Formalin-fixed Paraffin-embedded Tissue Research Articles

SPOT: spatial proteomics through on-site tissue-protein-labeling

BackgroundSpatial proteomics seeks to understand the spatial organization of proteins in tissues or at different subcellular localization in their native environment. However, capturing the spatial organization of proteins is challenging. Here, we present an innovative approach termed Spatial Proteomics through On-site Tissue-protein-labeling (SPOT), which combines the direct labeling of tissue proteins in situ on a slide and quantitative mass spectrometry for the profiling of spatially-resolved proteomics.Materials and MethodsEfficacy of direct TMT labeling was investigated using seven types of sagittal mouse brain slides, including frozen tissues without staining, formalin-fixed paraffin-embedded (FFPE) tissues without staining, deparaffinized FFPE tissues, deparaffinized and decrosslinked FFPE tissues, and tissues with hematoxylin & eosin (H&E) staining, hematoxylin (H) staining, eosin (E) staining. The ability of SPOT to profile proteomes at a spatial resolution was further evaluated on a horizontal mouse brain slide with direct TMT labeling at eight different mouse brain regions. Finally, SPOT was applied to human prostate cancer tissues as well as a tissue microarray (TMA), where TMT tags were meticulously applied to confined regions based on the pathological annotations. After on-site direct tissue-protein-labeling, tissues were scraped off the slides and subject to standard TMT-based quantitative proteomics analysis.ResultsTissue proteins on different types of mouse brain slides could be directly labeled with TMT tags. Moreover, the versatility of our direct-labeling approach extended to discerning specific mouse brain regions based on quantitative outcomes. The SPOT was further applied on both frozen tissues on slides and FFPE tissues on TMAs from prostate cancer tissues, where a distinct proteomic profile was observed among the regions with different Gleason scores.ConclusionsSPOT is a robust and versatile technique that allows comprehensive profiling of spatially-resolved proteomics across diverse types of tissue slides to advance our understanding of intricate molecular landscapes.

Evaluation of a multiplex real-time PCR targeting the β-tubulin gene for the detection and differentiation of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, S. schenckii and S. brasiliensis. The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

PCR GeneScan Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

SnPATHO-seq, a versatile FFPE single-nucleus RNA sequencing method to unlock pathology archives

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Targeted microscopic rare cell retrieval yields sufficient deoxyribonucleic acid for molecular testing in fixed tissue with low tumor fraction

Abstract Introduction/Objective DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) blocks is often utilized for molecular oncologic testing. Cases with low tumor fraction often fail to obtain adequate tumor for molecular testing. We hypothesized that targeted retrieval of visualized tumor cells from FFPE sections, including those with low tumor fraction, could yield sufficient DNA for molecular testing. Methods/Case Report Unstained sections of various thicknesses were cut from low tumor fraction blocks and visualized by light microscopy and the RareCyte CyteFinder II (Seattle, WA). Microscopic cell retrieval was performed on the RareCyte using 40 µm diameter needles from identified regions of interest. Samples were deparaffinized using Zymo Research (Tustin, CA) solution, and digested and purified using Zymo-Spin Column technology. Sample DNA was quantified with NanoDrop Spectrophotometer (Chino, CA). Results (if a Case Study enter NA) Regions of interest were morphologically identifiable on 4-10 µm thick sections and harder to visualize on 12 µm thick sections. Targeted microscopic cell retrieval was performed with 40 µm diameter needles. Retrieval was performed from 26 to 34 regions of interest per sample and pooled for molecular testing. Samples (n=4) yielded a mean of 81.0 ng of DNA ± 18.4 ng (1.8 ng/µL ± 0.4 ng/µL) and a median of 87.75 ng (1.95 ng/µL). Conclusion Targeted microscopic cell retrieval from low tumor fraction FFPE slides using the RareCyte CyteFinder II can provide sufficient DNA material for molecular testing. Application of this technology has the possibility of recovering sufficient tumor DNA for molecular testing from specimens that may be deemed unsuitable for molecular testing according to current standards. We aim to focus on increasing sample size and completing comprehensive molecular analysis as we continue to expand this work.

Reliable RNA-seq analysis from FFPE specimens as a means to accelerate cancer-related health disparities research.

Whole transcriptome sequencing (WTS/ RNA-Seq) is a ubiquitous tool for investigating cancer biology. RNA isolated from frozen sources limits possible studies for analysis of associations with phenotypes or clinical variables requiring long-term follow-up. Although good correlations are reported in RNA-Seq data from paired frozen and formalin fixed paraffin embedded (FFPE) samples, uncertainties regarding RNA quality, methods of extraction, and data reliability are hurdles to utilization of archival samples. We compared three different platforms for performing RNA-seq using archival FFPE oropharyngeal squamous carcinoma (OPSCC) specimens stored up to 20 years, as part of an investigation of transcriptional profiles related to health disparities. We developed guidelines to purify DNA and RNA from FFPE tissue and perform downstream RNA-seq and DNA SNP arrays. RNA was extracted from 150 specimens, with an average yield of 401.8 ng/cm 2 of tissue. Most samples yielded sufficient RNA reads >13,000 protein coding genes which could be used to differentiate HPV-associated from HPV-independent OPSCCs. Co-isolated DNA was used to identify patient ancestry. Utilizing the methods described in this study provides a robust, reliable, and standardized means of DNA & RNA extraction from FFPE as well as a means by which to assure the quality of the data generated.

