Formalin-fixed Paraffin-embedded Tissue Research Articles

Identification of microbial antigens in liver tissues involved in the pathogenesis of primary biliary cholangitis using 16S rRNA metagenome analysis.

Multiple factors are involved in the pathogenesis of primary biliary cholangitis (PBC), a chronic cholestatic liver disease, characterized by intrahepatic cholangiopathy. In particular, studies have suggested that environmental factors such as the presence of granulomas in the portal vein region are important for the development of PBC. This study aimed to comprehensively analyze and identify foreign-derived antigens in PBC liver tissue to confirm their involvement in PBC pathogenesis. Portal areas and hepatocyte regions were selectively dissected from formalin-fixed paraffin-embedded PBC liver tissue samples using the microlaser method, followed by total DNA extraction. We then validated whether the bacterial strains identified through 16S rRNA metagenomic analysis were detected in PBC liver tissues. The most frequently detected bacterial genera in the PBC liver tissue samples were Sphingomonas panacis, Providencia, and Cutibacterium. These bacterial genera were also detected in the other PBC samples. Validation for the detection of S. panacis, the most abundant genus, revealed polymerase chain reaction bands extracted from the portal areas of all samples. They were also more highly expressed than bands detected in the hepatocyte region. S. panacis antigen was specifically detected in the portal areas of PBC liver tissues. The introduction of foreign-derived antigens into the liver as an environmental factor could be a possible mechanism for the development of PBC.

Exploring Therapeutic Challenges in Patients with HER2-Positive Breast Cancer-A Single-Center Experience.

Breast cancer is one of the most common forms of neoplasia worldwide. The purpose of our observational study was to evaluate the status of HER2 overexpression among new cases of breast neoplasia with an impact on the natural history of breast cancer disease and therapeutic personalization according to staging. This study included 45 breast cancer patients which have an overexpression of HER2 through the mutation of the EGFR-ERBB2 receptor. Immunohistochemical staining was performed on sections of formalin-fixed paraffin-embedded breast tissue. The patients were evaluated demographically and therapeutically in all stages. The post-surgical histopathological examination revealed complete pathological responses in 19 patients and pathological responses with residual disease either at the tumor level or lymphatic or both variants in a percentage of 44% (15 cases). The disease-free interval (DFI) under anti-HER2 therapy was recorded in 41 patients, representing 91% of the study group. Anti-HER2 therapy in any therapeutic stage has shown increased efficiency in blocking these tyrosine kinase receptors, evidenced by the high percentage of complete pathological responses, as well as the considerable percentage (47%) of complete remissions and stationary disease, in relation to the HER2-positive patient group.

Microsporum canis Causes Cutaneous and Extracutaneous Feline Dermatophytic Pseudomycetomas: Molecular Identification and Clinicopathological Characteristics.

Dermatophytic pseudomycetoma (DPM) is a rarely reported invasive fungal infection of humans and animals, especially cats. This study aimed to identify dermatophytes, breed associations, and the frequency of extracutaneous (EC) involvement in feline DPM. Electronic records and formalin-fixed paraffin-embedded tissue (FFPET) from 32 suspected DPM cases in 30 cats were retrieved from a diagnostic laboratory between 2018 and 2024. To confirm DPM and molecular identity, DNA was extracted from FFPET for ITS2 sequencing, and immunohistochemistry was performed on PCR-negative cases. All cases were confirmed as DPM. Microsporum canis was the only dermatophyte identified. The sensitivity and specificity of ITS2 sequencing for M. canis identification in FFPET were 22/32 (68.8%) and 21/22 (95.5%), respectively. Exotic (36.7%) and Persian (23.3%) but not British breeds (26.3%) were over-represented compared to feline admissions at an affiliated veterinary hospital (8.5%, p < 0.001; 3%, p < 0.001; 21.6%, p = 0.817, respectively). Five cases (16.7%) had EC lesions; two had intra-abdominal masses; two had oral cavity masses, including one which extended into the cranial vault; and one had superficial cervical lymph node invasion. Exotic and Persian breeds are over-represented for DPM and M. canis is the primary cause. EC lesions of DPM may occur more commonly than previously thought.

