Formalin-fixed Paraffin-embedded Research Articles

P53 marker expression in epithelial ovarian tumours in a centre in Nigeria – a descriptive study

Backgroundp53 is a tumor suppressor gene. p53 expression in epithelial ovarian tumors (EOTs) is correlated with their biological behavior and predicts patient overall survival. However, there is a dearth of knowledge regarding p53 expression in these tumors among women from southwest Nigeria. Our study aimed to determine the patterns of p53 expression in various types of epithelial ovarian tumours.MethodsWe conducted a retrospective study of epithelial ovarian tumours. We retrieved formalin-fixed, paraffin-embedded (FFPE) tissue blocks of previously diagnosed epithelial tumors from the departmental archive. We performed immunohistochemical analysis using p53 antibodies. We scored the expression and staining intensity of p53 as follows: negative (0), focal/weakly positive (1 +), and diffuse/strongly positive (2 +) on the basis of the recommended Cytomation scoring system.ResultsThe spectrum of p53 expression in the 51 histologically diagnosed cases revealed that 29 cases had no expression, consisting of 21 benign EOTs, two borderline EOTs, and six malignant EOTs. Nine cases exhibited wild-type expression, including six serous carcinomas, two mucinous carcinomas, and one signet ring cell carcinoma. p53 overexpression was observed in 13 patients overall, with 12 having serous carcinomas and one having endometrioid carcinoma. Among the 21 serous carcinoma patients, 28.6% (6 patients) presented with wild-type p53 expression, 57.1% (12 patients) presented with p53 overexpression, and 14.3% (three patients) presented negative p53 expression. There was a significant association between p53 expression and the histological grade of serous carcinoma.ConclusionMost epithelial ovarian carcinomas in our hospital are high grade, with many serous carcinomas showing either p53 overexpression or loss of expression. This may contribute to the poor patient survival rate.

Genetic validation of a TSC2 immunohistochemistry assay in TSC/mTOR-pathway altered renal tumors.

Pathogenic mutations in the genes associated with tuberous sclerosis complex (TSC)/mTOR pathway are linked to histologically diverse renal cell neoplasms, including eosinophilic solid and cystic renal cell carcinoma (ESC RCC), low grade oncocytic tumor (LOT), eosinophilic vacuolated tumor (EVT), and xanthomatous giant cell renal cell carcinoma (XGC RCC). Here, we validate a TSC2 immunohistochemistry (IHC) assay by comparison to genomic data in these neoplasms. Automated TSC2 IHC was performed on formalin-fixed paraffin embedded (FFPE) tissues from 38 genetically-confirmed TSC/mTOR-associated renal tumors (6ESCs, 16 EVTs, 13 LOTs, 2 XGC and 1 clear cell RCC) and visually scored in a semi-dichotomous fashion compared to internal control tissue. The positive predictive value (PPV) of TSC2 protein loss for underlying pathogenic mutation in TSC2 was 92% (11/12), while the negative predictive value (NPV) of intact TSC2 by IHC for lack of underlying pathogenic mutation in TSC2 was 81% (21/26). Intact TSC2 by IHC was 95% (21/22) specific for absence of underlying pathogenic TSC2 mutation. All the cases lacking TSC2 mutation with intact TSC2 protein had an underlying mutation in TSC1, MTOR or PIK3CA. Loss of TSC2 was 77% (10/13) sensitive for underlying TSC2 truncation mutations and 33% (1/3) sensitive for underlying TSC2 missense mutations. Overall, 73% (8/11) tumors with TSC2 IHC loss and underlying pathogenic alterations in TSC2 showed heterogeneous protein loss, with rare interspersed positively staining tumor cells. These data support TSC2 IHC as a potentially useful assay for the diagnostic workup of renal tumors suspected to belong to the TSC/mTOR-associated subgroups.

Enriched Methylomes of Low-input and Fragmented DNA Using Fragment Ligation EXclusive Methylation Sequencing (FLEXseq).

