Formalin-fixed Paraffin-embedded Sections Research Articles

A universal pipeline to combine spatial transcriptomics, proteomics, and diagnostic H&E assays on a single tissue section to study tissue microenvironment.

e14657 Background: Spatial multi-omics approaches, including spatial transcriptomics (ST) and spatial proteomics (SP), that elucidate the complex tissue microenvironment (TME) are gaining traction in cancer research studies. However, combining these approaches onto one tissue section remains challenging. ST methods visualize, locate and quantify RNA targets, evaluating the transcriptome of the TME at the subcellular level while SP methods stain and visualize protein biomarkers at the cellular level. Here we show a pipeline that combines these ST and SP methods with Hematoxylin and Eosin (H&E) approach on a single tissue section to achieve single slide multi-omics data with high resolution tissue morphology images. Methods: 5µm formalin-fixed paraffin-embedded sections of human lung cancer samples were placed on a Xenium (10x Genomics, Pleasanton, CA) slides and treated to expose RNA molecules. Probe hybridization was performed with a human lung panel kit containing 289 barcoded DNA padlock probes. Downstream ligation and amplification generated copies of the barcode and thereafter, the Xenium Analyzer performed imaging and data collection. The same tissue section was then stained and imaged in COMET (Lunaphore Technologies SA, Switzerland) using sequential immunofluorescence (seqIF) method to detect 40 protein targets in situ. Serial sections of the specimens were used for direct H&E and seqIF staining for comparative validation. Results: Multi-omics images generated from Xenium and COMET were overlayed, jointly analyzed and interpreted. Staining specificity of the protein targets for the post-Xenium slides remain comparable with the direct COMET-stained slide. 40-plex COMET staining intensity in post-Xenium slide was minimally weaker compared to direct COMET-stained slide. Additionally, H&E staining of the post-Xenium/COMET slide and direct COMET-stained slide were comparable to the direct H&E-stained section, demonstrating discernable tissue landscape features despite a fainter hematoxylin stain. Overall, tissue integrity and cell morphology were well-preserved across the different workflows performed on each section, and the protein targets remain relatively stable for labelling after in situ RNA detection process. Conclusions: We present a pipeline that combines two spatial omics approaches with the diagnostic standard H&E staining on a single tissue section, allowing the same cells to be assessed for their histological, proteomic, and transcriptomic information. A visual comparison of images showed minimal compromise to cellular morphology and protein stability at the end of the pipeline compared to direct staining of serial sections. This pipeline could be potentially generalized to most of the ST and SP approaches, thus advancing multi-omics cancer research and potentially clinical diagnostics in the future.

Exploiting spatial transcriptomics to investigate the molecular mechanisms involved in cardiac metastasis development

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): IEO-Monzino Foundation Background Cardiac metastasis is considered a relatively rare malignancy, although an increasing prevalence is expected in the future due to the increasing life expectancy. Several cases of cardiac metastases remain untreatable, given the anatomical location that often hampers both surgical eradication and bioptic collection for biological characterization. Here, we use spatial transcriptomics to provide an unbiased molecular characterization of cardiac metastases, and identify novel targets for their treatment. Material and methods We performed spatial transcriptomics on samples of primary tumor, cardiac, and extra-cardiac metastasis from three different patients. First, we checked RNA integrity by means of DV200 analysis. Spatial transcriptomic analysis was performed by using the GeoMX technology (Nanostring), which provides whole-transcriptome data along with spatial distribution information of the preselected samples from the candidate regions of interest (ROIs). ROIs were selected on the formalin-fixed paraffin-embedded sections by means of geometric shapes in combination with cell markers specific for tumor cells, cardiomyocytes and endothelial cells, respectively. Differential gene expression analysis and Gene Set Enrichment Analysis (GSEA) were performed on the entire transcriptome. Cell-cell interactions between cardiac cells and cancer cells were then evaluated by means of InterCellaR. Results Differential expression analysis between cardiac metastases and extra-cardiac tumors (primary tumors + extra-cardiac metastases) identified 92 differentially expressed genes (DEGs). Of these, 53 genes were significantly overexpressed in cardiac metastases, while 39 genes were overexpressed in the extra-cardiac samples, regardless of the site of origin of the tumor. Unsupervised hierarchical clustering of protein-coding genes clearly separated the samples according to their anatomical location, in particular cardiac metastasis from extra-cardiac tumors. GSEA identified several pathways enriched in cardiac metastases compared to the extra-cardiac tumors, including mechanotransduction (e.g. keratinization and cell-matrix interaction) and triglyceride metabolism, suggesting a potential role of these signaling pathways in the development of cardiac metastasis. Interactome analysis revealed a rich plethora of receptor-ligand interactions in cardiac metastasis, especially between tumor cells and cardiomyocytes. Conclusions These results start shedding light on the mechanisms that allow cancer cell growth in the heart and pave the way to the development of novel therapies to treat this malignancy.

