Formalin-fixed Tissue Samples Research Articles

Use of Carrot Oil, Olive Oil, Pine Oil, Rose Oil in Comparison to Xylene as Clearing Agent During Tissue Processing: an in Vitro Study

ABSTRACT: Background: Clearing plays a vital role in histological section preparation. Xylene has long been valued for its ability to efficiently deparaffinize and clear tissue samples. However, its hazardous properties have raised health and environmental concerns, leading researchers to seek alternative solvents for tissue processing. This study utilized safer substitutes to address these concerns. Aim: To assess the clearing ability of four different oils i.e., Carrot oil, Olive oil, Pine oil, and Rose oil in comparison with that of xylene. Materials and Methods: Thirty different formalin-fixed tissue samples were cut into five parts and cleared using Carrot oil, Olive oil, Pine oil, Rose oil, and xylene followed by routine processing, sectioning, and staining steps. Presence or absence of shrinkage, ease of cutting, nucleus, cytoplasm details and staining quality were assesed and comparison was done between each of them cleared with the 4 different oils with that cleared using xylene. Results: The results showed that all four oils cleared tissues as effectively as xylene, with Pine oil superior in nucleus and cytoplasm details, as well as staining quality insignificant number of samples. Conclusion: These oils not only more affordable and eco-friendly than xylene but can also serve as effective clearing agents.

Phenotypic and Genotypic Features of the FAN1 Mutation-Related Disease in a Large Hungarian Family.

Pathogenic variants in the FAN1 gene lead to a systemic disease with karyomegalic interstitial nephritis (KIN) at the forefront clinically. The phenotypic-genotypic features of a FAN1 mutation-related disease involving five members of a Hungarian Caucasian family are presented. Each had adult-onset chronic kidney disease of unknown cause treated with renal replacement therapy and elevated liver enzymes. Short stature, emaciation, latte-colored skin, freckles, and a hawk-like nose in four patients, a limited intellect in two patients, and chronic restrictive lung disease in one patient completed the phenotype. Severe infections occurred in four patients. All five patients had ceased. Four patients underwent autopsy. KIN and extrarenal karyomegaly were observed histologically; the livers showed no specific abnormality. The genotyping using formalin-fixed tissue samples detected a hitherto undescribed homozygous FAN1 mutation (c.1673_1674insT/p.Met558lfs*4; exon 5) in three of these patients and a heterozygous FAN1 mutation in one patient. The reason for the heterozygosity is discussed. In addition, 56 family members consented to the screening for FAN1 mutation from which 17 individuals proved to be heterozygous carriers; a blood chemistry evaluation of their kidney and liver function did not find any abnormality. The clinical presentation of FAN1-related disease was multifaceted, and not yet described manifestations were observed besides kidney and liver disease. Mutation in this gene should be suspected in adults with small kidneys of unknown cause, elevated liver enzymes, and recurrent infections, even without a family history.

Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

Alterations in tumor immune microenvironment and progressive immune exhaustion with advancing penile squamous cell carcinoma using multiplex immunofluorescence and image analysis.