Methylome profiling is an emerging clinical tool for tumor classification and liquid biopsies. Here, we developed FLEXseq, a genome-wide methylation profiler that enriches and sequences the fragments of DNA flanking the CCGG motif. FLEXseq strongly correlates (Pearson's r = 0.97) with whole genome bisulfite sequencing (WGBS) while enriching 18-fold. To demonstrate the broad applicability of FLEXseq, we verified its usage across cells, body fluids, and formalin-fixed paraffin-embedded (FFPE) tissues. DNA dilutions down to 250 pg decreased CpG coverage, but bias in methylation remained low (Pearson's r ≥ 0.90) compared to a 10 ng input. FLEXseq offers a cost-efficient, base-pair resolution methylome with potential as a diagnostic tool for tissue and liquid biopsies.

AI-Assisted High-Throughput Tissue Microarray Workflow

Immunohistochemical (IHC) studies of formalin-fixed paraffin-embedded (FFPE) samples are a gold standard in oncology for tumor characterization, and the identification of prognostic and predictive markers. However, despite the abundance of archived FFPE samples, their research use is limited due to the labor-intensive nature of IHC on large cohorts. This study aimed to create a high-throughput workflow using modern technologies to facilitate IHC biomarker studies on large patient groups. Semiautomatic constructed tissue microarrays (TMAs) were created for two tumor patient cohorts and IHC stained for seven antibodies (ABs). AB expression in the tumor and surrounding stroma was quantified using the AI-supported image analysis software QuPath. The data were correlated with clinicopathological information using an R-script, all results were automatically compiled into formatted reports. By minimizing labor time to 7.7%—compared to whole-slide studies—the established workflow significantly reduced human and material resource consumption. It successfully correlated AB expression with overall patient survival and additional clinicopathological data, providing publication-ready figures and tables. The AI-assisted high-throughput TMA workflow, validated on two patient cohorts, streamlines modern histopathological research by offering cost and time efficiency compared to traditional whole-slide studies. It maintains research quality and preserves patient tissue while significantly reducing material and human resources, making it ideal for high-throughput research centers and collaborations.

Blood Plasma Methylated DNA Markers in the Detection of Lymphoma: Discovery, Validation, and Clinical Pilot.

Lymphoma is one of the leading causes of cancer and cancer deaths and yet has not been amenable to population screening. The role of methylated DNA markers (MDMs) in the detection of lymphoma has not been extensively studied. We aimed to discover, validate, and test tissue-derived MDMs of lymphoma in archival plasma specimens. Reduced representation bisulfite sequencing (RRBS) was performed on a discovery set of frozen tissues. MDMs identified were converted to methylation-specific PCR assays and validated on independent formalin-fixed, paraffin-embedded (FFPE) tissues. Target enrichment long-probe quantitative-amplified signal (TELQAS) assays were developed and assayed in plasma-extracted, bisulfite-converted DNA from independent treatment-naïve lymphoma patients and healthy controls. Prediction of cancer status was modeled using random forest model with in silico cross-validation. After discovery and validation in tissue, 16 TELQAS assays (ZNF503, VWA5B1, HOXA9, GABRG3, ITGA5, MAX.chr17.7190, BNC1, CDK20, MAX.chr4.4069, TPBG, DNAH14, SYT6, CACNG8, FAM110B, ADRA1D, and NRN1) were selected for testing in plasma. These detected 78% (95% CI, 74%-82%) of lymphoma cases at 90% specificity. Excluding marginal zone and T-cell lymphomas, sensitivity increased to 84% (80%-88%). MDMs in plasma show promise to detect lymphoma and are candidates for inclusion in multi-cancer detection studies.

High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue.

