature.

Odontogenic myxoma is a rare, benign, and locally aggressive tumor that develops in the tooth-bearing areas of the jaw. The molecular mechanisms underlying odontogenic myxomas are unknown and no diagnostic markers are available to date. The aim of this study was to analyze DNA methylation and copy number variations in odontogenic myxomas to identify new molecular signatures for diagnostic decision-making. We collected a cohort of 16 odontogenic myxomas from 2006 to 2021 located in the mandible (n = 10) and maxilla (n = 6) with available formalin-fixed paraffin-embedded or fresh frozen tumor tissue from a biopsy or resection material. Genome-wide DNA methylation and copy number variation data were generated from 12 odontogenic myxomas using the Illumina Infinium Methylation EPIC array, interrogating >850,000 CpG sites. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) revealed that odontogenic myxomas formed a distinct DNA methylation class. Copy number profiling showed recurrent whole-chromosome gains (trisomies) of chromosomes 5, 8, and 20 in all cases, and of chromosomes 10, 12, and 17 in all except one case. In conclusion, odontogenic myxomas harbor recurrent copy number patterns and a distinct DNA methylation profile, which can be used as an additional diagnostic tool in the appropriate clinical and radiologic context. Further research is needed to explain the genetic mechanisms caused by these alterations that drive these locally aggressive neoplasms.

Systematic Modeling of Risk-Associated Copy Number Alterations in Cancer.

The determination of the cancer prognosis is paramount for patients and medical personnel so that they can devise treatment strategies. Transcriptional-based signatures and subtypes derived from cancer biopsy material have been used in clinical practice for several cancer types to aid in setting the patient prognosis and forming treatment strategies. Other genomic features in cancer biopsies, such as copy number alterations (CNAs), have been underused in clinical practice, and yet they represent a complementary source of molecular information that can add detail to the prognosis, which is supported by recent work in breast, ovarian, and lung cancers. Here, through a systematic strategy, we explored the prognostic power of CNAs in 37 cancer types. In this analysis, we defined two modes of informative features, deep and soft, depending on the number of alleles gained or lost. These informative modes were grouped by amplifications or deletions to form four single-data prognostic models. Finally, the single-data models were summed or combined to generate four additional multidata prognostic models. First, we show that the modes of features are cancer-type dependent, where deep alterations generate better models. Nevertheless, some cancers require soft alterations to generate a feasible model due to the lack of significant deep alterations. Then, we show that the models generated by summing coefficients from amplifications and deletions appear to be more practical for many but not all cancer types. We show that the CNA-derived risk group is independent of other clinical factors. Furthermore, overall, we show that CNA-derived models can define clinically relevant risk groups in 33 of the 37 (90%) cancer types analyzed. Our study highlights the use of CNAs as biomarkers that are potentially clinically relevant to survival in cancer patients.

Dendritic cells and type I and II interferon production in the local microenvironment of sinonasal tumors

The subpopulations of dendritic cells and their functional potential determined by endogenous interferon status in tumor microenvironment is an important stage in tumor-specific T-lymphocytes effector reactions development. In this regard, understanding the patterns of sinonasal neoplasms immune cells changes is important in the context of the search for biomarkers of the tumor process which may open up new opportunities for targeted therapy. This study is the first to provide a comparative description of dendritic cells subsets and the type I and type II interferon production in patients with malignant and benign sinonasal tumors.Tumor tissue from 30 patients with neoplasms – main group (15 patients with malignant tumors and 15 patients with inverted papilloma) and biopsy material of the mucous membrane from 15 patients with polypous rhinosinusitis (comparison group) were used. Tumor-infiltrating immune cells were isolated from tissue using automated and enzymatic cell dissociation. The surface and intracellular cells phenotype was assessed using monoclonal antibodies and flow cytometry. The production of α- and γ-interferons was determined by enzyme-linked immunosorbent assay. Statistical data analysis was proceeded in Statistica 10.0.An increase of tumor-infiltrating myeloid dendritic cells number was found in patients with malignant tumors as compared to control group what correlated with the stage of the pathological process (R = -0.67; p = 0.01). There was an increase in both myeloid and plasmacytoid dendritic cells in the neoplasm tissue in patients with inverted papilloma relative to the comparison group. The microenvironment of malignant growth was characterized by an increase in α- and γ-interferons production in combination with a pronounced decrease in IFNγ-producing T cells percent while a decrease in both intracellular and extracellular production of γ-interferons was detected in tumor tissue of patients with inverted papilloma as compared with control group.In patients with sinonasal tumors changes in dendritic cells subpopulations as well as a decrease in the reserve capacity of γ-interferon synthesis were revealed what may characterize the involvement of these mechanisms in the formation of the antitumor immune response failure and requires further research to establish their pathogenetic role.