Molecular and ultrastructural morphological analyses of highly metamorphosed Aspergillus fumigatus on human formalin-fixed paraffin-embedded tissue.

Invasive fungal infections including invasive pulmonary aspergillosis (IPA) generally have a poor prognosis, because the fungi spread throughout various organs. Therefore, it is important to accurately identify the fungal species for treatment. In this article, we present the results of pathological and molecular morphological analyses that were performed to elucidate the cause of respiratory failure in a patient who died despite suspicion of IPA and treatment with micafungin (MCFG). Pathological analysis revealed the existence of cystic and linear fungi in lung tissue. The fungi were identified as Aspergillus fumigatus (A. fumigatus) by partial sequencing of genomic DNA. Correlative light microscopy and electron microscopy (CLEM) analysis confirmed that fungi observed with light microscopy can also be observed with scanning electron microscopy (SEM) using formalin-fixed paraffin-embedded tissue sections. SEM revealed an atypical ultrastructure of the fungi including inhomogeneous widths, rough surfaces, and numerous cyst-like structures of various sizes. The fungi showed several morphological changes of cultured A. fumigatustreated with MCFG that were previously reported. Our results indicate that integrated analysis of ultrastructural observation by SEM and DNA sequencing may be an effective tool for analyzing fungi that are difficult to identify by conventional pathological analysis.

Aspergillosis coinfection in patients with proven mucormycosis.

Although research on aspergillosis and mucormycosis confection is important to optimize antifungal therapy, data on this issue is scarce. Thus, we systematically investigated aspergillosis coinfection in patients with proven mucormycosis. Medical records of adult patients with proven mucormycosis whose formalin-fixed paraffin-embedded (FFPE) tissue sections were available, in a tertiary hospital from August 2007 to July 2023 were retrospectively reviewed to assess coinfection with aspergillosis. We noted cultures of fungi from sterile and non-sterile sites and performed polymerase chain reaction (PCR) assays on FFPE tissues to detect Aspergillus- and Mucorales-specific DNA. Sixty-seven patients with proven mucormycosis, including 12 (18%) with a positive culture of the mucormycosis agent from sterile site cultures, were enrolled. Fungal cultures from sterile and non-sterile sites revealed Aspergillus spp. growth in nine (13%) of the 67 patients, including two sterile and seven non-sterile cultures. The fungal PCR analysis from the FFPE sections was positive for Aspergillus-specific PCR in five (7%) and positive for both Aspergillus- and Mucorales-specific PCR results in eight (12%). Overall, 21 (31%) of the 67 patients with proven mucormycosis had microbiologic and/or molecular evidence of aspergillosis coinfection. Positive blood or bronchoalveolar lavage fluid galactomannan results were more common in the coinfection group (67% [14/21]) than in the mucormycosis group (37% [17/46], P=.024). No significant difference in mortality between the two groups was observed. Approximately one-third of patients with proven mucormycosis exhibited molecular and/or microbiologic evidence of aspergillosis coinfection. Further research is needed to identify patients with aspergillosis and mucormycosis coinfections, for optimal antifungal therapy.

Association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma: Experience from a North Indian tertiary care hospital

