Formalin-fixed Paraffin-embedded Tissue Samples Research Articles

KRAS G12C mutation in NSCLC in a small genetic center: insights into sotorasib therapy response potential

Lung cancer remains a significant health challenge, characterized by aberrant tissue growth within the pulmonary system. Early carcinogenic events often involve genomic instability and the emergence of a mutator phenotype. In this study, we aimed to explore the mutator phenotype in 89 patients diagnosed with non-small-cell lung cancer (NSCLC). RNA isolation from formalin-fixed paraffin-embedded (FFPE) tissue samples was performed using the Promega ReliaPrep RNA Miniprep System, facilitating gene amplification relevant to cancer through the Archer® FusionPlexComprehensiveThyroid and Lung (CTL) kit. Next-generation sequencing (NGS) on the Illumina NextSeq platform enabled comprehensive analysis of target areas. Utilizing Archer Analysis software, secondary analyses involving data cleansing, alignment, and variant/fusion identification were executed against the human reference genome hg19 (GRCh37). Expression patterns were visualized using HeatMap graphics. Our findings revealed a notable presence of KRAS gene mutations in approximately 20% of NSCLC patients. Among these mutations, the G12C variant was predominant at 50%, followed by G12V and G12D variants at 11.2% each. Notably, patients harboring the G12C variant responded favorably to sotorasib medication. These results underscore the importance of mutational profiling and targeted therapeutic approaches in managing NSCLC, particularly highlighting the promising efficacy of sotorasib in G12C-mutated cases.

High chromosomal instability is associated with higher 10-year risks of recurrence for hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer patients: clinical evidence from a large-scale, multiple-site, retrospective study.

Long-term survival varies among hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients and is seriously impaired by metastasis. Chromosomal instability (CIN) was one of the key drivers of breast cancer metastasis. Here we evaluate CIN and 10-year invasive disease-free survival (iDFS) and overall survival (OS) in HR+/HER2-- breast cancer. In this large-scale, multiple-site, retrospective study, 354 HR+/HER2- breast cancer patients were recruited. Of these, 204 patients were used for internal training, 70 for external validation, and 80 for cross-validation. All medical records were carefully reviewed to obtain the disease recurrence information. Formalin-fixed paraffin-embedded tissue samples were collected, followed by low-pass whole-genome sequencing with a median genome coverage of 1.86X using minimal 1 ng DNA input. CIN was then assessed using a customized bioinformatics workflow. Three or more instances of CIN per sample was defined as high CIN and the frequency was 42.2% (86/204) in the internal cohort. High CIN correlated significantly with increased lymph node metastasis, vascular invasion, progesterone receptor negative status, HER2 low, worse pathological type, and performed as an independent prognostic factor for HR+/- breast cancer. Patients with high CIN had shorter iDFS and OS than those with low CIN [10-year iDFS 11.1% versus 82.2%, hazard ratio (HR) = 11.12, p < 0.01; 10-year OS 45.7% versus 94.3%, HR = 14.17, p < 0.01]. These findings were validated in two external cohorts with 70 breast cancer patients. Moreover, high CIN could predict the prognosis more accurately than Adjuvant! Online score (10-year iDFS 11.1% versus 48.6%, HR = 2.71, p < 0.01). Cross-validation analysis found that high consistency (83.8%) was observed between CIN and MammaPrint score, while only 45% between CIN and Adjuvant! Online score. In conclusion, high CIN is an independent prognostic indicator for HR+/HER2- breast cancer with shorter iDFS and OS and holds promise for predicting recurrence and metastasis.

Targeted Therapy in Salivary Gland Cancer: Prevalence of a Selected Panel of Actionable Molecular Alterations in a German Tertiary Referral Center Patient Cohort.