9 Background: Penile squamous cell carcinoma (PSCC) is a rare and aggressive malignancy with limited treatment options in the advanced or recurrent settings. Immunotherapy can yield clinical responses in PSCC, but less than 20% of patients benefit. Herein, using multiplex immunofluorescence (MIF), we analyzed the composition of the tumor immune microenvironment as it varies with human papillomavirus (HPV) status and across advancing disease stages with an aim to further our understanding of how immune composition may impact clinical outcome, potential role in disease progression and response to immune therapies. Methods: Formalin-fixed and paraffin-embedded tissue samples from men with PSCC treated at Moffitt cancer center (Tampa, FL) were immunostained for CD3, CD4, CD8, CD68, PD-1, PD-L1, CD163 and CD 206 using OPAL TM 7 kit (AKOYA Biosciences) for MIF. Densities of various cell phenotypes (expressed as cells/mm2) were quantified using an automated quantitative image analysis system (InForm). Immune cell phenotype densities were stratified by HPV status and by tumor stage. We will apply the nearest neighbor cell analysis to identify the spatial orientation of immune cells in the tumor microenvironment. Results: 57 PSCC patients (median age, 60 years [IQR (interquartile range) 31-92]; 60% HPV negative) had MIF analysis. More than half (31/57) had lymph nodes involved (6 N1, 25 N2-3). The immune composition did not differ significantly with HPV status. When investigated by individual clinical stage, we observed an increase in median density of total (CD3+), helper (CD3+CD4+), cytotoxic (CD3+CD8+) and CD3+PD-L1+ T cells from N0 to N1 stage (pN0: CD3+ 44.23, CD3+CD4+ 14.77, CD3+CD8+ 16.15 cells/mm2; pN1: CD3+ 188.36, CD3+CD4+ 71.69, CD3+CD8+ 40.22 cells/mm2) followed by a decrease in N2-N3 stage (CD3+CD4+ 18.45 and CD3+CD8+ 28.22 cells/mm2).The median density of total macrophages increased with stage (pN0: 306.38; pN1: 502.47; pN2-3: 648.27 cells/mm2). While the activated M1 macrophages increased from N0 to N1 and decreased in the more advanced N2-3 stage, M2 macrophages steadily increased across stages and became the dominant type in N2-3. Conclusions: This study describes the interplay of T cells and macrophages across disease stages using MIF. We observed an initial T cell and myeloid immune response in the early locoregional stage, followed by the emergence of immune exhaustion, marked by a decline in the density of cytotoxic T cells, rising PD-L1 expression, and the progressive replacement of anti-tumor M1 macrophages with pro-tumorigenic M2 macrophages across stages. Our findings can inform treatments utilizing immune manipulation. Geospatial analysis exploring the proximity relationships of different immune cell types to each other and to the tumor is ongoing and will be presented at the meeting.

Evaluation Of Surrogate Markers to Define H3 G34R/V-Mutant Gliomas

Background: H3 G34-mutant glioma was a new high-grade glioma, characterized by mutation of histone3.3 codon34. Currently, standard gene sequencing methods are helping in tumor diagnosis, but less expensive and faster H3 G34R/V staining method has not been studied yet in Thailand. Objectives: The purpose of this research is to find surrogate makers for diagnosing this tumor by uncomplicated immunohistochemical method. Methods: Formalin-fixed and paraffin-embedded tissue samples of 30 diffuse hemispheric gliomas were collected H3 G34R, H3 G34V, Olig2, p53 were evaluated using immunohistochemistry. Comparisons of immunohistochemical method and sequencing standard technique were made. Cost-effectiveness was analyzed. Results: Two cases of H3 G34-mutant gliomas were confirmed by mutation analysis. One case was positive for H3 G34V antibody (1/1) while another case was negative for H3 G34R (0/1). The cost-effectiveness showed that the sequencing technique prolonged turnaround time but gave a cost saving 23 USD (17%). Conclusions: G34R and G34V antibodies are not sensitive surrogate markers for diagnosing H3.3 G34-mutant gliomas. However, more samplings are yet to be tested in the future.

Elevated expression of Axin2 in intestinal metaplasia and gastric cancers.

The Wnt signaling pathway regulates crucial cellular processes, including stem cell development and tissue repair. Dysregulation of this pathway, particularly β-catenin stabilization, is linked to colorectal carcinoma and other tumors. Axin2, a critical component in the pathway, plays a role in β-catenin regulation. This study examines Axin2 expression in normal gastric mucosa and various gastric pathologies. Formalin-fixed and paraffin-embedded tissue samples from normal stomach, gastritis, intestinal metaplasia (IM), and gastric carcinoma were collected. Axin2 and β-catenin expression were evaluated using RNA in situ hybridization and immunohistochemistry, respectively. Histo-scores (H-scores) were calculated to quantify expression levels of Axin2. Associations between Axin2 expression and clinicopathological variables were examined. Axin2 expression was examined in normal stomach, gastritis, and IM tissues. Axin2 expression was mainly observed in the surface and isthmus areas in the normal stomach and gastritis, whereas Axin2 expression was markedly higher at the bases of IM. Axin2 H-scores were significantly elevated in IM (mean ± standard deviation [SD], 87.0 ± 38.9) compared to normal (mean ± SD, 18.0 ± 4.5) and gastritis tissues (mean ± SD, 33.0 ± 18.6). In total, 30% of gastric carcinomas showed higher Axin2 expression. Axin2 expression did not have significant associations with age, sex, Lauren classification, histological differentiation, invasion depth, and lymph node metastasis. However, a strong positive correlation was observed between Axin2 and nuclear β-catenin in gastric carcinomas (p < .001). Axin2 expression was significantly increased in IM compared to normal and gastritis cases. In addition, Axin2 showed a strong positive association with nuclear β-catenin expression in gastric carcinomas, demonstrating a close relationship with abnormal Wnt/β-catenin signaling pathway.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Fatal acute undifferentiated febrile illness among clinically suspected leptospirosis cases in Colombia, 2016-2019.