Inflammation-Associated Long Non-Coding RNAs (lncRNAs) in Chronic Viral Hepatitis- Associated Hepatocellular Carcinoma.

This study aimed to identify the expression profile and prognostic significance of inflammation-associated lncRNAs in chronic viral hepatitis (CVH) and CVH-associated hepatocellular carcinoma (CVH-HCC). In the first step, lncRNA expression analysis was performed by real-time polymerase chain reaction (RT-PCR) using an array panel of 84 inflammation-associated lncRNAs in 48 formalin-fixed paraffin-embedded (FFPE) tissue samples (12 CVH-HCC, 12 peritumoral cirrhotic parenchyma, 12 nontumoral cirrhotic CVH parenchyma, 12 normal liver samples). In the second step, 7 lncRNAs (DLEU2, HOTAIR, LINC00635, LINC00662, RP11-549J18.1, SNHG16 and XIST) were chosen for RT-PCR assay testing in 72 samples (24 CVH-HCC, 24 peritumoral cirrhotic parenchyma, 24 nontumoral cirrhotic CVH parenchyma samples). Fifty-six inflammation-associated lncRNAs were significantly up-regulated in the peritumoral cirrhotic parenchyma compared to the normal liver. Expression of 71 lncRNAs was significantly higher in peritumoral cirrhotic parenchyma compared to cirrhotic CVH parenchyma. DLEU2 and SNHG16 were up-regulated both in the tumor and peritumoral cirrhotic parenchyma compared to cirrhotic CVH parenchyma. Expression of LINC00662 was significantly higher in CVH-HCC than in cirrhotic CVH parenchyma. Expression of XIST was also increased in both tumor and peritumoral parenchyma samples, albeit without statistical significance. No significant association was found between lncRNA expressions and survival. Inflammation-associated lncRNAs DLEU2, SNHG16, LINC00662, and XIST are candidate diagnostic biomarkers in CVH-HCC. More evidence is needed to prove their utility as prognostic markers.

Immunohistochemical expression of CYP11A1, CYP11B, CYP17, and HSD3B2 in functional and nonfunctional canine adrenocortical tumors.

Functionality of human adrenal tumors is inferred by CYP11B1 (cortisol synthase) expression, CYP11B2 (aldosterone synthase) expression, or both. Nonfunctional canine adrenal tumors have low expression of steroidogenic enzymes, whereas aldosterone-producing tumors express CYP11B, and cortisol-producing tumors express both CYP11B and CYP17. Twenty-two client-owned dogs with adrenocortical tumors (ACT) (8 nonfunctional, 7-cortisol producing, 2 aldosterone-producing and 5 functional noncortisol producing) and 2 dogs with normal adrenal glands. Retrospective case series. Adrenal functionality was determined from clinical signs and endocrine testing. CYP11A1, CYP11B, CYP17, and HSD3B2 expression was detected by immunohistochemistry on formalin-fixed paraffin-embedded adrenal tissue. Protein expression was semiquantified by 2 blinded observers using H-scoring (results reported as median [range]) and compared in nonfunctional and cortisol-producing adrenal tumors by Mann-Whitney U tests. CYP11A1, CYP11B, and HSD3B2 were present within all cortical layers of normal adrenal glands, and CYP17 was expressed within the zona fasciculata and zona reticularis. Expression of CYP11A1 (191.25 [97.5-270] vs. 175 [102.5-295] P = .69), CYP11B (190 [130-265] vs. 147.5 [95-202.5]; P = .07), CYP17 (177.5 [87.5-240] vs. 247.5 [55-292.5]; P = .40), and HSD3B2 (230 [47.5-295] vs. 277.5 [67.5-295]; P = .34) were not significantly different between cortisol-producing and nonfunctional ACT. Our findings suggest it is not possible to determine functionality of canine ACT by immunohistochemistry for steroidogenic enzymes. Tumor size cannot be used to infer functionality of adrenal tumors.

