Formalin-fixed And Paraffin-embedded Research Articles

Large-scale analysis of whole genome sequencing data from formalin-fixed paraffin-embedded cancer specimens demonstrates preservation of clinical utility

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England’s 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an “FFPEImpact” score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.

Large field of view and spatial region of interest transcriptomics in fixed tissue

Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and formalin fixation and paraffin embedding (FFPE) tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis to promote the development of transcriptomics.

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3–4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Expression of somatostatin receptors in canine and feline meningioma.

The standard treatment for canine and feline meningiomas includes radiotherapy, surgical excision or combined therapy. However, new therapeutic approaches are required due to the possible recurrence or progression of meningiomas despite initial therapy. Adjunctive therapy with synthetic long-acting somatostatin (SST) analogues has been described in humans with SST-expressing tumours. The expression of SST receptors (SSTRs) by feline meningiomas is currently unknown, and there are little data about canine meningiomas. We hypothesized that SSTR is expressed by canine and feline meningiomas (S1). Seven canines and 11 felines with histologically confirmed meningiomas underwent STTR screening. RNA expressions of SSTR1, SSTR2, SSTR3 and SSTR5 (canine) and SSTR1-SSTR 5 (feline) in fresh frozen and formalin-fixed and paraffin-embedded (FFPE) samples were investigated using real-time (RT)-qPCR. The expression of SSTR1 and SSTR2 in FFPE samples was evaluated using immunohistochemistry (IHC). The specificity of applied antibodies for canine and feline species was confirmed by western blotting. In canine meningiomas (n=7), RNA expression of SSTR1, SSTR2 and SSTR5 was detected in all samples; SSTR3 RNA expression was detected in only 33% of samples. In feline meningiomas (n=12), RNA expression of SSTR1, SSTR4, SSTR5 and SSTR2 was detected in 91%, 46%, 46% and 36% of samples, respectively; SSTR3 was not expressed. Overall, the detection rate was lower in FFPE samples. IHC revealed the expression of SSTR1 and SSTR2 in all samples from both species. However, it is important to exercise caution when interpreting IHC results due to the presence of diffuse background staining. SSTRs are widely expressed in canine and feline meningiomas, thereby encouraging further studies investigating SSTR expression to conduct trials about the effect of adjunctive therapy with long-acting SST-analogues.

Poster 352: Histopathological Etiology of Mucoid Degeneration of the Anterior Cruciate Ligament