Acute undifferentiated febrile illness is a common challenge for clinicians, especially in tropical and subtropical countries. Incorrect or delayed diagnosis of febrile patients may result in medical complications or preventable deaths. Common causes of acute undifferentiated febrile illness in Colombia include leptospirosis, rickettsioses, dengue fever, malaria, chikungunya, and Zika virus infection. In this study, we described the acute undifferentiated febrile illness in postmortem patients reported as suspected cases of leptospirosis through the national leptospirosis surveillance in Colombia, 2016-2019. We retrospectively analyze human fresh and formalin-fixed tissue samples from fatal suspected leptospirosis cases reported by the Public Health Laboratories in Colombia. Leptospirosis confirmation was made by immunohistochemistry, real-time polymerase chain reaction (PCR) in the tissue samples. In some cases, the serum sample was used for confirmation by Microagglutination test (MAT). Simultaneously, tissue samples were tested by PCR for the most common viral (dengue, Zika, and chikungunya), bacterial (Brucella spp., and Rickettsia spp.), and parasitic (malaria). Fresh tissue samples from 92 fatal suspected leptospirosis cases were reported to the National Reference Laboratory from 22/32 departments in Colombia. We confirmed leptospirosis in 27% (25/92) of cases. Other pathogens identified by real-time PCR were Brucella spp. (10.9%), Rickettsia spp. (14.1%), and dengue (2.2%). Dengue (6.9%), hepatitis (3.5%), and Yellow Fever cases (2.2%) were detected by the pathology. All patients were negative for chikungunya and Plasmodium spp. Most cases were classified as undifferentiated febrile illnesses (45.7%; 42/92). This study underscores the importance of early and accurate recognition of leptospirosis to prevent mortalities. Moreover, it draws attention to the existence of other febrile syndromes in Colombia, including rickettsiosis and brucellosis, that currently lack sufficient human surveillance and regular reporting. Expanding laboratory surveillance to include viruses such as Hantavirus, Mayaro virus, Oropouche virus, and West Nile virus is crucial.

Cross-species evaluation of fibroblast activation protein alpha as potential imaging target for soft tissue sarcoma: a comparative immunohistochemical study in humans, dogs, and cats.