RNA Polymerase II hypertranscription in cancer FFPE samples.

Hypertranscription is common in human cancers and predicts poor prognosis. However detection of hypertranscription is indirect, relying on accurately quantifying mRNA levels and estimating cell numbers. Previously, we introduced FFPE-CUTAC, a genome-wide method for mapping RNA Polymerase II (RNAPII) in formalin-fixed paraffin-embedded (FFPE) sections. Here we use FFPE-CUTAC to demonstrate genome-wide hypertranscription both in transgene-driven mouse gliomas and in assorted human tumors at active regulatory elements and replication-coupled histone genes with reduced mitochondrial DNA abundance. FFPE-CUTAC identified RNAPII-bound regulatory elements shared among diverse cancers and readily categorized human tumors despite using very small samples and low sequencing depths. Remarkably, RNAPII FFPE-CUTAC identified de novo and precisely mapped HER2 amplifications punctuated by likely selective sweeps including genes encoding direct positive regulators of RNAPII itself. Our results demonstrate that FFPE-CUTAC measurements of hypertranscription and classifications of tumors using small sections provides an affordable and sensitive genome-wide strategy for personalized medicine.

Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections.

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

Feasibility of intraoperative pathologic examination for sentinel lymph nodes during sentinel node navigation surgery in early gastric cancer: results of pathologic protocol for SENORITA trial.

During sentinel node navigation surgery in patients with gastric cancer, intraoperative pathologic examination of sentinel nodes is crucial in determining the extent of surgery. In this study, we evaluated the feasibility and accuracy of intraoperative pathologic protocols using data from a prospective, multicenter, randomized trial. A retrospective analysis was conducted using data from the SEntinel Node ORIented Tailored Approach trials from 2013 to 2016. All sentinel lymph nodes were evaluated during surgery with hematoxylin-eosin (HE) staining using a representative section at the largest plane for lymph nodes. For permanent histologic evaluation, sentinel basin nodes were stained with HE and cytokeratin immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) sections and examined with HE for three deeper-step sections at 200-μm intervals. The failure rate of identification by frozen section and the metastasis rate in non-sentinel basins were investigated. Of the 237 patients who underwent sentinel node basin dissection, 30 had lymph node metastases on permanent pathology. Thirteen patients had macrometastasis confirmed in frozen sections as well as FFPE sections (failure rate: 0%). Patients with negative sentinel nodes in frozen sections but micrometastasis in FFPE sections had no lymph node recurrence during the follow-up period (0%, 0/6). However, in cases with tumor-positive nodes in frozen sections, metastases in non-sentinel basins were detected in the paraffin blocks (8.3%, 2/24). The single-section HE staining method is sufficient for detecting macrometastasis via intraoperative pathological examination. If a negative frozen-section result is confirmed, sentinel basin dissection can be performed safely. Otherwise, standard surgery is required.

Neural-net-based cell deconvolution from DNA methylation reveals tumor microenvironment associated with cancer prognosis.

DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.