Objectives: Mucoid degeneration of the anterior cruciate ligament (ACL) is an uncommon clinical condition with reported prevalence below 0.5%. There is a paucity of histopathological description in the literature, which has limited our understanding of the underlying pathophysiology. Such limited understanding has resulted in treatment options limited to mechanical debridement rather than biologic interventions to prevent development or progression of mucoid degeneration. The purpose of this study is to define the histopathology of mucoid degeneration of the ACL to better guide future biologic and surgical management options. Methods: This is a case control study where a retrospective series of 10 patients were identified to have mucoid degeneration of the ACL from 2002 to 2023. They were evaluated and treated by two senior attending orthopedic sports medicine surgeons. Magnetic resonance images (MRI) were evaluated for multiple anatomic and morphometric measures. Tissue samples were obtained at the time of arthroscopic partial debridement of the ACL for all patients. Intra-operative ACL samples and cadaveric control tissues were prepared for histologic examination of microstructure and composition, and gene expression using multiplex transcriptional profiling. For the gene expression analysis RNA was isolated from archived formalin-fixed and paraffin-embedded (FFPE) sections. Three consecutive 6mm sections were pooled to generate one RNA sample and total RNA was extracted using a High Pure FFPET RNA Isolation Kit per the manufacturer’s protocol and stored at -80°C until further analysis. RNA concentration and integrity was evaluated using an Agilent BioAnalyzer 2100 and a corrected input of 50ng of total RNA was used for NanoString analyses using the NanoString nCounter® Human Fibrosis V2 Panel for multiplex molecular analysis of 770 genes. Analysis was performed using nSolver 4.0 Software and only probes with counts above background were included following the manufacturer instructions. Results: All 10 patients reported pain and limited knee flexion. MRI exhibited a bulbous appearance of the ACL. There was an increased posterior tibial slope (PTS), narrow notch width index, and increased signal intensity of the ACL. Medial meniscus tear and cartilage degeneration were reported with high prevalence. The histological analysis showed significant differences in the architecture of the tissue compared with normal ACL controls, with disorganized collagen matrix and increased glycosaminoglycan content. NanoString analysis demonstrated a total of 155 differentially expressed genes (DEGs) between the mucoid degeneration and control groups after adjustments for multiple comparisons (p<0.05), with the top 20 DEGs summarized in Table 1. Among all the DEGs identified the 5 most upregulated ones include Fibronectin 1 (FN1), COL5A1, COL6A3, COL3A1 and COL1A2. There were also significant differences in the pathway scores for epithelial-to-mesenchymal transition (EMT), Extracellular matrix Degradation and Synthesis, Collagen Biosynthesis, Focal Adhesion Kinase, PDGF Signaling and PI3K-Akt (Fig. 1). The histology findings demonstrate distinct changes in the structure and composition of the ACL, and we hypothesize that repetitive microtrauma of the ACL may lead to cumulative damage that ultimately results in mucoid degeneration. Increased posterior tibial slope and narrow notch width index may contribute to cumulative loading of the ligament and subsequent mucoid degeneration. Identification of biomechanical risk factors may help to identify high risk patients. Associated injury of the medial meniscus and medial compartment cartilage also suggests that mucoid degeneration of the ACL may be part of an early degenerative process in the knee. Conclusions: Mucoid degeneration of the ACL is an uncommon condition that may represent the result of repetitive microtrauma to the ligament. Current interventions address the condition after it has progressed to an irreversible biological state. By defining the cellular and molecular mechanism(s) of the condition, we aim to broaden treatment options to potentially identify biologic interventions. The association of mucoid degeneration and medial compartment osteoarthritis suggests that patients may benefit from earlier interventions. [Figure: see text][Figure: see text]

Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

Prediction model for pheochromocytoma/paraganglioma using nCounter assay.

World Health Organization defined pheochromocytomas/paragangliomas (PPGL) as malignant tumors in 2017 because the existing classification system could not reflect locally aggressive behavior sufficiently. However, predicting the likelihood of metastasis remains a crucial part of the treatment strategy. From one tertiary care hospital and onesecondary hospital, 97 PPGL cases were selected. Medical records of PPGL cases with the presence of formalin-fixed and paraffin-embedded (FFPE) tissue of primary lesion were reviewed. For FFPE tissues, a nCounter assay was conducted to determine differently expressed genes between metastatic and non-metastatic PPGL groups. Performances of prediction models for the likelihood of metastasis were calculated. Of a total of 97 PPGL cases, 39, 20, and 38 were classified as benign, malignant, and validation, respectively. In the nCounter assay, CDK1, TYMS, and TOP2A genes showed significant differences in expression. Tumor size was positively correlated with CDK1 expression level. The Lasso regression model showed supreme performance of sensitivity 91.7% and specificity 95.5% when those significant factors were considered. Machine learning of multi-modal classifiers can be used to create a prediction model for metastasis of PPGL with high sensitivity and specificity using nCounter assay. Moreover, CDK1 inhibitors could be considered for developing drug treatment.

Virtual formalin-fixed and paraffin-embedded staining of fresh brain tissue via stimulated Raman CycleGAN model.

Intraoperative histology is essential for surgical guidance and decision-making. However, frozen-sectioned hematoxylin and eosin (H&E) staining suffers from degraded accuracy, whereas the gold-standard formalin-fixed and paraffin-embedded (FFPE) H&E is too lengthy for intraoperative use. Stimulated Raman scattering (SRS) microscopy has shown rapid histology of brain tissue with lipid/protein contrast but is challenging to yield images identical to nucleic acid-/protein-based FFPE stains interpretable to pathologists. Here, we report the development of a semi-supervised stimulated Raman CycleGAN model to convert fresh-tissue SRS images to H&E stains using unpaired training data. Within 3 minutes, stimulated Raman virtual histology (SRVH) results that matched perfectly with true H&E could be generated. A blind validation indicated that board-certified neuropathologists are able to differentiate histologic subtypes of human glioma on SRVH but hardly on conventional SRS images. SRVH may provide intraoperative diagnosis superior to frozen H&E in both speed and accuracy, extendable to other types of solid tumors.