Microplastic in Gastric Fasting Liquid and Associated Gastric Pathology.

To determine the presence of microplastics in the stomach, and the relationship between pathological changes in stomach tissue and microplastics. An analytical study. Place and Duration of the Study: Department of Internal Medicine, Sorgun State Hospital, Yozgat, Turkiye, from December 2022 to November 2023. Fasting gastric fluid sampling and endoscopic sampling including mucosal and submucosal layers from the antrum were performed. The pH values of the gastric fluids were recorded. Samples were analysed gradually by adding iron solution, hydrogen peroxide, and sodium chloride (NaCl) in a beaker at 75 degrees for 30 minutes. Biopsy materials obtained from antrum were examined histopathologically and reported according to the Sydney classification. The relationship between gastric biopsy results and the presence of microplastic was evaluated using Chi-square test. The significance level was taken as p <0.005. The study included 61 individuals. The presence of microplastics was detected in 17 (27.86%) gastric fluid samples obtained from the individuals. A significant correlation was found between increased activity and inflammation in antrum biopsy and the presence of microplastic (χ2 = 8.55 p = 0.014; χ2 = 25.75, p = 0.001). The relationship between atrophy, metaplasia, and Helicobacter pylori in gastric tissue and the presence of microplastic was statistically insignificant (p >0.05). Microplastics were detected in gastric fasting fluid. These materials can cause histopathologic changes and inflammation in the gastric antrum. H. pylori, Intestinal metaplasia, Inflammation, Microplastic, Plastic, Sydney classification.

Predictors of intrahepatic cholangiocarcinoma recurrence after surgical treatment

Purpose of the study. To study the CT semiotics of intrahepatic cholangiocarcinoma (ICC) to determine the prognostic markers of recurrence. To analyze the association between CT characteristics of ICC and mutations in IDH1/2, MET, KRAS, BRAF, ERBB2, EGFR, FGFR genes. Materials and methods. We analyzed databases and diagnostic images of Vishnevsky National Medical Research Center of Surgery and Loginov Moscow Clinical Research Center for the period from April 2016 to January 2022 using the key queries «intrahepatic cholangiocarcinoma», «liver», «hepatocellular carcinoma», «metastases», «radio genomics». 142 patients with liver neoplasms were identified, including 90 cases of ICC, 31 cases of hepatocellular carcinoma and 21 cases of metastatic liver lesions, all morphologically verified (histologic and immunohistochemical analysis of biopsy material). Results. Associations between CT features and mutations of MET and IDH1/2 genes were determined. According to the results of statistical analysis all four CT-signs, such as bile duct dilatation, capsule retraction, presence of dropout foci and tissue volume changes, are correlated with the probability of recurrence (death) in patients with ICC. Conclusion. In a retrospective study, our results emphasize the potential prognostic significance of CT signs of ICC. We identified CT signs that allow differential diagnosis of ICC with hepatocellular carcinoma and colorectal cancer metastases. We also identified associations between CT signs of ICC and mutations of IDH1/2 and MET genes, which may allow us to non-invasively obtain data on clinically significant molecular markers of tumors to apply a personalized approach to patient treatment.