Immunohistochemical and molecular profiles of heterogeneous components of metaplastic breast cancer: a squamous cell carcinomatous component was distinct from a spindle cell carcinomatous component

Metaplastic breast carcinoma (MBC), a category of breast cancer, includes different histological types, which are occasionally mixed and heterogeneous. Considering the heterogeneity of cancer cells in a tumour mass has become highly significant, not only from a biological aspect but also for clinical management of recurrence. This study aimed to analyse the immunohistochemical and molecular profiles of each MBC component of a tumour mass. Twenty-five MBC tumours were histologically evaluated, and the most frequent MBC component (c) was squamous cell carcinoma (SCC), followed by spindle cell carcinoma (SpCC). A total of 69 components of MBC and non-MBC in formalin-fixed paraffin-embedded sections were examined for 7 markers by immunohistochemistry. SCC(c) were significantly PTEN negative and CK14 positive, and SpCC(c) were significantly E-cadherin negative and vimentin positive. Multivariate analyses revealed that immunohistochemical profiles of normal/intraductal (IC)(c), no special type (NST)(c), and MBC(c) differed; moreover, SCC(c) and SpCC(c) were distinctly grouped. PTEN gene mutation was detected only in SCC(c) (2/7), but not in SpCC(c). Next-generation sequence analyses for 2 cases with tumours containing SCC(c) demonstrated that PTEN gene mutation increased progressively from IC(c) to NST(c) to SCC(c). In conclusion, the immunohistochemical and molecular profiles of the SCC(c) of MBC are distinct from those of the SpCC(c).

Modification of the Hi-C Technology for Molecular Genetic Analysis of Formalin-Fixed Paraffin-Embedded Sections of Tumor Tissues.

Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development; it is also necessary for the choice of targeted therapy. The Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements, such as inversions and translocations. We propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues. The developed protocol allows to generate high-quality Hi-C data and detect all types of chromosomal rearrangements. We have analyzed various databases to compile a comprehensive list of translocations that hold clinical importance for the targeted therapy selection. The practical value of molecular genetic testing is its ability to influence the treatment strategies and to provide prognostic insights. Detecting specific chromosomal rearrangements can guide the choice of the targeted therapies, which is a critical aspect of personalized medicine in oncology.

Using the DNA Integrity Number to Analyze DNA Quality in Specimens Collected from Liquid-Based Cytology after Fine-Needle Aspiration of Breast Tumors and Lesions

Introduction: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. Methods: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. Results: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. Conclusion: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

Abstract 3753: Co-expression proteomic analysis of antibody drug conjugate receptor and payload biomarkers in breast cancer

Abstract The use of antibody drug conjugates (ADC) targeting HER2, and Trop 2 has made a great difference in the treatment of breast cancer. Such agents consist of an antibody targeting the receptor which is conjugated to a payload chemotherapy agent. Chemotherapy involves the use of anti-tubulin agents (e.g. docetaxel), topoisomerase inhibitors etc. We examined 507 breast cancer samples using targeted proteomics to quantitate biomarkers for ADC targets such as HER2, HER3, EGFR and Trop2 in our clinical proteomics (CLIA) laboratory. We also measured biomarkers that are associated with response and biomarkers that are associated with resistance to chemotherapeutic agents. Resistance markers include TUBB3 (taxanes), and response markers include TOPO1 (irinotecan, topotecan). Methods: Formalin fixed, paraffin embedded (FFPE) tissues blocks of breast cancer were sectioned, marked for tumor, laser capture microdissected, digested and analyzed by LC-selected reaction monitoring mass spectrometry. Seventy-two proteins were quantitated. Discussion:In our previous work, we have determined that >740 amol/µg HER2 corresponds to IHC 3+/2+ and FISH +. Based on the HER2 quantitation for each sample, the samples were segregated into three groups. Negative (Group A, <150 amol/µg), High expressors (Group C, >740 amol/µg), and Detected (Group B, 151-739 amol/µg). Groups A, B, and C were 39%, 46%, and 43% of the cases respectively. In Group A (HER2 negative), HER3 was detected in 14% of cases with a 3x range (181-602 amol/µg), EGFR was detected in 53% of samples with 139x range (108-14986 amol/µg), Trop2 was detected in 59% of cases ranging from 256 to 11900 amol/µg, Topo1 was detected in 86% of cases with a 12x range (402-4870 amol/µg) and TUBB3 was detected in 63% of cases at levels ranging from 755 to 11750 amol/µg. In Group B (HER2 detected), HER3 was detected in 38% with a 3x range (178-565 amol/µg), EGFR was detected in 34% at levels ranging from 104-7040 amol/µg, Trop2 was detected in 63% ranging from 157 to 12808 amol/µg, Topo1 was detected in 94% with a 12x range (413-5100 amol/µg), and TUBB3 was detected in 51% of cases at levels ranging from 757 to 9311 amol/µg.In Group 3 (HER2 highly expressed), HER3 was detected in 43% with a 5x range (189- 890 amol/µg), EGFR was detected in 30% at levels ranging from 101 to 565 amol/µg, Trop2 was detected in 68% ranging from 310 to14650 amol/µg, Topo1 was detected in 95% with a 6x range (487- 2885 amol/µg), and TUBB3 was detected in 64% of samples at levels ranging from 829 to 2300 amol/µg. Conclusion:The capacity to multiplex 72 protein biomarkers from 2-3 FFPE sections will help in design of ADCs bispecific antibodies and/or bispecific ADCs. Citation Format: Negin Ghafourian, Sheeno P. Thyparambil, Richard Mortensen, James Chou, Robert Heaton, Xuefeng B. Ling. Co-expression proteomic analysis of antibody drug conjugate receptor and payload biomarkers in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3753.