IntroductionNasopharyngeal carcinoma (NPC), arising from nasopharyngeal epithelium is caused by Epstein-Barr virus (EBV). It is common in South China, South East Asia and North East India. The aim and objectives of this study were to determine the prevalence of EBV in formalin-fixed paraffin-embedded (FFPE) tissue sections of clinically suspected NPC patients, correlate the results of polymerase chain reaction (PCR) with histopathology findings, and to determine the utility of tissue EBV DNA as a diagnostic bio-marker. Materials and methods31 FFPE tissue samples were collected from clinically suspected NPC patients from April 2018–December 2019. Histopathological diagnosis was done by examination of Hematoxylin and Eosin stained slides. Presence of EBV was detected by EBNA-1 PCR. IHC was performed using EBV Latent Membrane Protein 1. ResultsOf the 31 clinically suspected NPC cases, 15 (48.4 %) were histopathological confirmed NPC. Of these15, 13 (86.6 %) were non-keratinising undifferentiated NPC, and one each were keratinising NPC and non-keratinising differentiated NPC respectively.EBV EBNA1 PCR was positive in 35.5 % (11/31) of clinically suspected NPC cases. Of the 11 PCR positive cases, 9 (81.8 %) were histopathological confirmed NPC.Of the 31 clinically suspected NPC cases, IHC was indicated in 23 biopsies. Of which, 12 (52.2 %) were positive for LMP1 in the abnormal cells. Of the 12 IHC positive samples, 10 were NPC cases. ConclusionEBV DNA as an indicator towards NPC among clinically suspected cases had a sensitivity of 60 % and specificity of 87.5 %. In this study, addition of EBV DNA detection by PCR from FFPE tissue sections could confirm EBV association in 20 % of cases where it was not detected by EBV LMP1 IHC, thus helped in increasing the detection of EBV positivity in NPC cases. Early diagnosis of NPC will improve the cure rate and hence reduce the morbidity and mortality rates.

High success rate of first proficiency testing for RET fusions and RET mutations in lung and thyroid cancer samples by various methods.

This study describes the external quality assessment (EQA) scheme for molecular testing of RET alterations in non-small cell lung cancer (NSCLC), medullary thyroid carcinomas (MTC), and non-MTC. The lead panel institute and Quality Assurance Initiative in Pathology (Qualitätssicherungs-Initiative Pathologie [QuIP] GmbH) selected formalin-fixed paraffin-embedded (FFPE) tissue from MTC for RET mutation testing by next-generation sequencing (NGS) methods and FFPE tissue from NSCLC and non-MTC for RET gene fusion testing using either in situ hybridisation (ISH) or NGS methods, forming 3 sub-schemes of the EQA scheme. Tissue material underwent an internal validation phase followed by an external testing phase. The internal validation phase served as a cross-validation step conducted by panel institutes. In the external testing phase, the number of participating institutes in the RET point mutation sub-scheme, RET fusion (ISH) sub-scheme, and RET fusion (NGS) sub-scheme was 32, 24, and 38, respectively. The reported success rates for external testing were 96.0%, 89.5%, and 93.5% for the RET point mutation, the ISH RET fusion, and the NGS RET fusion EQA sub-schemes, respectively. These findings confirm the reliability of the NGS method in detecting RET alterations and align with current screening recommendations. Overall, 31 institutes were certified for RET point mutation testing by NGS methods, 22 institutes were certified for RET fusion testing by ISH, and 36 institutes were certified for RET fusion testing by NGS methods. Results can be employed to inform real-world diagnostic decisions in Germany, Austria, and Switzerland.

Systematic review and feasibility study on pre-analytical factors and genomic analyses on archival formalin-fixed paraffin-embedded breast cancer tissue

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Quantitative Comparison of Deparaffinization, Rehydration, and Extraction Methods for FFPE Tissue Proteomics and Phosphoproteomics.

Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The choice of the sample preparation method influences the number, intensity, and reproducibility of identifications. By comparing four deparaffinization and rehydration methods, including heptane, histolene, SubX, and xylene, we found that heptane and methanol produced the lowest coefficients of variation (CVs). Using this, five extraction methods from the literature were modified and evaluated for their performance using kidney, leg muscle, lung, and testicular rat organs. All methods performed well, except for SP3 due to insufficient tissue lysis. Heat n' Beat was the fastest and most reproducible method with the highest digestion efficiency and lowest CVs. S-Trap produced the highest peptide yield, while TFE produced the best phosphopeptide enrichment efficiency. The quantitation of FFPE-derived peptides remains an ongoing challenge with bias in UV and fluorescence assays across methods, most notably in SPEED. Functional enrichment analysis demonstrated that each method favored extracting some gene ontology cellular components over others including chromosome, cytoplasmic, cytoskeleton, endoplasmic reticulum, membrane, mitochondrion, and nucleoplasm protein groups. The outcome is a set of recommendations for choosing the most appropriate method for different settings.