Formalin-fixed, paraffin-embedded (FFPE) patient tissues are a valuable resource for proteomic studies with the potential to associate the derived molecular insights with clinical outcomes. Here we present an optimized, partially automated workflow for FFPE proteomics combining pathology-guided macro-dissection with Adaptive Focused Acoustics (AFA) sonication for lysis and decrosslinking, S-Trap digestion to peptides, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using Orbitrap, Astral or timsTOF HT instrumentation. The workflow enables analysis of up to 96 dissected FFPE tissue samples or whole 10 µM scrolls, identifying 8,000-10,000 unique proteins with <20% median CVs. Key optimizations include improved tissue lysis, protein quantification for normalization, and peptide cleanup prior to LC-MS/MS analysis. Application to lung adenocarcinoma (LUAD) FFPE blocks confirms the platform's effectiveness in processing complex samples, achieving deep proteome coverage and quantitative robustness comparable to TMT-based methods. Using the newly released Orbitrap Astral with short, 24-minute gradients, the workflow identifies up to ∼10,000 unique proteins and ∼11,000 localized phosphosites in LUAD FFPE tissue. This high-throughput, scalable workflow advances biomarker discovery and proteomic research in archival tissue samples.

Towards routine proteome profiling of FFPE tissue: insights from a 1,220-case pan-cancer study.

Proteome profiling of formalin-fixed paraffin-embedded (FFPE) specimens has gained traction for the analysis of cancer tissue for the discovery of molecular biomarkers. However, reports so far focused on single cancer entities, comprised relatively few cases and did not assess the long-term performance of experimental workflows. In this study, we analyze 1220 tumors from six cancer entities processed over the course of three years. Key findings include the need for a new normalization method ensuring equal and reproducible sample loading for LC-MS/MS analysis across cohorts, showing that tumors can, on average, be profiled to a depth of >4000 proteins and discovering that current software fails to process such large ion mobility-based online fractionated datasets. We report the first comprehensive pan-cancer proteome expression resource for FFPE material comprising 11,000 proteins which is of immediate utility to the scientific community, and can be explored via a web resource. It enables a range of analyses including quantitative comparisons of proteins between patientsand cohorts, the discovery of protein fingerprints representing the tissue of origin or proteins enriched in certain cancer entities.

P16 expression in patient with Loop Electrosurgical Excision Procedure (LEEP)

HIGHLIGHTS Clinicopathologic characteristic of patient with LEEP procedures dominated by women above 35 years old, with total parity less than 3 and without history of contraceptive usage. The p16 expression analyzed by immunohistochemistry technique. The positive and negative reactivity of p16 ratio was 3:5 on LEEP specimen which microscopically appear as chronic cervicitis, low-grade and high-grade squamous intraepithelial lesion. ABSTRACT Objective: The study aim was to reveal p16 expression with the clinicopathological characteristics of patients who underwent cervical biopsy using the Loop electrosurgical procedure (LEEP) at Udayana University Hospital for the period 2020 – 2023. Materials and Methods: The research was conducted at Anatomic Pathology Laboratory of Udayana University Hospital, Denpasar, Bali, Indonesia. The samples were selected based on inclusion criteria such as the formalin fixed paraffin embedded (FFPE) sample from positive IVA patient and continue for LEEP procedure. Otherwise, the exclusion criteria were moldy FFPE sample and incomplete clinical data. Then, p16 immunostaining procedure was carried out manually. The interpretation of p16 results was analyzed using SPSS software, version 25 by International Business Machines (IBM) Corporation. Results: The positive p16 expression was revealed in 12 samples (38.7%), in contrast the negative staining appeared in 19 samples (61.3%). Unfortunately, p16 expression was not significant statistically based on age, parity, and contraceptive history, with p-values of 1.00, 0.45, and 0.65, respectively. Meanwhile, a statistically significant association was found between p16 expression and histopathologic diagnostic (p = 0.02, 95% CI 1.4 – 38.3). In addition, 22.2% of the variation of p16 expression based on multivariate analysis demonstrating a significant correlation (p = 0.01). Conclusion: p16 expression with histopathology diagnostic characteristic in patient who underwent Loop Electrosurgical Excision Procedure (LEEP) was found statistically significant. Moreover, clinical application of p16 in daily practice should be performed with consideration especially for pre-cancer lesion in LEEP biopsy specimen procedure and clinicopathological approach is essential.