Abstract 94: Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling

Abstract The STING (Stimulator of Interferon Genes) pathway is a pivotal player in the innate immune response, responsible for detecting cytosolic accumulation of endogenous DNA and initiating a cascade of events leading to the production of pro-inflammatory type I interferons, which promote an antitumor environment through activation of T cells, dendritic cells, and natural killer cells. Therefore, STING agonists are currently being explored as a potential therapeutic for cancer treatment, often in combination with immune checkpoint inhibitors. While the STING pathway can be stimulated in multiple cell types, activation in antigen presenting cells, such as dendritic cells (DC), is crucial for antitumor activity in the tumor microenvironment (TME). Additionally, the type 1 interferon family consists of multiple subtypes, including IFNb and IFNa, which can produce differential biological responses. Although the STING pathway is known to induce expression of multiple cytokines, combined spatial characterization of STING and type I interferons in the TME has not been performed. To characterize STING-interferon expression in the TME, we use the Integrated MultiOmyx-RNAscope platform. MultiOmyxTM (NeoGenomics Laboratories, Inc) is a proprietary multiplex immunofluorescence (mIF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section and offers high-resolution spatial and quantitative analysis of protein expression in tissue samples. RNAscopeTM (Bio-Techne) Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. The Integrated MultiOmyx-RNAscope assay allows for simultaneous detection of both protein and RNA markers in a single sample. Herein we report the design and use of a novel panel of commercially-available antibodies and ISH probes broad enough to characterize various immune subpopulations and cytokine expressing cells, including DCs and interferons, in the TME of a variety of tumor indications including melanoma and head and neck squamous cell carcinoma. Quantification and analysis will be performed using NeoLYTXTM, the proprietary MultiOmyx Analytics pipeline, to examine the spatial distribution and expression levels of key STING pathway induced cytokines to elucidate the dynamic communication of signaling molecules and their localization within specific cell populations. Understanding of the variety and phenotype of STING/cytokine expressing cells in the TME is crucial to define the populations being targeted by therapies for cancer treatment. Citation Format: Sandra Lam, Courtney Todorov, Jiong Fei, Harry Nunns, Eric Leones, Marianne Thio, Flora Sahafi, Erinn Parnell, Qingyan Au. Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 94.