Transcriptomic analyses of lung tissues reveal key genes associated with progression of systemic sclerosis-interstitial lung disease (SSc-ILD)

Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in SSc, affecting around 50% of the patients. Lung tissue of patients with early-stage SSc-ILD is characterized by a predominant inflammatory response with inconspicuous fibrosis, which may progress to honeycombing fibrosis. Hence, a better understanding of the molecular mechanisms underpinning SSc-ILD pathogenesis is needed to improve treatment options and progression prediction. This transcriptomic study aims to reveal the differential gene expression between control (ctrl) lung tissue and inflammatory, prefibrotic and fibrotic lung tissue to capture progression of early to late phase SSc-ILD. Twelve explanted lungs from patients with SSc-ILD were used to analyze gene expression from formalin-fixed paraffin-embedded lung tissues with varying stages of ILD (n=18) and control lung tissue (n=6). The SSc-ILD tissues were stratified into three ROIs: inflammatory, prefibrotic, and fibrotic using histological assessments to define a longitudinal simulation of early to late phases of SSc-ILD. The nanoString (nS) nCounter Human Fibrosis Panel was used to profile the transcriptome in the regions of interest. Validation of potential targetswas performed with immunohistochemistry in the same tissues that were used for transcriptome analysis. To validate our simulation model, we performed subgroup analysis that showed an incremental increase in pathway scores related to the severity of fibrosis. Ctrl vs SSc-ILD comparison demonstrated 24 differentially expressed genes, two of which had the most pronounced p-values. Cyclin-dependent kinase inhibitor (cdkn2c) was overexpressed (P=0.00052) in SSc-ILD compared to ctrl, while expression of Pellino E3 ubiquitin-protein ligase 1 (peli1) showed lower expression (P=0.0012). Additionally, in all four groups, cdkn2c and peli1 gene expression showed an incremental increase and decrease, respectively. Immunohistochemistry of cdkn2c showed consistent results with the nS analysis. More cdkn2c and less peli1 expression were associated with more advanced stages of SSc-ILD on histologic assessment. We report the potential of the cell cycle inhibitor and senescence marker, cdkn2c (p18) to be associated with fibrosis progression.

Immunohistochemistry for YAP1 N-terminus and C-terminus highlights metaplastic thymoma and high-grade thymic epithelial tumors by different staining patterns.

Metaplastic thymoma (MT), a rare subtype of thymic epithelial tumors (TETs), harbors YAP1::MAML2 fusions. Poroma, a skin tumor, also carries these fusions and exhibits a unique staining pattern for YAP1 immunohistochemistry (IHC), namely, a YAP1 N-terminus (YAP1[N])-positive but YAP1 C-terminus (YAP1[C])-negative pattern. In this context, MT was recently reported to lack YAP1(C) expression exclusively among TET subtypes. However, a lack of information about YAP1(N) expression in that study and another report that wild-type YAP1 expression was diminished in type B3 thymoma and thymic carcinoma warrants further studies for YAP1 expression in TETs. Thus, we immunohistochemically examined YAP1(N) and YAP1(C) staining patterns in our TET samples, including 14 cases of MT. In addition, 11 of the 14 MT cases were genetically analyzed with the formalin-fixed paraffin-embedded tissues if they harbored YAP1::MAML2 fusions. MT consistently exhibited YAP1(N)-positive and YAP(C)-negative staining, whereas type B3 thymoma and thymic carcinoma showed relatively heterogeneous staining patterns for YAP1(N) and YAP1(C) and were sometimes negative for both antibodies. Furthermore, a lower expression of YAP1 was found in type B3 compared to B2 thymomas. Among genetically analyzed 11 MT cases, 6 cases showed YAP1::MAML2 fusions, whereas the analysis failed in 5 very old cases due to poor RNA quality. These results indicate that IHC of both YAP1(N) and YAP1(C) is recommended to obtain staining patterns almost unique to MT. The biological significance of YAP1 in high-grade TETs warrants further investigation.