Predominance of T-bet-positive Th1 cells in infiltrating T-lymphocytes in most of active arteritis lesions of giant cell arteritis.

Immunological mechanisms through the activation of CD4-positive T-cells have been assumed to be involved in the pathogenesis of giant cell arteritis (GCA). Many studies employing frozen tissues of temporal artery biopsy, peripheral blood lymphocytes, and plasma of GCA patients have revealed the contribution of interferon-γ and interleukin-17 in both protein and mRNA levels. However, the analyses using formalin-fixed and paraffin-embedded (FFPE) tissue specimens, in which the correlation between histopathologic pictures and immunological circumstances would be elucidated, have been limited. Here, we performed the immunohistochemical analyses of infiltrating small lymphocytes in GCA lesions using FFPE specimens, especially of the subsets of CD4-positive T-cells by immunohistochemistry with antibodies against T-bet, GATA-3, RORγT, and Foxp3, which is the differentiation-specific transcription factor for Th1, Th2, Th17, and Treg cells, respectively. In these slides, the nuclear-positive staining is much more clearly and easily identifiable than the cytoplasmic staining for cytokines. The results indicate the predominance of T-bet-positive Th1 cells in infiltrating T-cells in most of active arteritis lesions of GCA. Furthermore, our data suggest the possible immunosuppressive microenvironment induced by T-reg cells and M2-type macrophages in the arteritis lesions throughout the course of GCA inflammation.

Characteristics of innate and adaptive immune disorder in IgA nephropathy based on integrated bioinformatics.

Our study aims to investigate the immunological pathogenesis underlying immunoglobulin A nephropathy (IgAN) and explore potential biomarkers for IgAN diagnosis. Differentially expressed genes (DEGs) of formalin-fixed and paraffin-embedded (FFPE) samples were screened between IgAN patients and healthy people based on GSE115857. Gene oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment was performed to identify related biological processes and pathways. CIBERSORT was utilized to seek the relationship of immune cell infiltration with IgAN. Finally, the expression of paraoxonase 2 (PON2) related to innate immune response was verified in FFPE samples of minimal change disease and IgAN patients by immunohistochemistry and PAS staining. 25 down-regulated genes and 12 up-regulated genes were identified in IgAN patients, which mainly responded to endothelial cell proliferation, inflammatory response, and angiogenesis. Toll-like receptor signaling pathway and Epstein-Barr virus (EBV) infection might be involved in IgAN pathogenesis. In addition, the infiltration of macrophages M0, naïve B cells, and follicular helper T (Tfh) cells was positively correlated in IgAN patients. Macrophages M1 and M2 infiltration were up-regulated in IgAN patients, which indicated that innate immune response was closely associated with IgAN. Besides, the results of immunohistochemistry showed that PON2 was obviously positively expressed in acute and chronic lesions of IgAN patients. In addition to abnormalities in the adaptive immune response, macrophages M1/M2 and innate immune disorder may participate in IgAN pathogenesis. PON2 may become the feasible targets for further investigation of IgAN.

Pathological investigation of high pathogenicity avian influenza H5N8 in captive houbara bustards (Chlamydotis undulata), the United Arab Emirates 2020

At the end of 2020, an outbreak of HPAI H5N8 was registered in captive African houbara bustards (Chlamydotis undulata) in the United Arab Emirates. In order to better understand the pathobiology of this viral infection in bustards, a comprehensive pathological characterization was performed. A total of six birds were selected for necropsy, histopathology, immunohistochemistry, RNAscope in situ hybridization and RT-qPCR and nanopore sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue blocks. Gross lesions included mottled and/or hemorrhagic pancreas, spleen and liver and fibrinous deposits on air sacs and intestine. Necrotizing pancreatitis, splenitis and concurrent vasculitis, hepatitis and fibrino-heterophilic peritonitis were identified, microscopically. Viral antigens (nucleoprotein) and RNAs (matrix gene) were both detected within necro-inflammatory foci, parenchymal cells, stromal cells and endothelial cells of affected organs, including the myenteric plexus. Molecular analysis of FFPE blocks successfully detected HPAI H5N8, further confirming its involvement in the lesions observed. In conclusion, HPAI H5N8 in African houbara bustards results in hyperacute/acute forms exhibiting marked pantropism, endotheliotropism and neurotropism. In addition, our findings support the use of FFPE tissues for molecular studies of poorly characterized pathogens in exotic and endangered species, when availability of samples is limited.