O237: Conversion of human kidney tissue to Bombay type using FucOB enzyme

Abstract Introduction The Bombay phenotype (Oh) is characterised by an absence of H antigen expression on red blood cells and vascular endothelium. The Oh phenotype is rare but most prevalent in the Indian subcontinent where it poses significant challenges in performing blood transfusions and kidney transplants, as Oh recipients can only receive from Oh blood type donors. The enzyme α-1,2-fucosidase (FucOB) derived from Akkermansia muciniphila bacteria has been shown to hydrolyse blood group H antigens. This study examined the efficiency of the FucOB enzyme on O type human kidney tissue to obtain Bombay phenotype. Methods H antigen removal was investigated in formalin-fixed paraffin embedded (FFPE) human kidney cortical biopsies from blood group O-type donors (n=8). Biopsies were incubated at 37OC for 1h with FucOB enzyme at concentrations 0µg/ml, 0.05µg/ml, 0.1µg/ml, 0.5µg/ml and 5µg/ml and inactive enzyme (iFucOB; 5µg/ml). Quantitative immunofluorescence was used to determine co-localisation of H antigen and vascular endothelium (Ulex). Results Increasing concentrations of FucOB progressively removed H antigen from FFPE sections of renal cortex. When compared to untreated control and iFucOB, a FucOB concentration of 5µg/ml caused 95% loss of H antigen expression on renal vascular endothelium (p<0.0001; Friedman test with Dunn’s multiple comparisons test). Conclusion This is the first description of enzymatic cleavage of H antigen from human O-type kidney tissue. The next step is to deliver FucOB enzyme to human kidneys during normothermic machine perfusion. This strategy has the potential to dramatically increase the availability of ABO compatible kidneys from individuals with Bombay phenotype.

Effects of melatonin and metformin on the ovaries of rats with polycystic ovary syndrome

To study the combined and isolated effects of melatonin and metformin in the ovarian tissue of rats with PCOS. Experimental study using a rat model of PCOS induced by continuous light exposure. Forty adult female rats were divided into 5 groups: physiological estrus phase (Sham); permanente estrus with PCOS induced by continuous lighting exposure for 60 consecutive days (control); with PCOS treated with melatonin; with PCOS treated with metformin; with PCOS treated with melatonin + metformin. After 60 days of treatments, all rats were killed, and ovaries were collected and processed for paraffin-embedding. Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin or subjected to immunohistochemistry for proliferation (Ki-67) and apoptosis (cleaved caspase 3) detection markers. Federal University of São Paulo, Brazil. Forty adult female Wistar rats (Rattus norvegicus albinus). Number of corpus luteum and ovarian cysts, number of ovarian follicles (primary and antral follicles), number of interstitial cells, percentage of ovarian follicles (primary and antral follicles), and of interstitial cells immunostained to cleaved caspase-3 and Ki-67. Absence of corpus luteum, a higher number of cysts, and increased nuclear volume and area of interstitial cells, along with a decrease in primary and antral follicle numbers, were noticed in the control group compared with the Sham group. Melatonin and metformin treatments attenuated these effects, although the combined treatment did not mitigate the increased number of cysts and ovaries induced by PCOS. An increase in theca interna cell apoptosis was observed in the control group, whereas melatonina and metformin treatments reduced it significantly. A higher percentage of caspase-3-immunostained granulosa cells was noted in the Sham and all treated groups compared with the control group; no aditive effects on ovarian cell apoptosis were observed in the combined treatment. The percentage of Ki-67- immunostained granulosa cells was significantly higher in the control group compared with the Sham group. However, the combined treatment, not melatonin and metformin alone, mitigated this effect. A higher percentage of Ki-67-immunostained interstitial cells was observed in all treated groups compared with the Sham and control groups, whereas no additive effects in that immunoreactivity were observed in the combined treatment. Melatonin and metformin may improve ovarian function in rats with PCOS. The combined melatonin and metformin treatment is more effective in attenuating excessive granulosa cell proliferation, but it is not more effective in improving ovarian function than these drugs applied alone in rats with PCOS.