Abstract B004: Identification of distinct subpopulations of cancer associated fibroblasts in oral squamous cell carcinoma by imaging mass cytometry

Abstract Background: Although cancer associated fibroblast (CAF) heterogeneity is recognized in oral squamous cell carcinoma (OSCC), the phenotype and role of CAF subpopulations in OSCC is still unknown. Objectives: This study aimed to dissect CAF heterogeneity and spatial location by using imaging mass cytometry on formalin fixed paraffin embedded (FFPE) tissues from primary OSCC. Methods: Imaging mass cytometry (Hyperion technology) was used to identify 24 markers (E-cadherin, EGFR, Ki-67, α-SMA, CD140β, FAP, FSP-1/S100A4, CD90/Thy-1, integrin α-11, collagen-1, tenascin-C, caveolin-1, CD4, CD8a, CD16, CD20, CD68, CD163, FoxP3, granzyme B, CD31, podoplanin, CD146, and YAP-1) in HPV-negative OSCC lesions (n=10). Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R language. Results: Unsupervised clustering identified several major cell types, with E-cadherin+ cells being the most abundant (30%) and CD146+ being the least abundant cell type (0.62%). Within the immune compartment CD4+ cells were most abundant (11.72%) followed by CD8+ cells (10.6%) and CD68+ cells (10.25%). Cells negative for other major lineage markers and positive for stromal markers were considered as CAFs and they clustered in 6 clusters defined as CAF1 to CAF6. CAF1-3 were high in FAP and CD140β while CAF 4-6 were low expressing FAP and CD140β. CAF1 and 6 expressed high αSMA, while CAF 3 and 5 expressed high collagen-1. CAF6 had high expression of integrin α-11. CAF1 and 3 subtypes were found closer to immune cells whereas CAF2 and 6 were closely located to cancer cells. Conclusions: We identified six distinct subpopulations of CAFs in OSCC by using imaging mass cytometry. Their clinical implications can be further revealed by using this method on larger patient cohorts with FFPE tissues available in diagnostic biobanks. Citation Format: Stian Tornaas, Siren Fromreide, Dimitrios Kleftogiannis, Hans Jørgen Aarstad, Olav K. Vintermyr, Lars Akslen, Harsh Dongre, Daniela Elena Costea. Identification of distinct subpopulations of cancer associated fibroblasts in oral squamous cell carcinoma by imaging mass cytometry [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B004.

Abstract C040: Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders

Abstract Spatial transcriptomics is revolutionizing our understanding of cellular dynamics and interactions in the tumor microenvironment (TME). By mapping distinct cellular populations and their interactions, we can gain valuable insights into tumor behavior. To investigate these dynamics in breast cancer, we conducted Xenium In-situ spatial transcriptomics on twenty formalin-fixed paraffin-embedded (FFPE) samples taken from eight patients undergoing neoadjuvant chemotherapy. These samples have been taken pre-treatment, mid-treatment and post-treatment from four cases with pathological complete response (pCR), and four cases with residual disease (RD) at time of surgery. Our analyses have found distinct cellular composition changes between pCR and RD cases. Patients with RD demonstrated an increase in endothelial, perivascular-like (PVL) and epithelial cells from pre- to post-treatment. This was accompanied by a decrease in myeloid and malignant epithelial cells at mid-treatment. In contrast, patients with pCR demonstrated increases in cancer-associated fibroblasts (CAFs) and myeloid cells, with reductions in plasmablasts and cancer epithelial cells at mid-treatment. This suggests that an increase in CAF and myeloid cells observed between pre-treatment and mid-treatment samples may be predictive of response. We performed ligand-receptor (LR) analyses, which demonstrated diverse myeloid interactions in RD cases and diverse CAF interactions in pCR cases in pre-treatment samples. Mid-treatment samples exhibited a decrease in interactions for CAFs, myeloid and T cells in pCR and RD cases. The most frequently identified CAF LR interactions, as senders, were between TGFB1/TGFB3 and TGFBR1/TGFBR2, while the most frequently identified myeloid LR interactions were between PDCD1LG2 - PDCD1 (encoding PDL2 &amp; PD1), CCL8 - ACKR1 (encoding Atypical Chemokine Receptor 1) and CCL2 - ACKR1. Our findings indicate that the dynamic changes in cellular composition and interactions within the TME differs between responders and non-responders. To further explore these differences, we plan to perform spatially constrained analyses to identify unique cellular niches that may serve as potential biomarkers for informing novel therapeutic strategies. Citation Format: Jeremy Mo, Hanyun Zhang, Kate Harvey, Alexander Swarbrick. Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C040.