Abstract B107: Spatial transcriptomics analysis of PanIN reveals loss of pro-inflammatory signaling and the presence of cancer-associated fibroblasts

Abstract Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that potentially progresses into pancreatic ductal adenocarcinoma (PDAC). Since PanIN diagnosis is restricted to formalin-fixed and paraffin-embedded (FFPE) samples, little is known about PanIN interactions with the surrounding microenvironment due to the technical limitations to perform genome-wide single-cell analysis. We developed whole transcriptome spatial technology for FFPE to provide the ideal tool to profile PanINs in their microenvironment. We performed FFPE spatial transcriptomics (ST) using the Visium (10x Genomics) approach in a cohort of 14 PanIN lesions (9 low-grade and 5 high-grade). To overcome experimental limitations, we developed an artificial intelligence method to integrate imaging, spatial transcriptomics, and single-cell RNA-seq (scRNA-seq) analyses. Imaging analysis of the same stained sections used for ST was applied before gene expression exploration. Using machine learning to annotate cell types, we were able to deconvolve and quantify the cell types per ST spot using cell morphology instead of a scRNA-seq gene expression reference. Subsequently, we applied transfer learning to a scRNA-seq PDAC atlas that was built from multiple published datasets. The whole transcriptome ST findings were confirmed using targeted single-cell transcriptomics (Xenium, 10x Genomics) and proteomics (imaging mass cytometry, IMC). The analysis revealed that PanINs already exhibit molecular and cellular features of PDAC, including expression of the PDAC classical subtype signature and the presence of the same cancer-associated fibroblast (CAF) subtypes enriched in invasive carcinoma stages. The CAF subtypes, including the recently described antigen-presenting CAFs, were confirmed at single-cell resolution using Xenium and IMC. Integrating multi-omics data with a PDAC scRNA-seq atlas through transfer learning, revealed that an inflammatory pattern gradually decreases during PanIN progression, accompanied by a compensatory increase in proliferation pathways in the PanIN cells relative to normal duct cells. This trend between inflammation and proliferation was confirmed specifically in PanIN cells using Xenium. Our experimental and computational approach provides valuable insights into the dynamics of pancreatic tumor progression from PanIN lesions using innovative methods to provide a unique spatial multi-omics reference of PanINs. Citation Format: Alexander T.F. Bell, Jacob T. Mitchell, Ashley L. Kiemen, Melissa Lyman, Kohei Fujikura, Jae W. Lee, Erin Coyne, Sarah M. Shin, Pei-Hsun Wu, Jacquelyn W. Zimmerman, Denis Wirtz, Won J. Ho, Neeha Zaidi, Elizabeth Thompson, Elizabeth M. Jaffee, Laura D. Wood, Elana J. Fertig, Luciane T. Kagohara. Spatial transcriptomics analysis of PanIN reveals loss of pro-inflammatory signaling and the presence of cancer-associated fibroblasts [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 B107.

A Multi-Perspective Self-Supervised Generative Adversarial Network for FS to FFPE Stain Transfer.