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Descriptive Analysis of Common Fusion Mutations in Papillary Thyroid Carcinoma in Hungary

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto’s disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto’s disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

A COMPARATIVE STUDY OF CLINICOHISTOPATHOLOGICAL CORRELATION IN PATIENTS OF PSORIASIS AND PSORIASIFORM LESIONS WITH KI67 AND CD34 IMMUNOHISTOCHEMICAL EXPRESSION

Objectives: Psoriasis, characterized by chronic inflammation of the skin, stands as a notable example of a psoriasiform tissue pattern. In dermatology, the term “psoriasiform dermatitis” frequently appears across various inflammatory skin conditions, presenting challenges for dermatologists and pathologists alike in accurate differentiation. Relying solely on clinical features may prove insufficient in distinguishing between psoriasis and other psoriasiform dermatoses. Therefore, comprehensive differentiation requires consideration of clinical, histopathological, and immunohistochemical factors. Our study is to compare the clinicohistopathological features of psoriasis and psoriasiform dermatitis and to evaluate the immunohistochemical expression of CD34 and Ki67 in psoriasis and psoriasiform dermatitis. Methods: This retrospective study was conducted at the Department of Pathology, Mediciti Institute of Medical Sciences, Ghanpur, focusing on cases between January 1, 2020, and December 31, 2022. Clinical histories were extracted from requisition forms submitted by the Department of Dermatology. The study analyzed paraffin blocks from 50 patients each diagnosed with psoriasis and psoriasiform dermatitis. Formalin-fixed paraffin-embedded tissue samples were processed into 4-micron sections and initially stained with hematoxylin and eosin. The immunohistochemical analysis included primary antibodies against Ki-67 and CD34, with positive controls sourced from strongly positive samples of squamous cell carcinoma and capillary hemangioma, respectively. Appropriate negative controls were employed to ensure the accuracy of our findings. Results: In our study, we analyzed a total of 100 cases, comprising 50 cases each of psoriasis and psoriasiform dermatitis. Among the psoriasis cases, there was a male predominance with 29 males and 21 females, while psoriasiform dermatitis also showed male predominance with 32 males. The mean age was 33.8 years for psoriasis and 34.8 years for psoriasiform dermatitis. The majority of psoriasis cases fell in the 21–30 years age range, whereas for psoriasiform dermatitis, it was between 31 and 40 years. Significantly, our immunohistochemical analysis revealed that the mean staining intensity of the two markers was notably higher in psoriasis compared to psoriasiform dermatitis. Conclusions: In our study, we observed that the expression levels of Ki-67 and CD34 biomarkers were significantly elevated in psoriasis compared to psoriasiform dermatitis. Notably, four cases of psoriasiform dermatitis demonstrated positivity for CD34 in our analysis. This finding suggests a potential avenue for further research to explore the implications of CD34 positivity in psoriasis.

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Evaluation of GeneXpert MTB/Rif Ultra assay performance on formalin-fixed paraffin-embedded tissues for Mycobacterium tuberculosis detection.

We evaluated the Xpert MTB/Rif Ultra assay performance for Mycobacterium tuberculosis (MTB) detection in formalin-fixed paraffin-embedded tissue (FFPET) compared to mycobacterial culture or laboratory-developed MTB PCR test (LDT). FFPET samples with histological features suggestive of tuberculosis from 2018 to 2023 were selected. Five hundred microlitres of tissue lysis buffer was added to FFPET scrolls and incubated at 75 °C for 5 min. After adding 50 µl of proteinase K and overnight incubation at 56 °C, sample aliquots were processed as per the manufacturer's instructions. MTB culture or LDT assay results were used as a reference for sensitivity and specificity calculations. Of 51 eligible FFPET, 32 were positive for MTB either by culture or LDT PCR on FFPET. Xpert MTB/Rif Ultra detected MTB in 23/32 positive specimens [71.9%, 95% confidence interval (CI) 54.6-84.4%]. Of nine discordant specimens, seven were MTB positive by culture and two were identified by LDT MTB PCR only, as no specimen was submitted for MTB culture. Of 19 negative samples, 100% specificity (95% CI 83.2-100.0%) was attained via Xpert MTB/Rif Ultra. Implementation of Xpert MTB/Rif Ultra on FFPET within clinical laboratories is promising, given its improved turnaround time compared to MTB culture and ability to detect MTB in cases where no tissue is available for culture.

Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA

Quantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probe-based multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143bp; nDNA = 71 and 181bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69bp mtDNA target and the 71bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice.

Mutational Landscape of Gastric Adenocarcinoma of the Fundic Gland Type Revealed by Whole Genome Sequencing.