Histopathological analysis potential for unveiling hormone signaling in endocrine-related tumors.

Elucidating the mechanisms of action of steroid hormones will contribute to the development of therapeutic strategies for hormone-dependent tumors. Recent advances in genetic engineering have revealed the complex and diverse mechanisms of steroid hormone signaling; however, these techniques are limited to in vitro or animal experiments. It is believed that verifying hormone signals elucidated using human pathological tissue specimens will directly aid in treatment and diagnosis. However, pathological tissue specimens are generally formalin-fixed paraffin-embedded (FFPE), and protein/gene analyses of FFPE tissues are limited. Protein detection using immunohistochemistry with specific antibodies in FFPE tissues is a classical technique essential for diagnosis and treatment decisions in various types of cancer. In steroid hormone signaling, the expression and localization of receptors, hormone-related enzymes, and proteins encoded by response genes can be clarified using immunohistochemistry. Although protein-protein interactions such as receptor dimers and DNA-binding proteins are mainly detected in vitro, they can be examined in FFPE tissues using in situ proximity ligation assays and southwestern histochemistry, respectively. Using these detection methods, including immunohistochemistry, it is possible to analyze each hormone signaling pathway in hormone-related tumors histopathologically. Although FFPE tissues still suffer from gene and protein denaturation, their advantages include the ability to retrospectively study target factors/signals and obtain spatial information through microscopy. This review describes a visualization method for elucidating steroid hormone signaling in hormone-dependent tumors using FFPE tissues.

Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC–MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Epidemiological distribution of high-risk human papillomavirus genotypes and associated factors among patients with esophageal carcinoma at Bugando medical center in Mwanza, Tanzania

BackgroundEsophageal carcinoma is a growing concern in regions that have a high incidence of human papillomavirus (HPV) infection such as East Africa. HPV, particularly the high-risk genotypes, is increasingly recognized as a risk factor for esophageal carcinoma. We set out to investigate the prevalence and associated factors of high-risk HPV in formalin-fixed paraffin-embedded (FFPE) tissue blocks with esophageal carcinoma at Bugando Medical Center, a tertiary referral hospital in Mwanza, Tanzania, East Africa.MethodsA total of 118 esophageal carcinoma FFPE tissue blocks, collected from January 2021 to December 2022, were analyzed. Genomic DNA was extracted from these tissues, and multiplex polymerase chain reaction (PCR) was performed to detect HPV using degenerate primers for the L1 region and type-specific primers for detecting HPV16, HPV18, and other high-risk HPV genotypes. Data were collected using questionnaires and factors associated with high-risk HPV genotypes were analyzed using STATA version 15 software.ResultsOf the 118 patients’ samples investigated, the mean age was 58.3 ± 13.4 years with a range of 29–88 years. The majority of the tissue blocks were from male patients 81/118 (68.7%), and most of them were from patients residing in Mwanza region 44/118 (37.3%). Esophageal Squamous Cell Carcinoma (ESCC) was the predominant histological type 107/118 (91.0%). Almost half of the tissue blocks 63/118 (53.3%) tested positive for high-risk HPV. Among these, HPV genotype 16 (HPV16) was the most common 41/63 (65.1%), followed by HPV genotype 18 (HPV18) 15/63 (23.8%), and the rest were other high-risk HPV genotypes detected by the degenerate primers 7/63 (11.1%). The factors associated with high-risk HPV genotypes were cigarette smoking (p-value < 0.001) and alcohol consumption (p-value < 0.001).ConclusionA substantial number of esophageal carcinomas from Bugando Medical Center in Tanzania tested positive for HPV, with HPV genotype 16 being the most prevalent. This study also revealed a significant association between HPV status and cigarette smoking and alcohol consumption. These findings provide important insights into the role of high-risk HPV in esophageal carcinoma in this region.