Complete surgical tumor resection is paramount in the management of soft tissue sarcoma (STS) in humans, dogs, and cats alike. Near-infrared targeted tracers for fluorescence-guided surgery (FGS) could facilitate intraoperative visualization of the tumor and improve resection accuracy. Target identification is complicated in STS due to the rarity and heterogeneity of the disease. This study aims to validate the expression of fibroblast activation protein alpha (FAP) in selected human, canine, and feline STS subtypes to assess the value of FAP as a target for FGS and to validate companion animals as a translational model. Formalin-fixed and paraffin-embedded tissue samples from 53 canine STSs (perivascular wall tumor (PWT), canine fibrosarcoma (cFS), and STS not further specified (NOS)), 24 feline fibrosarcomas, and 39 human STSs (myxofibrosarcoma, undifferentiated pleomorphic sarcoma, dermatofibrosarcoma protuberans, and malignant peripheral nerve sheath tumor) as well as six canine and seven feline healthy controls and 10 inflamed tissue samples were immunohistochemically stained for their FAP expression. FAP labeling in tumor, peritumoral, healthy skin, and inflamed tissue samples was quantified using a visually assessed semiquantitative expression score and digital image analysis. Target selection criteria (TASC) scoring was subsequently performed as previously described. Eighty-five percent (85%) of human (33/39), 76% of canine (40/53), and 92% of feline (22/24) STSs showed FAP positivity in over 10% of the tumor cells. A high expression was determined in 53% canine (28/53), 67% feline (16/24), and 44% human STSs (17/39). The average FAP-labeled area of canine, feline, and human STSs was 31%, 33%, and 42%, respectively (p > 0.8990). The FAP-positive tumor area was larger in STS compared to healthy and peritumoral tissue samples (p < 0.0001). TASC scores were above 18 for all feline and human STS subtypes and canine PWTs but not for canine STS NOS and cFS. This study represents the first cross-species target evaluation of FAP for STS. Our results demonstrate that FAP expression is increased in various STS subtypes compared to non-cancerous tissues across species, thereby validating dogs and cats as suitable animal models. Based on a TASC score, FAP could be considered a target for FGS.

Clinical and morphological features of bladder cancer course in HPV-infected patients

Purpose of the study. To study the histological type, grade of tumor differentiation in patients with primary and recurrent clinically non-muscle-invasive bladder cancer (NMIBC) with highly carcinogenic human papillomavirus (HPV) infection.Patients and methods. Formalin-fixed and paraffin-embedded bladder tumor tissue samples have been studied in 159 patients who underwent transurethral resection (TUR) of the bladder, for the presence of HPV DNA. To detect, quantify and differentiate DNA of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 genotypes in the samples, the AmpliSense® HPV HRC genotype-titer-FL was used. The result of the study was taken into account when the amount of DNA of the β-globin gene was at least 1000 copies per reaction. In order to statistically analyze our data we used the Fisher exact test and also calculated the odds ratio (OR) and 95 % CI.Results. According to the results of the study, out of 159 patients, high-risk HPV DNA was detected in the tumor tissue in 59 (37.1 %), of which HPV type 16 was found in 52 patients (89.4 %), HPV 18 was detected in 4 patients type (6.7 %) and type 35 in 3 (5.08 %). In a morphological study of the tissues of HPV-positive patients, the grade of tumor differentiation was G2 in 18 cases (30.5 %), G3 in 37 blocks, and G1 was detected only in 4 cases (6.7 %). In the presence of HPV, the chance of detecting a stage G3 tumor increases by 4.3 times. According to the received data, we can assume that there is a close relationship between detection in HPV patients of high-risk genotypes with moderately differentiated and low-differentiated forms of bladder cancer.Conclusion. this study may indicate that HPV infection affects the grade of tumor differentiation, and this, in turn, may allow the use of the HPV test to assess the nature of the development of relapse and/or progression of the disease.

TMA-Mate: An open-source modular toolkit for constructing tissue microarrays of arbitrary layouts

Biomedical research and quality control procedures often demand a variety of microscopic analysis of numerous formalin-fixed and paraffin-embedded (FFPE) tissue samples from different individuals of both healthy and diseased regions of interest. Depending on the number of samples to be analyzed, conventional processing of each FFPE block separately can be laborious or impracticable. This effort can be drastically reduced by using tissue microarrays (TMAs). TMAs have a wide range of applications and can be considered as a high-throughput method to process up to hundreds of miniaturized tissue samples simultaneously on a single microscopy slide, in order to reduce labor, costs and sample consumption, and to increase results comparability. Several commercial and self-made solutions to fabricate TMAs with varying degrees of automation are available. However, these solutions may not be suitable for every situation, either due to high costs, high complexity, lack of precision or lack of flexibility, especially when diagnostically oriented pathology institutes or laboratories with constrained resources are considered. This article introduces the TMA-Mate, an open-source 3D printable modular toolkit for constructing high-density TMAs of arbitrary layouts, providing an affordable, lightweight, and accessible procedure to implement TMAs into existing histology processing pipelines. Step-by-step demonstrations for replicating the hardware and constructing TMAs are included.