Gastric adenocarcinoma of the fundic gland type (GA-FG) is a newly described variant of gastric adenocarcinoma with lack of knowledges regarding its genetic features. We performed whole-genome sequencing (WGS) in formalin-fixed paraffin-embedded (FFPE) tumor tissues and matched adjacent noncancerous specimens from 21 patients with GA-FG, and integrated published datasets from 1105 patients with traditional gastric adenocarcinoma with the purpose of dissecting genetic determinants both common to conventional gastric adenocarcinoma and unique to GA-FG disease. We characterized the genomic architecture of GA-FG disease, revealing the predominant proportion of C > T substitution among the six types of SNVs. GNAS was the most significantly mutated driver gene (14.29%). 42.8% of samples harbored "Kataegis." Distinct genomic alterations between GA-FG and conventional gastric cancer were identified. Specifically, low mutational burden and relatively moderate mutational frequencies of significantly mutated driver genes, coupled with the absence of non-silent alterations of formerly well-known drivers such as TP53, PIK3CA and KRAS were identified in GA-FG patients. Oncogenic signaling pathway analysis revealed mutational processes associated with focal adhesions and proteoglycans in cancer, highlighting both common and specific procedures during the development of GA-FG and conventional gastric cancer. Our study is the first to comprehensively depict the genomic landscape highlighting the multidimensional perturbations in GA-FG patients. These discoveries offered mechanistic insights for novel diagnostic and therapeutic strategies for patients with such disease.

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.

Telomere Length Measurement in Human Tissue Sections by Quantitative Fluorescence In Situ Hybridization (Q-FISH).

Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.

Highly-Multiplexed Immunofluorescence PhenoCycler Panel for Murine FFPE Yields Insight into Tumor Microenvironment Immunoengineering

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is Co-Detection-by-indEXing (CODEX), recently renamed as the PhenoCycler system. This is a highly-multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh-frozen tissues. Formalin-fixed paraffin-embedded (FFPE) blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine FFPE tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of three gene-delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift towards intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of IFNγ, in addition to 4-1BBL and IL-12, further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help to explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of IFNγ therapeutic efficacy.

Autophagy-associated biomarkers ULK2, UVRAG, and miRNAs miR-21, miR-126, and miR-374: Prognostic significance in glioma patients.

As the pioneering study from Pakistan, our research distinctly focuses on validating the roles of autophagy-associated genes and MicroRNAs (miRs) in the unique context of our population for glioma prognosis. The study delves into the nuanced interplay of autophagy within a miR-modulated environment, prompting an exploration of its potential impact on glioma development and survival. Employing real-time PCR (qPCR), we meticulously assessed the expression profiles of autophagy genes and miRs in glioma tissues, complemented by immunohistochemistry on Formalin-fixed paraffin-embedded tissues from the same patients. Our comprehensive statistical analyses, including the data normality hypothesis Shapiro-Wilk test, the Mann-Whitney U-test, Spearman correlation test, and Kaplan-Meier survival analysis, were tailored to unravel the intricate associations specific to low- and high-grade glioma within our population. Clinicopathological analysis revealed a predominance of male patients (66%) with a median age of 35 years. Glioblastoma (32%) and Astrocytoma (36%) were the most prevalent histopathological subtypes. Molecular analysis showed significant correlations between prognostic markers (Ki-67, IDH-1, p53) and clinicopathological factors, including age, histological type, radiotherapy, and chemotherapy. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 in low-grade glioma, unveiling distinctive molecular signatures unique to our study. Furthermore, a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma, providing further insights into the autophagy pathway within our population. The enrichment analysis for miR-21, miR-126, and miR-374 showed MAPK pathway as a common pathway along with Ras, PI3K, and mTOR pathway. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients, validating it in our population. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas within the specific context of the Pakistani population.

Higher Microbial Abundance and Diversity in Bronchus-Associated Lymphoid Tissue (BALT) Lymphomas than in Non-Cancerous Lung Tissues.

It is well known that the majority of the extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) are associated with microbiota, e.g., gastric MALT lymphoma with Helicobacter pylori. In general, they are very sensitive to low-dose radiotherapy and chemotherapeutic agents. The microbiota profile is not clearly elucidated in bronchus-associated lymphoid tissue (BALT) lymphoma, a rare type of MALT lymphoma in the lung. Thus, this study aimed to clarify the intratumor microbiome in BALT lymphoma using the third-generation NGS method. DNAs were extracted from 12 formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from BALT lymphoma patients diagnosed between 1990 and 2016. 16S rRNA gene was amplified by polymerase chain reaction. Amplicons were sequenced using a Nanopore platform. Next-generation sequencing analysis was performed to assess microbial profiles. For comparison, FFPE specimens from nine non-cancerous lung tissues were also analyzed. Specific bacterial families including Burkholderiaceae, Bacillaceae, and Microbacteriaceae were associated with BALT lymphoma by a linear discriminant analysis effect size approach. Although the number of specimens was limited, BALT lymphomas exhibited significantly higher microbial abundance and diversity with distinct microbial composition patterns and correlation networks than non-cancerous lung tissues. This study provides the first insight into intratumor microbiome in BALT lymphoma using the third-generation NGS method. A distinct microbial composition suggests the presence of a unique tumor microenvironment of BALT lymphoma.