Abstract A022: Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues

Abstract Despite recent breakthroughs in spatial biology, either imaging-based or sequencing-based spatial transcriptomics is largely equivalent to spatial messenger RNA (mRNA) expression quantification. An RNA molecule experiences a complex life cycle involving transcription, splicing, maturation, translation, and degradation . It is highly desirable to profile all these RNA species spanning their life cycle to explore the biology of RNA at genome scale and cellular level. Furthermore, formalin-fixed paraffin-embedded (FFPE) tissues are essential in clinical practice, being the backbone of human disease histopathological diagnoses . Pathology departments have accrued vast collections of FFPE blocks over time, creating a rich, yet underutilized compendium of materials that, accompanied by clinical data, stands as a treasure trove for human biology and translational research . In this evolving landscape, we present “Pathology-compatible Deterministic Barcoding in Tissue” (Patho-DBiT) not only enabling spatial full-coverage base-by-base whole transcriptome sequencing but also crafted to address the challenges of clinically archived FFPE tissues. Patho-DBiT integrates in situ polyadenylation, microfluidic in tissue barcoding, and computational innovations to decode rich RNA biology inherent in FFPE samples. The platform capitalizes on RNA fragmentation naturally occurring in FFPE specimens and appends poly(A) tails to a broad spectrum of RNA species, thereby overcoming traditional barriers associated with FFPE samples and even outperforming the assays conducted with frozen tissues. Patho-DBiT permits spatial co-profiling of gene expression and alternative splicing, unveiling region-specific isoforms in the mouse brain. High-sensitivity transcriptomics is constructed from 5-year archived T-cell lymphoma tissues, with cross-validation conducted using super-resolution spatial phenotyping technology (CODEX). Furthermore, genome-scale single nucleotide RNA variants (SNVs) are captured to distinguish malignant subclones from non-malignant cells in human B-cell lymphomas, dissecting spatial clonal architectures based on both SNVs and copy number variation (CNV) profiles. Patho-DBiT also enables spatially resolved co-profiling of large and small RNAs, facilitating the analysis of a microRNA-mRNA regulatory network and tRNA utilization within clinical biopsies and elucidating their roles in tumorigenesis. With superior intronic read capture efficiency, Patho-DBiT spatially mapped RNA splicing dynamics associated with the developmental trajectory of tumor B cells. High resolution Patho-DBiT with a 10-μm spot size reveals the heterogeneities of human lymphomas within a spatial neighborhood and traces the spatiotemporal molecular kinetics driving tumorprogression at the cellular level. Patho-DBiT represents a first-of-its-kind technology, enabling the spatial exploration of rich RNA biology in FFPE tissues to aid in clinical pathology research. Citation Format: Zhiliang Bai, Dingyao Zhang, Yan Gao, Bo Tao, Mingyao Li, Yi Xing, Jun Lu, Mina Xu, Rong Fan. Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A022.