Biopsy Proteome Scoring to Determine Mucosal Remodeling in Celiac Disease

Histologic evaluation of gut biopsy specimens is a cornerstone for diagnosis and management of celiac disease (CeD). Despite its wide use, the method depends on proper biopsy orientation, and it suffers from interobserver variability. Biopsy proteome measurement reporting on the tissue state can be obtained by mass spectrometry analysis of formalin-fixed paraffin-embedded tissue. Here we aimed to transform biopsy proteome data into numerical scores that give observer-independent measures of mucosal remodeling in CeD. A pipeline using glass-mounted formalin-fixed paraffin-embedded sections for mass spectrometry-based proteome analysis was established. Proteome data were converted to numerical scores using 2 complementary approaches: a rank-based enrichment score and a score based on machine-learning using logistic regression. The 2 scoring approaches were compared with each other and with histology analyzing 18 patients with CeD with biopsy specimens collected before and after treatment with a gluten-free diet as well as biopsy specimens from patients with CeD with varying degree of remission (n= 22). Biopsy specimens from individuals without CeD (n= 32) were also analyzed. The method yielded reliable proteome scoring of both unstained and H&E-stained glass-mounted sections. The scores of the 2 approaches were highly correlated, reflecting that both approaches pick up proteome changes in the same biological pathways. The proteome scores correlated with villus height-to-crypt depth ratio. Thus, the method is able to score biopsy specimens with poor orientation. Biopsy proteome scores give reliable observer and orientation-independent measures of mucosal remodeling in CeD. The proteomic method can readily be implemented by nonexpert laboratories in parallel to histology assessment and easily scaled for clinical trial settings.

Histomorphometric and molecular characterization of stromal and mineralized components in fibro-osseous lesions.

Indistinct and analogous histopathological features of various fibro-osseous lesions make establishing a definitive diagnosis a challenge. There is a need for additional molecular and histochemical tools to support and differentiate these lesions in order to establish a concrete diagnosis. A retrospective analysis of biopsied lesions in formalin-fixed paraffin-embedded sections (10 cases each of fibrous dysplasia, ossifying fibroma, and cement-osseous dysplasia) retrieved from the archives was studied for immunoexpression of osteocalcin (quantitative analysis in osteocytes), collagen characterization using Azan, Picrosirus, and Toluidine blue stain for evaluating intensity and localization of collagen fibers, and morphometric analysis of vasculature (for evaluating mean vessel density as square microns). Positive immunostaining of osteocalcin suggested mutations of the GNAS-1 gene found in fibrous dysplasia indirectly, as it is a negative regulator of bone formation. Osteocalcin immunopositivity was quantitatively measured in the fibro-osseous lesions, with fibrous dysplasia measuring 14.47 ± 3.628 as compared to ossifying fibroma measuring 5.23 ± 1.33, followed by cemento-osseous dysplasia measuring 2.30 ± 1.409. Toluidine blue suggests the presence of oxytalan fibers (resistant to acid hydrolysis) in ossifying fibroma and cemento-osseous dysplasia, pointing toward the pathogenesis of the lesion. Azan stain and Picrosirus (under a polarizing microscope) helped in distinguishing hard tissue characteristics (70% of cases of fibrous dysplasia showed only a magenta component followed by intermixed magenta with a blue component in 20% of cases and only 10% of cases showed magenta with blue borders whereas for ossifying fibroma, 40% of cases depicted magenta with blue borders along with the other 40% with intermixed magenta with blue component). The mean vessel density was also highest in fibrous dysplasia measuring 7.90 ± 1.079 (in Sq. micron area), followed by ossifying fibroma and cemento-osseous dysplasia. The diagnosis of fibro-osseous lesions by hematoxylin and eosin alone is confusing and thus should be supported by relatively simple histomorphometric analysis for better treatment outcomes. At the diagnostic stage of fibro-osseous lesions, evaluation of intralesional vessel size, reliable molecular marker, and histochemical nature can aid in differentiating fibrous dysplasia from central ossifying fibroma and cemento-osseous dysplasia alongside, other clinical, radiographic and pathological criteria. These parameters help in the diagnostic decision-making of fibro-osseous lesions.