High keratin 15 expression reflects favorable prognosis in early cervical cancer patients.

Keratin 15 (KRT15) exhibits inconsistent prognostic roles in different cancers, and its prognostic value in early cervical cancer patients who receive tumor resection remains unknown. This study aimed to assess the relationship of KRT15 expression with prognosis in these patients. Totally, 147 early cervical cancer patients who received tumor resection were reviewed in this retrospective study. KRT15 was detected in formalin-fixed paraffin-embedded tumor tissue by immunohistochemistry (IHC). KRT15 IHC scores were computed by multiplying the percentage of positively stained cells (scored as 0-4) and corresponding staining intensity (scored as 0-3), ranging from 0 to 12. Elevated KRT15 IHC score was linked with moderate to well differentiation (P = 0.005), tumor size ≤ 4cm (P = 0.017), and International Federation of Gynecology and Obstetrics (FIGO) stage Ia/Ib (P < 0.001). KRT15 IHC score was inversely associated with adjuvant radiotherapy (P = 0.025) and adjuvant chemotherapy (P = 0.016). KRT15 IHC score ≥ 1 was linked with increased disease-free survival (DFS) (P = 0.003) and overall survival (OS) (P = 0.049). Meanwhile, KRT15 IHC score ≥ 1 independently predicted increased DFS (hazard ratio = 0.213, P = 0.017), but not OS (P > 0.05). KRT15 IHC score ≥ 3 and KRT15 IHC score ≥ 6 could not predict DFS or OS (all P > 0.05). By subgroup analyses, KRT15 IHC score ≥ 1 forecasted favorable DFS in patients with age > 45years, human papillomavirus-positive, squamous carcinoma, and tumor size ≤ 4cm (all P < 0.05). KRT15 IHC score ≥ 1 and KRT15 IHC score ≥ 3 predicted ascended DFS in patients without adjuvant radiotherapy or adjuvant chemotherapy (all P < 0.05). High KRT15 expression reflects favorable tumor features and longer survival in early cervical cancer patients who receive tumor resection.

Proximity ligation-based sequencing for the identification of human papillomavirus genomic integration sites in formalin-fixed paraffin embedded oropharyngeal squamous cell carcinomas.

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

The clinical significance and anti-tumor role of PRKG1 in bladder cancer.

cGMP-dependent protein kinase 1 (PRKG1) has shown to be associated with some tumorigenesis, while the role of PRKG1 in bladder cancer is unclear. To investigate the biological and clinical significance of PRKG1 in bladder cancer, we detected the expression of PRKG1 and explored the function of PRKG1 in bladder cancer cells. The PRKG1 transcripts data was downloaded from The Cancer Genome Atlas (TCGA) database, and immunohistochemistry staining was conducted on formalin-fixed paraffin-embedded (FFPE) sample tissues. Relationship between clinical characteristics of patients and expression of PRKG1 was analyzed in FFPE samples, TCGA database, and GSE19423 dataset. PRKG1 was over-expressed, and cell proliferation, migration, invasion, apoptosis, and spheroidizing ability were then detected. Chemosensitivity to cisplatin was detected with cell viability, and half-maximal drug inhibitory concentration (IC50) was calculated. In addition, the relation between PRKG1 expression and the infiltration level of tumor immune cells in tumor microenvironment were analyzed. The results showed expression of PRKG1 was lower in bladder cancer, compared with normal tissues both at protein and transcript levels. Lower PRKG1 expression was related to higher tumor grade, T stage, and muscle invasion, also predicted worse overall survival and recurrence free survival in patients treated with Bacillus Calmette-Guerin (BCG) intravesical immunotherapy. Analysis of tumor immune cells infiltration showed lower PRKG1 was associated with non-inflamed tumor microenvironment. The present study firstly identified the anti-tumor role and tumor immune regulatory role of PRKG1, also found loss of PRKG1 could be used as a prognosis factor. The present study provided a potential biomarker and therapy target to bladder cancer.