The role of histology in tissue engineering: Significance of complex morphological characterization of decellularized foreskin scaffolds

Decellularization is a technique of cell removal that prepares vacant extracellular matrices (ECMs), which can be reseeded with patient-specific cells and used in regenerative medicine. Histological methods are vital for protocol standardization and efficacy assessment, which are necessary for preparing such bioscaffolds. Foreskins from male subjects (n=20) aged three to fifteen years were harvested and decellularized using detergent-based and enzyme-based protocols. To acquire a standardized protocol that would provide the optimal balance between effective cell removal and ECM preservation, a total of seven protocols were evaluated using the most basic formalin-fixed paraffin-embedded (FFPE) tissue blocks, which were sectioned and stained with HE, Masson’s blue trichrome and orcein. After achieving the optimal result, we further morphologically characterized the scaffolds using scanning electron microscopy (SEM) and immunohistochemical (IHC) detection of fibronectin and collagen IV. Histology proved to be a quick, relatively easy, and cheap method to achieve protocol standardization and assess decellularization efficacy and ECM preservation. Morphological analysis via normal histology, SEM, and IHC showed that both definitive protocols are effective in cell removal but, at the same time, don’t compromise the structural integrity of the ECM. Morphological characterization of foreskin-derived bioscaffolds via normal histology, IHC, and SEM is irreplaceable for standardizing decellularization protocols, assessing the efficacy of cell removal, and evaluating ECM preservation.

Abstract A013: Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays

Abstract The availability of high-quality matched biological samples (tumor-blood) is the major limiting factor for research and development of novel, liquid biopsy-based assays in oncology. Frequently, ongoing specifically timed collections of blood samples are required. De-identified tissue blocks (formalin-fixed, paraffin embedded (FFPE) or flash frozen) matched with patient's blood samples collected in fixative-containing (Streck) of EDTA containing tubes, were collected at worldwide geographic locations and transferred to biobanking facility (Reference Medicine, Phoenix, AZ). Additional blood samples were collected at various times regarding the cancer surgery. Plasma and buffy coat samples were locally prepared and shipped to the biobank. A subset of blood samples without matched tissues from healthy donors were also collected. All samples were shipped under strict transport conditions. Quality control performed at the biobanking facility included pathology review and nucleic acids quality assessment. A total of over 3,000 cases were collected and provided to end-users working on various assays based on cfDNA (e.g. monitoring for minimal residual disease or multi-cancer early detection). Immuno-reactivity to various antigens in samples collected in Streck and EDTA tubes (from 50 patients) was assessed by peptide microarray binding assay. Feedback information on the quality of samples was shared in 900 cases. Approximately 4% of cases had failed internal pathology review, due to poor preservation, lack of adequate tissue composition, or low tumor volume and 11% failed internal nucleic acid quality assessment (DNA concetration and integrity). The blood collection tubes had no significant impact on serologic testing using peptide microarary assay. Customer feedback data showed that overall success rate exceeded 85%. Type of blood collection tubes (Streck vs EDTA) had no impact on serologic testing and studies looking at predicted neoantigens based on NGS data can be confirmed using peptide epitope arrays. Blood collected for NGS works without compromise on peptide arrays. Future validation of other collection tubes for blood or other types of fluids is needed to confirm their adequacy for specific assays. Citation Format: Zoran Gatalica, Phillip Stafford, Chris Diehnelt, Inga Rose. Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A013.

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome

BackgroundBreast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients.MethodsNinety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification.ResultsHierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683.ConclusionsOur data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

Imaging Mass Cytometry to Decipher the Maturation of Tertiary Lymphoid Structures.

In the realm of inflammation, including conditions like cancers, infections, and autoimmune diseases, the emergence of tertiary lymphoid structures (TLS) holds significant implications for prognosis and treatment response. Yet, traditional methodologies such as immunohistochemistry and immunofluorescence falter in capturing the nuanced complexities of TLS, necessitating innovative approaches, like imaging mass cytometry (IMC), to unravel their significance. With the capacity to concurrently assess nearly 40 markers within the same tissue section, IMC transcends the constraints of traditional approaches.This chapter delineates the IMC methodology tailored for TLS visualization and subsequent quantification. By leveraging IMC on formalin-fixed, paraffin-embedded (FFPE) sections of human colorectal cancer (CRC) tissues, we aim to establish a standardized protocol that elucidates the intricacies of TLS dynamics and composition.