Tissue of origin prediction for cancer of unknown primary using a targeted methylation sequencing panel

RationaleCancer of unknown primary (CUP) is a group of rare malignancies with poor prognosis and unidentifiable tissue-of-origin. Distinct DNA methylation patterns in different tissues and cancer types enable the identification of the tissue of origin in CUP patients, which could help risk assessment and guide site-directed therapy.MethodsUsing genome-wide DNA methylation profile datasets from The Cancer Genome Atlas (TCGA) and machine learning methods, we developed a 200-CpG methylation feature classifier for CUP tissue of origin prediction (MFCUP). MFCUP was further validated with public-available methylation array data of 2977 specimens and targeted methylation sequencing of 78 Formalin‐fixed paraffin‐embedded (FFPE) samples from a single center.ResultsMFCUP achieved an accuracy of 97.2% in a validation cohort (n = 5923) representing 25 cancer types. When applied to an Infinium 450 K array dataset (n = 1052) and an Infinium EPIC (850 K) array dataset (n = 1925), MFCUP achieved an overall accuracy of 93.4% and 84.8%, respectively. Based on MFCUP, we established a targeted bisulfite sequencing panel and validated it with FFPE sections from 78 patients of 20 cancer types. This methylation sequencing panel correctly identified tissue of origin in 88.5% (69/78) of samples. We also found that the methylation levels of specific CpGs can distinguish one cancer type from others, indicating their potential as biomarkers for cancer diagnosis and screening.ConclusionOur methylation-based cancer classifier and targeted methylation sequencing panel can predict tissue of origin in diverse cancer types with high accuracy.

CT imaging features of lung ground-glass nodule patients with upgraded intraoperative frozen pathology

PurposeIntraoperative frozen section pathology (FS) is widely used to guide surgical strategies while the accuracy is relatively low. Underestimating the pathological condition may result in inadequate surgical margins. This study aims to identify CT imaging features related to upgraded FS and develop a predictive model.MethodsCollected data from 860 patients who underwent lung surgery from January to December 2019. We analyzed the consistency rate of FS and categorized the patients into three groups: Group 1 (n = 360) had both FS and Formalin-fixed Paraffin-embedded section (FP) as non-invasive adenocarcinoma (IAC); Group 2 (n = 128) had FS as non-IAC but FP as IAC; Group 3 (n = 372) had both FS and FP as IAC. Clinical baseline characteristics were compared and propensity score adjustment was used to mitigate the effects of these characteristics. Univariate analyses identified imaging features with inter-group differences. A multivariate analysis was conducted to screen independent risk factors for FS upgrade, after which a logistic regression prediction model was established and a receiver operating characteristic (ROC) curve was plotted.ResultsThe consistency rate of FS with FP was 84.19%. 26.67% of the patients with non-IAC FS diagnosis were upgraded to IAC. The predictive model’s Area Under Curve (AUC) is 0.785. Consolidation tumor ratio (CTR) ≤ 0.5 and smaller nodule diameter are associated with the underestimation of IAC in FS.ConclusionCT imaging has the capacity to effectively detect patients at risk of upstaging during FS.

Abstract C109: Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment

Abstract The prognostic significance of basal-like and classical subtypes of pancreatic ductal adenocarcinoma (PDAC) is well-established, and ongoing clinical trials are poised to validate the predictive utility of such subtypes in guiding first-line treatment decision-making. While the Purity-independent (PurIST)-based subtyping strategy provides a method for PDAC subtype determination based on the Nanostring gene expression quantification platform, the utility of this platform has not been tested in a real-world patient setting. Here, we leverage a retrospective dataset of patients with resectable PDAC (n=299) that received Nanostring sequencing in combination with comprehensive clinical metadata curation and histopathological analysis. All sequencing analyses were performed using formalin-fixed paraffin-embedded (FFPE) tumor whole sections. The proportion of basal-like cases (15%) was comparable with other resectable PDAC cohorts, and patients with basal-like tumors showed reduced overall survival (p=3.9e-4). Histopathological quantification of epithelial, stromal and normal components in each sample revealed significantly (p=0.007) reduced classical gene expression in stroma-high samples. Out of 29 patients with repeat sampling from different FFPE blocks derived from the same tumor and surgery, 31% of tumors received discordant subtypes calls and increases in the amount of stroma sequenced between two samples from the same tumor was negatively correlated with expression of classical subtype genes (p=0.02). Taken together, these data demonstrate the presence of intra-tumoral subtype heterogeneity, highlight the quantity of stroma sequenced as a major contributing factor in PurIST-based subtype determination and generate insight into the reproducibility of this subtyping strategy in a real-world clinical setting. Citation Format: James T. Topham, Steve E. Kalloger, Joanna M. Karasinska, Jenny E. Chu, Hassan Ali, Dongxia Gao, Christine Chow, Andrew Metcalfe, Jonathan M. Loree, David F. Schaeffer, Daniel J. Renouf. Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C109.