Differential expression of host oncogenes in human papillomavirus-associated nasopharyngeal and cervical epithelial cancers.

Human papillomavirus (HPV)-related cervical and nasopharyngeal cancers differ in molecular mechanisms underlying the oncogenic processes. The disparity may be attributed to differential expression of oncoproteins. The current study investigated the host oncogenes expression pattern in HPV-associated cervical and nasopharyngeal cancer. Formalin-fixed paraffin-embedded tissues originating from the nasopharyngeal and cervical regions were screened using Hematoxylin and Eosin staining. Genomic DNA and total RNA were extracted from confirmed cancer biopsies and non-cancer tissues (NC). HPV was detected by PCR using MY09/GP5+/6+ primers. Protein expression levels of AKT, IQGAP1, and MMP16 in HPV-infected cancers and controls were determined by immunohistochemistry. RT-qPCR was used to profile mRNAs of the oncogenes. AKT and IQGAP1 proteins were highly expressed in the epithelial cancers compared with the non-cancer tissues (p < 0.05). IQGAP1 and MMP16 mRNAs level was significantly higher in the cancers than in the NC (p < 0.05), but not AKT mRNA levels. MMP16 protein was ubiquitously expressed in all tissues. AKT mRNA level was significantly elevated in CC compared with NPC (p < 0.001). However, the difference in AKT, IQGAP1 and MMP16 proteins level between CC and NPC was not significant (p > 0.05). The oncoproteins expression level between the HPV-positive and HPV-negative cancer biopsies showed no significant difference (p < 0.05). Current study reports AKT but not IQGAP1 and MMP16 mRNAs differentially expression in cervical and nasopharyngeal cancers, independent of HPV infection status.

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.

Diagnostic Potential of miR-143-5p, miR-143-3p, miR-551b-5p, and miR-574-3p in Chemoresistance of Locally Advanced Gastric Cancer: A Preliminary Study.

Gastric cancer (GC) is one of the most frequently diagnosed cancers in the world. Although the incidence is decreasing in developed countries, the treatment results are still unsatisfactory. The standard treatment for locally advanced gastric cancer (LAGC) is gastrectomy with perioperative chemotherapy. The association of selected microRNAs (miRNAs) with chemoresistance was assessed using archival material of patients with LAGC. Histological material was obtained from each patient via a biopsy performed during gastroscopy and then after surgery, which was preceded by four cycles of neoadjuvant chemotherapy (NAC) according to the FLOT or FLO regimen. The expression of selected miRNAs in the tissue material was assessed, including miRNA-21-3p, miRNA-21-5p, miRNA-106a-5p, miRNA-122-3p, miRNA-122-5p, miRNA-143-3p, miRNA-143-5p, miRNA-203a-3p, miRNA-203-5p, miRNA-551b-3p, miRNA-551b-5p, and miRNA-574-3p. miRNA expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The response to NAC was assessed using computed tomography of the abdomen and chest and histopathology after gastrectomy. The statistical analyses were performed using GraphPad Prism 9. The significance limit was set at p < 0.05. We showed that the expression of miR-143-3p, miR-143-5p, and miR-574-3p before surgery, and miR-143-5p and miR-574-3p after surgery, decreased in patients with GC. The expression of miR-143-3p, miR-143-5p, miR-203a-3p, and miR-551b-5p decreased in several patients who responded to NAC. The miRNA most commonly expressed in these cases was miRNA-551b-5p. Moreover, it showed expression in a patient whose response to chemotherapy was inconsistent between the histopathological results and computed tomography. The expression of miR-143-3p, miR-143-5p, miR-203a-3p, and miR-551b-5p in formalin-fixed paraffin-embedded tissue (FFPET) samples can help differentiate between the responders and non-responders to NAC in LAGC. miR-143-3p, miR-143-5p, and miR-574-3p expression may be used as a potential diagnostic tool in GC patients. The presence of miR-551b-5p may support the correct assessment of a response to NAC in GC via CT.