Advancement of Techniques for Precise Visualization and Quantification of Tertiary Lymphoid Structure-Associated Immune Cells in Tissue Samples.

Tertiary Lymphoid Structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or the tumor microenvironment has been associated with the local development of an active immune response. While high densities of TLS are correlated with disease severity in autoimmune diseases or during graft rejection, it has been associated with longer patient survival in many cancer types and more recently with positive responses to anti-PD-1 immunotherapy. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. In clinical settings, the use of single-marker immunohistochemistry (IHC) protocols prevails in immune cell infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. In contrast, the development of automated multiplex immunofluorescence markings, i.e., 40-plex, requires very costly investments in equipment and analysis stations. Yet, employing two or more markers can enhance the characterization of immune infiltrates, particularly in the context of TLS. Besides the growing development of multiplex labeling approaches, imaging can also be used to overcome some technical difficulties encountered during the immunolabeling of tissues with several markers.This chapter describes IHC methods to visualize in human tissue (tumoral or not) the presence of TLS. These methods are based on the immunostaining of four TLS-associated immune cell populations, namely, follicular B cells, follicular dendritic Cells (FDCs), mature Dendritic Cells (mDCs), and Follicular Helper T cells (TFH), together with non-TFH T cells. Methodologies for subsequent quantification of TLS density are also proposed, as well as a virtual multiplexing method based on image registration using the open-source software ImageJ (IJ), aiming at co-localizing several immune cell populations from different IHC stainings performed on serial tissue sections.

The prognostic impact of CDKN2A/B hemizygous deletions in IDH-mutant glioma.

Homozygous deletions of CDKN2A/B are known to predict poor prognosis in gliomas, but the impact of hemizygous deletions is less clear. This study aimed to evaluate the prognostic significance of hemizygous CDKN2A/B deletions in IDH-mutant low-grade astrocytomas and oligodendrogliomas. Tissue samples diagnosed as astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3 were collected from the archives of the Institute of Neuropathology in Heidelberg. DNA methylation analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples. Evaluation of the CDKN2A/B locus was performed by visual inspection of copy-number plots derived from methylation-array data for each case. Hemizygous and homozygous losses were assessed in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan-Meier method. A total of 334 low-grade glioma cases were identified, including 173 astrocytomas and 161 oligodendrogliomas. Hemizygous deletions in CDKN2A/B (37/173 in astrocytomas, 15/161 in oligodendrogliomas) did not confer significantly worse survival outcomes compared to CDKN2A/B wildtype cases in neither low grade astrocytoma (log-rank p= 0.2556; HR 2.29, 95% CI [0.76; 6.40], p= 0.135) nor oligodendroglioma (log-rank p= 0.2760; HR 0.17; 95% CI [0.01; 5.05]; p= 0.305), regardless of CNS WHO grade, which was further demonstrated on a subgroup of astrocytoma, IDH mutant CNS WHO 4 cases (log-rank p= 0.1680; HR 4.55, 95% CI [0.88; 24.51], p= 0.0689). Hemizygous CDKN2A/B deletions do not significantly worsen OS or PFS in IDH-mutant astrocytomas and oligodendrogliomas, CNS WHO grade 2 and 3.

Automated Quantification of Tertiary Lymphoid Structures in Human Tumor Samples Using Immunofluorescence and AI-Powered Analysis Pipeline.