XPERT® breast cancer STRAT4 as an alternative method of identifying breast cancer phenotype in Cape Verde (preliminary results).

Breast cancer (BC) is a public health problem in developing countries, including Cape Verde. Immunohistochemistry (IHC) is the gold standard technique used for BC phenotypic characterisation to support efficient therapeutic decisions. However, IHC is a demanding technique that requires knowledge, trained technicians, expensive antibodies and reagents, controls, and results validation. The low number of cases in Cape Verde increases the risk of expiring the validity of the antibodies, and manual procedures often jeopardise the quality of the results. Thus, IHC is limited in Cape Verde, and an alternative technically easy solution is needed. A point-of-care messenger RNA (mRNA) STRAT4 BC assay to assess estrogen (ER), progesterone (PR), hormone growth factor 2 receptor (HER2), and Ki67, using the GeneXpert platform, has been recently validated on tissues from internationally accredited laboratories, showing excellent concordance with IHC results.To assess whether this technology can be implemented in Cape Verde to guide BC treatment we decided to study the level of agreement between the findings yielded by BC STRAT4 and the results are the same cases obtained by IHC. Formalin-fixed and paraffin-embedded (FFPE) tissue samples from 29 Cabo Verdean BC patients diagnosed in Agostinho Neto University Hospital were analysed by applying IHC and BC STRAT4 assay. The time between sample collection and pre-analytic procedures is unknown. All the samples were pre-processed in Cabo Verde (fixed in formalin and embedded in paraffin). IHC studies were performed in referenced laboratories in Portugal. STRAT4 and IHC result concordance was assessed by calculating the percentage of results agreement and Cohen's Kappa (K) statistics. STRAT4 assay failed in 2 out of the 29 analysed samples. Of the 27 successfully analysed samples, STRAT4/IHC results for ER, PR, HER2, and Ki67 were concordant in 25, 24, 25, and 18 cases, respectively. Ki67 was indeterminate in three cases, and PR was indeterminate once.The percentage of agreement between STRAT4 and IHC results for ER, PR, HER2, and Ki67 was 92.59%, 92.31%, 92.59% and 81.82%, respectively. The Cohen's K statistic coefficients for each biomarker were 0.809, 0.845, 0.757 and 0.506, respectively. According to our preliminary results, a point-of-care mRNA STRAT4 BC assay may be an alternative in laboratories unable to provide quality and/or cost-efficient IHC services. However, more data and improvement on sample pre-analytic processes are required to implement this BC STRAT4 Assay in Cape Verde.

Abstract 993: Epigenetic immune cell profiling in B-non-Hodgkin lymphoma biopsies

Abstract Early detection and differential diagnosis of Non-Hodgkin lymphoma (NHL) is essential for the initiation of suitable therapy options. Biopsy based diagnosis allows allocation to World Health Organization (WHO) sub-classification groups but prognosis often needs identification of specific genetic aberrations or gene expression patterns. Despite this strategy, prognosis is difficult, since diseases, like e.g. mantel cell lymphoma, can be indolent or aggressive. Here we report the characterization of NHL biopsies using epigenetic immune cell quantification. This approach is based on the identification of specifically demethylated regions in individual immune cell types. These regions are then quantified using methylation-sensitive qPCR after bisulfite conversion. This highly reproducible and objective method allows in depth immunophenotyping of the tumor microenvironment. Next to CD4+ and CD8+ T cells, B cells and NK cells more specialized immune cells like regulatory T cells (Tregs), follicular T helper cells, PD-1 expressing cells and Myeloid derived suppressor cells (MDSC) were quantitatively determined in formalin-fixed tissue samples of 251 patients with different B cell Non-Hodgkin lymphomas. As a result, characteristic immune cell distribution patterns were identified for the different lymphoma entities, including chronic lymphocytic leukemia (B-CLL) (n=41), mantle cell lymphoma (MCL) (n=32), follicular lymphoma (FL) (n=33), diffuse large B cell lymphoma (DLBCL) (n=74), marginal zone lymphoma (MZL) (n=40) and B cell acute lymphoblastic leukemia (B-ALL) (n=26). In addition, substantial heterogeneity within the entities was observed that may correlate with disease prognosis. Among others, this is particularly the case for MDSC in B-CLL patients as well as for NK cells and Tregs in MCL and PD-1+ cells in FL patients. Currently clusters within the different disease groups are analyzed with regard to the clinical outcome to investigate if disease prognosis can be predicted by epigenetic immune cell quantification. In conclusion, epigenetic immune cell quantification is a suitable method to analyze the tumor microenvironment of NHL patients with the potential to support patient’s prognosis. Citation Format: Jeannette Werner, Steffi Walter, Christoph Sachsenmaier, Janika Schulze, Deborah Phippard, Sven Olek, Barbara Seliger, Claudia Wickenhauser. Epigenetic immune cell profiling in B-non-Hodgkin lymphoma biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 993.

Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways

BackgroundThe pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases.MethodsIn this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology).ResultsIn total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE.ConclusionDIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Discrimination of Classical and Atypical BSE by a Distinct Immunohistochemical PrPSc Profile

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrPSc). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrPSc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrPSc-profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrPSc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrPSc profile for the different BSE-types, which included both the topographic and cellular pattern of PrPSc. This qualitative and quantitative analysis of PrPSc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Immunohistochemical Expression of IDH1, ATRX, Ki67, GFAP, and Prognosis in Indonesian Glioma Patients.

The current World Health Organization (WHO) 2021 classification of human glioma is based on key molecular biomarkers to define neoplastic entities. This review further delineates mutant IDH (isocitrate dehydrogenase) from wild-type IDH disease, a necessity given the large survival gap between mutant IDH and wild-type IDH tumors. In Indonesia, there are currently few reports on the distribution and significance of these mutations. Therefore, this research aims to determine the relationship between IDH mutations, as well as clinicopathological and prognostic factors in patients with gliomas. Other immunohistochemical markers including ATRX (alpha-thalassemia/mental retardation, X-linked), Ki67 and GFAP (glial fibrillary acidic protein) expression were also evaluated. Forty-two glioma samples were collected from patients who underwent surgery at Dr. Kariadi General Hospital in Semarang, Central Java, Indonesia. Fresh and paraffin-embedded, formalin-fixed tissue samples were removed and sectioned for hematoxylin and eosin staining, immunohistochemistry, and IDH analysis of mutation. Medical records were used to collect clinicopathological and survival data. IDH1 mutations were discovered in 32 (76,1%) patients, and those with IDH1 mutation had longer overall survival when corresponded to patients with IDH1-wild-type. Lower expression of Ki67 was discovered to be very associated with a better prognosis. IDH1 mutations status showed a significant relationship with prognosis in patients with glioma. Meanwhile, other markers (ATRX, Ki67, and GFAP) did not correlate with the prognosis.