A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.

In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.

Assessment of MDM2 Gene Locus Amplification by Fluorescence In-Situ Hybridization in Juvenile Ossifying Fibroma.

With IRB approval, a search of the institutional files of the archives of the Oral Pathology and Surgical Pathology biopsy services at the University of Florida Health was performed. The cases were re-evaluated by an oral pathology resident, an oral and maxillofacial pathologist, and a bone and soft tissue pathologist. Cases with consensus in diagnosis were selected (n = 9) for MDM2 testing. Testing by FISH for MDM2 gene locus amplification was applied to all retrieved cases. The examined cases were all negative for MDM2 gene locus amplification via FISH testing. In our small series, JOF did not demonstrate MDM2 gene locus abnormality, a characteristic of LGIOS. This finding suggests that JOF has a distinct underlying pathogenesis. If confirmed in a larger series, these findings may be useful in distinguishing these two entities in cases with overlapping features or when minimal biopsy material is available.

A Deep Learning Approach for the Identification of the Molecular Subtypes of Pancreatic Ductal Adenocarcinoma Based on Whole Slide Pathology Images

Delayed diagnosis and treatment resistance make pancreatic ductal adenocarcinoma (PDAC) mortality rates high. Identifying molecular subtypes can improve treatment, but current methods are costly and time-consuming. In this study, deep learning models were utilized to identify histological features that classify PDAC molecular subtypes based on routine hematoxylin-eosin (H&E)-stained histopathological slides. 97 histopathology slides associated with resectable PDAC from the Cancer Genome Atlas (TCGA) project were utilized to train a deep learning model and tested the performance on 44 needle biopsy material (110 slides) from a local annotated patient cohort. The model achieved balanced accuracy of 96.19% and 83.03% in identifying the classical and basal subtypes of PDAC in the TCGA and the local cohort, respectively. This study provides a promising method to cost-effectively and rapidly classifying PDAC molecular subtypes based on routine H&E slides, potentially leading to more effective clinical management of this disease.

A clinical case: gastric cancer after gastric bypass

Introduction: Gastric cancer is one of the most common oncological diseases in the world, occupying the 5th place of morbidity and the 3rd place in the structure of mortality from oncological diseases. For a long time, issues affecting the risk of developing cancer after bariatric surgery remain relevant. Our observation is devoted to the diagnosis and treatment of a patient with gastric cancer after gastric bypass. Description of the clinical case: A 62‑year‑old patient was operated on 13. 07. 2016 – laparoscopic Roux gastric bypass for morbid obesity, 11. 09. 2019 – laparoscopic installation of the Cardioplant plate on a small stomach due to recurrent weight gain. Since 2020 the patient had the phenomena of anastomositis, dysphagia and gastroesophageal reflux. Courses of conservative therapy, sessions of balloon dilation of gastroenteroanastomosis and anastomosis resection failed to show a significant effect. The patient underwent comprehensive examinations at each treatment, including abdominal MSCT, cancer markers and studies of biopsy material of the gastric mucosa and gastroenteroanastomosis. As a result of histological studies, no signs of cancer were found. After applying to the Avicenna Medical Center in 2022 a molecular genetic analysis was carried out, in which the mRNA panel most corresponded to a malignant neoplasm. 20. 12. 2022 extirpation of the stomach stump with resection of the esophagus was performed. The cancer diagnosis was confirmed by histological and immunohistochemical studies: low‑grade adenocarcinoma of the stomach with a cricoid component with germination into the esophagus and small intestine, with spread beyond the muscle layer. Conclusion: This clinical case highlights the complexity of oncological verification in patients after bariatric surgery. Prolonged dysphagia, anastomositis and recurrent GERD in such patients determine the need for a more detailed examination, including the latest achievements of molecular genetic analysis.