The tumor microenvironment (TME) is a complex entity comprising not only tumor cells but also immune, stromal, and endothelial cells. Preclinical and clinical studies indicate that the density, localization, function, and organization of immune infiltration can influence survival probability and treatment response in many cancers. Among these cell organizations, the clustering of T and B cells into tertiary lymphoid structures (TLS) has been associated with favorable clinical outcomes. In this protocol, we propose a protocol for 7-plex immunofluorescence staining for identifying six cell types enriched in TLS in formalin-fixed paraffin-embedded (FFPE) human tumor sections. Additionally, we provide a detailed methodology for quantifying these cell subtypes using the Halo-AI analysis software. This approach will enable a more precise and detailed characterization of TLS in the TME, opening new avenues for understanding their role in anticancer treatment response.

Single-cell spatial transcriptomics of fixed, paraffin-embedded biopsies reveals colitis-associated cell networks.

Imaging-based, single-cell spatial transcriptomics (iSCST) using formalin-fixed, paraffin-embedded (FFPE) tissue could transform translational research by retaining all tissue cell subsets and spatial locations while enabling the analysis of archived specimens. We aimed to develop a robust framework for applying iSCST to archived clinical FFPE mucosal biopsies from patients with inflammatory bowel disease (IBD). We performed a comprehensive benchmarking comparison of three iSCST platforms capable of analyzing FFPE specimens. We analyzed FFPE mucosal biopsies (n=57) up to 5 years old from non-IBD controls (HC; n=9) and patients with ulcerative colitis (UC;n=11). After platform-specific cell segmentation, we applied a uniform data processing pipeline to all datasets, including transcript detection, cell annotation, differential gene expression, and neighborhood enrichment. Transcriptomic signatures identified with iSCST were validated using external, publicly available bulk transcriptomic datasets. A custom 290-plex Xenium gene panel exhibited the highest sensitivity and specificity for transcript detection, enabling precise identification and quantification of diverse cell subsets and differentially expressed genes across cell types and disease states. We mapped transcriptionally distinct fibroblast subsets to discrete spatial locations and identified inflammation-associated fibroblasts (IAFs) and monocytes as a colitis-associated cellular neighborhood. We also identified signatures associated with Vedolizumab (VDZ) responsiveness. VDZ non-responders were characterized by an IAF-monocyte transcriptional signature, while responders exhibited enrichment of epithelial gene sets. Our optimized iSCST framework for archived FFPE biopsies provides unique advantages for assessing the role of colitis-associated cellular networks in routinely collected clinical samples. FFPE-based biomarkers could integrate with existing clinical workflows and potentially aid in risk-stratifying patients.

PATH-55. RAMAN-BASED MACHINE LEARNING PLATFORM REVEALS UNIQUE METABOLIC DIFFERENCES BETWEEN IDHMUT AND IDHWT GLIOMA

Abstract BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in Raman spectroscopy-based studies has been limited due to the background coming from embedding media. METHODS Spontaneous Raman spectroscopy was used for molecular fingerprinting of FFPE tissue from 46 patient samples with known methylation subtypes. Spectra were used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Support vector machine and random forest were used to identify the most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the frequencies identified. Mass spectrometry of glioma cell lines and TCGA were used to validate the biological findings. RESULTS Here we develop APOLLO (rAman-based PathOLogy of maLignant glioma) – a computational workflow that predicts different subtypes of glioma from spontaneous Raman spectra of FFPE tissue slides. Our novel APOLLO platform distinguishes tumors from nontumor tissue and identifies novel Raman peaks corresponding to DNA and proteins that are more intense in the tumor. APOLLO differentiates isocitrate dehydrogenase 1 mutant (IDH1mut) from wildtype (IDH1WT) tumors and identifies cholesterol ester levels to be highly abundant in IDHmut glioma. Moreover, APOLLO achieves high discriminative power between finer, clinically relevant glioma methylation subtypes, distinguishing between the CpG island hypermethylated phenotype (G-CIMP)-high and G-CIMP-low molecular phenotypes within the IDH1mut types. CONCLUSIONS Our results demonstrate the potential of label-free Raman spectroscopy to classify glioma subtypes from FFPE slides and to extract meaningful biological information thus opening the door for future applications on these archived tissues in other cancers.