Identification of Genetic Subtypes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma subtype and is characterized by heterogeneous clinical trajectories. While certain genetic abnormalities are highly prevalent, heterogeneity exists also at the molecular level. Yet, unlike diffuse large B-cell lymphoma, where genetic subtypes have been described in multiple studies, the taxonomy of FL has not been clearly established to date. While clusters defined by STAT6 mutations or aberrant somatic hypermutation have been described in FL by Crouch et al. (Blood Advances, 2022), its landscape of molecular alterations has been insufficiently dissected into biologically distinct subgroups. The recognition of such subtypes may pave the way for treatment individualization. Here, we sought to define novel genetic subtypes in FL by means of targeted DNA sequencing and clustering. Methods: Samples of FL grade 1 to 3A (i.e. classic FL) were accrued from biorepositories of participating centres in Canada, Northern Europe and Australia, as well as from two prospective clinical trials (E4402 and E2408). We performed hybridization-based targeted DNA-sequencing of 57 genes that are recurrently mutated in FL. Moreover, we included targeted DNA sequencing data from a previously published dataset (PMID 27959929). All cases were analyzed using a uniform computational pipeline. Samples were removed from the analysis if the mean on-target coverage was <50X. Single nucleotide variants were identified using Mutect2 and we applied a variant allele fraction cut-off of 0.1. Bernoulli mixture-model clustering was applied using variants as features for the identification of genetic subtypes. Cluster stability was assessed by bootstrap consensus. Clusters were tested for their association with outcome measures. As a further means to annotate genetic clusters, we generated genome-wide methylation data using the Illumina EPIC platform. Results: Following quality control, the total number of pre-treatment samples available for clustering was 715 (543 from formalin-fixed and paraffin-embedded tissue samples and 172 from fresh-frozen). The average number of mutations and mutated genes per sample were 5.9 (range 1-20) and 4.8 (range 0-14), respectively. An analysis of interactions between somatic mutations revealed both expected (e.g. mutual exclusivity of CREBBP and EP300 mutations) and unexpected patterns (e.g. co-occurring mutations of GNA13 and MEF2B), overall justifying the pursuit of unsupervised clustering. After consensus clustering, the optimal clustering solution resolved 5 genetically distinct groups, henceforth referred to as C1 to C5 (Figure). The C1 cluster was characterized by variants in genes involved in the mTOR signaling pathway (ATP6AP1, RRAGC, ATP6V1B2); the C2 cluster by low mutation burden; the C3 cluster by mutations involving TNFAIP3, TP53 and MYD88; the C4 cluster by GNA13 and MEF2B mutations; and the C5 cluster by STAT6, CREBBP, EZH2 and TNFRSF14 mutations. The number of mutations located in RGYW or WRCY motifs, suggestive of somatic hypermutation, was highest in the C4 and the C3 clusters (P < 0.001). Applying a previously reported methylation-based clock (epiCMIT) to the subset of samples with methylation data (n=345), we found that samples in the C2 cluster had the lowest proliferative history. Concordantly, the C2 cluster was characterized by a lower degree of global hypomethylation and lower average methylation of CpG islands, whereas samples in clusters C1, C3, C4 and C5 were characterized by not just longer proliferative histories, but also by higher mean methylation in CpG islands. In terms of clinical associations, the C2 cluster was significantly associated with limited-stage disease presentation. In an exploratory outcome analysis of 165 patients treated with immunochemotherapy, the C3 and C5 clusters were associated with inferior time to progression. Conclusion: Here, we identified novel genetic subtypes that are distinguishable not just by unique sets of mutations, but also by their clinical associations and methylation profiles. The novel genetic profiles shed light on core pathways distinctly tied to each subtype. Targeted DNA sequencing of a limited gene panel represents a potentially accessible method of subtyping FL in the clinical setting, with implications for the understanding of responses to targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Proteomics for Meningioma: An Integrated Workflow for Quantitative Proteomics and Biomarker Validation in Formalin-Fixed Paraffin-Embedded Tissue Samples.

Clinical proteomics is a rapidly emerging frontier in laboratory medicine. High-throughput proteomic investigations of biopsy tissues provide mechanistic insights into complex human diseases. For large-scale proteomics, formalin-fixed and paraffin-embedded (FFPE) tissue samples offer a viable alternative to fresh-frozen (FF) tissues that have restricted availability. In this context, meningioma is one of the most common primary brain tumors where innovation in diagnostics and therapeutic targets can benefit from clinical proteomics. We present here an integrated workflow for quantitative proteomics and biomarker validation of meningioma FFPE tissues. Applying label-free quantitative (LFQ) proteomics, we reproducibly (Pearson's correlation: 0.84-0.91) obtained an in-depth proteome coverage (nearly 4000 proteins per sample) from 120 min gradient of single unfractionated mass spectrometry run. Furthermore, building upon LFQ data and literature curated set of meningioma-associated proteins, we validated VIM, AHNAK, and CLU from FFPE tissues using selected reaction monitoring (SRM) assay and compared its performance with FF tissues. This study illustrates how knowledge from label-free proteomics can be integrated for selecting peptides for targeted validation and suggests that FFPE tissues are comparable to FF tissues for SRM assays. This quantitative clinical proteomics workflow is scalable for large-scale clinical diagnostics studies in the future, for example, utilizing the global repository of FFPE tissues in meningioma and possibly in other cancers.