In clinical practice, frozen section (FS) images can be utilized to obtain the immediate pathological results of the patients in operation due to their fast production speed. However, compared with the formalin-fixed and paraffin-embedded (FFPE) images, the FS images greatly suffer from poor quality. Thus, it is of great significance to transfer the FS image to the FFPE one, which enables pathologists to observe high-quality images in operation. However, obtaining the paired FS and FFPE images is quite hard, so it is difficult to obtain accurate results using supervised methods. Apart from this, the FS to FFPE stain transfer faces many challenges. Firstly, the number and position of nuclei scattered throughout the image are hard to maintain during the transfer process. Secondly, transferring the blurry FS images to the clear FFPE ones is quite challenging. Thirdly, compared with the center regions of each patch, the edge regions are harder to transfer. To overcome these problems, a multi-perspective self-supervised GAN, incorporating three auxiliary tasks, is proposed to improve the performance of FS to FFPE stain transfer. Concretely, a nucleus consistency constraint is designed to enable the high-fidelity of nuclei, an FFPE guided image deblurring is proposed for improving the clarity, and a multi-field-of-view consistency constraint is designed to better generate the edge regions. Objective indicators and pathologists' evaluation for experiments on the five datasets across different countries have demonstrated the effectiveness of our method. In addition, the validation in the downstream task of microsatellite instability prediction has also proved the performance improvement by transferring the FS images to FFPE ones. Our code link is https://github.com/linyiyang98/Self-Supervised-FS2FFPE.git.

The relationship between gastric microbiome features and responses to neoadjuvant chemotherapy in gastric cancer.

Emerging evidence demonstrates that the gastrointestinal microbiome has the potential to be a biomarker in neoadjuvant chemoradiotherapy for colorectal cancer (CRC). Yet studies on the impact of the gastric microbiome (GM) on the response to neoadjuvant chemotherapy (NACT) are still scarce. Forty-eight patients with gastric cancer participated in this retrospective study, and 16S rRNA sequencing was performed to evaluate formalin-fixed and paraffin-embedded (FFPE) tissue biospecimens and fresh-frozen tissues. In this study, 16 bacterial taxa at different levels, including Bacillus, Anaerococcus, and Chloroflexi, were identified to be enriched before NACT in response (R) patients in group FFPE. In contrast, 6 bacterial taxa, such as Haemophilus, Veillonellaceae (Veillonella), etc. were enriched after NACT, in which we reported for the first time that the phylum Chloroflexi was enriched before NACT in R patients. Thirty-one bacterial taxa of Coriobacteriaceae, Ruminococcaceae, Veillonellaceae, and Lachnospiraceae were identified in group mucosa as being enriched in R patients. In comparison, 4 bacterial taxa dominated by the phylum Proteobacteria were enriched in NR patients. Notably, the family Veillonellaceae was found in both tissue samples, and the metabolic pathways, including the citrate cycle (TCA cycle) and various amino acids, including alanine, were found to be potentially predictive in both sample species. There are differences in the features of the GM for different NACT response results. The causal relationship deserves to be confirmed by further investigations.

Label-free and non-destructive histology of unprocessed biological tissues with ultraviolet single-plane illumination microscopy

Histopathology has remained the gold standard for surgical margin assessment for decades. However, routine pathological examination based on formalin-fixed and paraffin-embedded (FFPE) tissues is laborious and time-consuming, failing to guide surgeons intraoperatively. Here, we propose a rapid, label-free, and non-destructive histological imaging method, termed microscopy with ultraviolet single-plane illumination (MUSI). With intrinsic fluorescence from deep ultraviolet excitation, MUSI enables both ex vivo and in vivo imaging of fresh and unprocessed tissues at the subcellular level with an imaging speed of 0.5 mm2/s, generating high-quality optically sectioned surface images from irregular surgical tissues with a long depth-of-field. We demonstrate that MUSI could differentiate between different subtypes of human lung adenocarcinomas (e.g., lepidic, acinar, papillary, and micropapillary), revealing diagnostically important features that are comparable to the gold standard FFPE histology. As an assistive imaging platform, MUSI can provide immediate feedback to surgeons and pathologists for intraoperative decision-making, holding great promise to revolutionize the current clinical practice in surgical pathology.

Immunohistochemistry for Cancer Stem Cell Detection: Principles and Methods.

Cancer stem cells (CSCs) are rare immortal cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity. CSCs play a pivotal role in the tumor development, progression, relapse, and resistance of anticancer therapy. The technique of choice to detect CSCs in formalin-fixed and paraffin-embedded (FFPE) samples is immunohistochemistry (IHC) since it is inexpensive and widespread in most laboratories. The main aims of this chapter are the description of the protocols and the automated immunohistochemical systems used for the identification of CSCs. Furthermore, a focus on the most common troubleshooting in CSC IHC is provided. Finally, an overview of the main markers of cancer stem cells in several cancer types will be provided.