Pilot testing of a simple alternative tissue processing method for immunofluorescence staining

Background: The image quality of tissues obtained with high-resolution imaging systems depends on several factors including the quality of sections prepared by different tissue processing methods. Fresh frozen sections and formalin-fixed paraffinembedded (FFPE) sections are the two routinely employed methods in histopathological examination having their own merits and demerits. Aims and Objectives: We tested the feasibility of a hybrid tissue processing method that could reduce the processing time without compromising the quality of processed and stained tissues. Materials and Methods: Bovine pancreatic tissue was utilized to standardize the process. The steps included fixation of tissue in formalin, followed by embedding in optimal cutting temperature compound and then cryo-sectioning to obtain 50 μm slices for immunostaining. The prepared thick slices were immunostained with islet markers and assessed by confocal imaging. Results: The images revealed that the staining quality was comparable with FFPE sections, though the tissue processing time was significantly curtailed. The modified Leica microsystems scoring system revealed a nearly 80% overall total score for the processed tissue specimens, indicating acceptable levels of quality of the tissues. We also observed that the staining quality of these formalin-fixed cryo-slices was preserved without tissue deterioration for up to 1 month when stored in formalin, indicating its storage potential for future applications. Conclusion: The proof of concept of the hybrid tissue processing method tested in this study will encourage researchers to extrapolate its use to the human pancreas, other solid organs, and other staining techniques for evaluating its general utility.

Drusen classification and quantification in pseudophakic postmortem eyes implanted with or without blue-light filtering intraocular lenses

Abstract Purpose: To investigate the effect of blue-light filtering intraocular lenses on drusen formation in postmortem eyes via histopathological analysis. Materials and Methods: One hundred and ninety-three postmortem pseudophakic eyes (100 with a clear intraocular lenses [cIOL] and 93 with a yellow, yellow intraocular lenses [yIOL]) were obtained from the Lions Gift of Sight eye bank. Sex, age-at-surgery, age-at-death, surgery-to-death time, and clinical history were collected. Eyes were sectioned on their coronal and sagittal axes, and formalin-fixed paraffin-embedded macular cross sections were obtained. Sections were stained with hematoxylin and eosin and scanned with the Zeiss Axio Scan. Z1 scanner. Drusen were classified by type, size or subtype, and quantity. Results: Large, soft drusen were present in 49% (n = 95) of eyes, 9% (n = 17) had cuticular drusen, 16% (n = 30) had hard drusen, and 26% (n = 51) had no drusen. There were significantly more cIOL eyes with large, soft drusen (P < 0.001). There were significantly more yIOL eyes with no drusen (P < 0.0001). No significant differences in the presence of hard or cuticular drusen were found. yIOL eyes had significantly higher mean age-at-surgery (P < 0.001) and mean age-at-death (P < 0.05), while cIOL eyes had a significantly higher mean surgery-to-death time (P < 0.05). Finally, significantly more yIOL eyes had a history of smoking (P < 0.01) and hypertension (P < 0.05), while significantly more cIOL eyes had a history of glaucoma (P < 0.05). Conclusions: Large, soft drusen were significantly less prevalent in yIOL eyes than in cIOL eyes and significantly more yIOL eyes had no drusen. These findings suggest that yIOLs may prevent the incidence and development of age-related macular degeneration after cataract surgery.