Morpho-phenotypic characterization of melanoma brain metastases immune microenvironment: A multicentre retrospective study

IntroductionThe recent rise of immunotherapy for melanoma brain metastases (MBM) has enhanced the interest in characterizing the composition of local immune microenvironment. Despite the central nervous system (CNS) is well known for harbouring a peculiar immunological milieu, its role in MBM is only partially understood. The aim of our study was to characterize tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs) distribution and density in a cohort of MBM and their potential relevance as prognostic biomarkers. Materials and methodsNinety-four MBM patients were retrospectively evaluated for morphological, molecular features and follow-up data. Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunostained with the following antibodies: CD4, CD8, FoxP3 CD68, CD163, PD-L1, HLA-ABC. Semiquantitative assessment of TILs and TAMs density and spatial distribution was performed and statistical analysis for prognostic correlations was carried out. ResultsThe distribution of CD4+ TILs was higher in the intratumoral region (p=0.012) while CD163+ TAMs in the peritumoral (p=0.016). An association was also found between tumour-associated astrogliosis (TAA) and CD163+ TAMs (p=0.021). Increased CD68+ TAMs correlated with BRAF V600 mutation (p=0.038). CD4+ TILs were significantly higher in PD-L1 <1 % group (p=0.030). CD68+ high expression group significantly correlated with PD-L1 ≥1 % (p=0.029). Median HLA-ABC H-score was significantly associated with intratumoral CD4+ (p=0.005) and peritumoral CD8+ infiltration (p=0.048). CD68+ TAMs intratumoral high expression showed a favorable association with OS in patients with multiple MBM (p=0.017), whereas a high HLA-ABC H-score with a prolonged OS in those with single MBM (p=0.007). ConclusionsOur findings suggest a peculiar topographical distribution of immune cells in both intratumoral and peritumoral areas, and correlations with both BRAF V600 mutation and PD-L1 expression in a large cohort of MBM. Moreover, CD68+ TAMs distribution and HLA-ABC favourably impacted prognosis in patients with multiple and single brain metastases, respectively.

Prevalence of Chlamydia infection detected by immunohistochemistry in patients with anorectal ulcer and granulation tissue

Anorectal ulcer with granulation tissue is typically associated with left-sided inflammatory bowel disease or infection. Due to emerging cases of Chlamydia proctitis, we aim to investigate the prevalence of Chlamydia infection using immunohistochemistry (IHC) in anorectal biopsies showing ulcer and granulation tissue. Seventy-seven patients including 60 males and 17 females with mean age of 51 years old were retrospectively identified in surgical pathology archives. Chlamydia IHC was validated with a monoclonal antibody on an index who was positive for Chlamydia by rectal swab nucleic acid amplification test (NAAT), then performed on formalin fixed and paraffin embedded (FFPE) tissue sections. Confirmative molecular test using real-time PCR was performed on DNA extractions of 14 IHC-positive and 14 IHC-negative FFPEs, 18 NAAT-positive, and 5 NAAT-negative cytology specimens. Chlamydia IHC showed strong intracytoplasmic or extracellular sphere morphology in 14 of 77 (18.2 %) FFPEs, including 11 of 60 (18.3 %) males and 3 of 17 (17.6 %) females (age 11–84 years). Eight of 14 (57.1 %) Chlamydia-IHC positive patients had known history of STDs, high-risk behavior, or immunosuppressive conditions. One of 14 (7.1 %) IHC-positive FFEP and 15 of 18 (83.3 %) NAAT-positive cytology cases were confirmed by real-time PCR. Chlamydia inclusions were detected in all 4 randomly selected NAAT and PCR-positive cytology specimens by IHC. Our data suggested that Chlamydia infection is more prevalent than we thought in patients with active proctitis and ulceration. Chlamydia IHC may be performed as a screening test in biopsies to facilitate early detection of this treatable proctitis in high-risk population.