Salivary gland carcinomas (SGC) are a heterogeneous group of malignancies, with 24 subtypes defined by the World Health Organization (WHO). The standard of therapy is surgical resection, with adjuvant radiotherapy in most cases. However, disease recurrence (R) or metastasis (M) is common and no active systemic therapies are currently available for RM-SGC resulting in a 5-year survival rate of only 20%. Overall, 55 SGC patients with seven different histological tumor subtypes were included in this study. formalin-fixed paraffin-embedded (FFPE) tissue samples were used for immunohistochemical (IHC) staining targeting HER2/neu, androgen receptor (AR), PD-L1, EGFR, panTRK, and TROP2. Fluorescence in situ hybridization (FISH) was performed for detecting HER2/neu amplifications and NTRK1/2/3 translocations in selected cases with relevant HER2/neu and panTRK protein expression, respectively. IHC and FISH results were correlated with patients' clinical and histopathological data. The overall prevalence of druggable molecular alterations, defined as an immunoreactive score ≥ 9 in at least one of the analyzed targets, was 54.4% with the highest percentage in oncocytic carcinomas (100%) and lowest percentage in acinic cell carcinomas (10%). EGFR overexpression proved to be the most common alteration (32.7% of cases) followed by overexpression of TROP2 (27.3%), AR (10.9%), HER2/neu (7.3%), PD-L1 (1.8%), and panTRK (1.8%). HER2/neu amplifications were found in 50% and NTRK translocations were found in 100% of all cases with elevated Her2/neu and panTRK protein expression, respectively. Our data indicate that targeted therapy using e.g., trastuzumab deruxtecan, bicalutamide, pembrolizumab, cetuximab, entrectinib or sacituzumab govitecan might be a promising option especially for a relevant subset of patients with RM-SGC not suitable for salvage surgery. However, evidence from clinical studies regarding response rates to these therapies remains sparse, which underlines the need of multicenter clinical trials.

Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.

Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection.

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

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

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

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

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

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

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

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

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

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

Human papillomavirus (HPV) and Epstein-Barr virus (EBV) in Penile Cancer in Thailand.

Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are important etiological factors for several cancers. This study aimed to determine the prevalence of HPV and EBV infection in penile cancer. Forty-three formalin-fixed paraffin-embedded penile cancer tissue samples were analyzed for the HPV-induced p16INK4A protein by immunohistochemistry and Epstein-Barr encoding region in situ hybridization. Demographic data and overall survival were analyzed. The median age of patients was 59 years, ranging from 23 to 91 years old. Most of the tumors (86%) were located at the tip of the penis. HPV infection was positive in 12/43 (27.9%) patients. EBV infection was observed in 2/43 (4.6%) of cases and there was no co-infection detected in this cohort. Patients who had p16INK4A overexpression had a trend toward longer survival compared to those without; the median survival time of 104.4 vs 89 months, the hazard ratio of 0.48 (95% CI: 0.16-1.42, p = 0.173). One-third of penile cancer patients were positive for HPV-induced p16INK4A expression and there was a trend toward better survival in HPV-positive patients. EBV infection was infrequent in penile cancer in Thailand.

A Proteomic Analysis of Nasopharyngeal Carcinoma in a Moroccan Subpopulation.

Nasopharyngeal carcinoma (NPC) is a distinct cancer of the head and neck that is highly prevalent in Southeast Asia and North Africa. Though an extensive analysis of environmental and genetic contributors has been performed, very little is known about the proteome of this disease. A proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissues can provide valuable information on protein expression and molecular patterns for both increasing our understanding of the disease and for biomarker discovery. To date, very few NPC proteomic studies have been performed, and none focused on patients from Morocco and North Africa. Label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was used to perform a proteomic analysis of FFPE tissue samples from a cohort of 41 NPC tumor samples of Morocco and North Africa origins. The LC-MS/MS data from this cohort were analyzed alongside 21 healthy controls using MaxQuant 2.4.2.0. A differential expression analysis was performed using the MSstats package in R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations were carried out using the DAVID bioinformatic tool. 3341 proteins were identified across our NPC cases, revealing three main clusters and five DEPs with prognostic significance. The sex disparity of NPC was investigated from a proteomic perspective in which 59 DEPs were found between males and females, with significantly enriched terms associated with the immune response and gene expression. Furthermore, 26 DEPs were observed between patients with early and advanced stages of NPC with a significant cluster related to the immune response, implicating up-regulated DEPs such as IGHA, IGKC, and VAT1. Across both datasets, 6532 proteins were quantified between NPC patients and healthy controls. Among them, 1507 differentially expressed proteins (DEPs) were observed. GO and KEGG pathway analyses showed enriched terms of DEPs related to increased cellular activity, cell proliferation, and survival. PI3K and MAPK proteins as well as RAC1 BCL2 and PPIA were found to be overexpressed between cancer tissues and healthy controls. EBV infection was also one of the enriched pathways implicating its latent genes like LMP1 and LMP2 that activate several proteins and signaling pathways including NF-Kappa B, MAPK, and JAK-STAT pathways. Our findings unveil the proteomic landscape of NPC for the first time in the Moroccan population. These studies additionally may provide a foundation for identifying potential biomarkers. Further research is still needed to help develop tools for the early diagnosis and treatment of NPC in Moroccan and North African populations.

Challenges and solutions in FISH for formalin-fixed paraffin-embedded tissue: A scoping review.

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.

Abstract C094: Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is a significant public health issue in Colombia, It occupies third place in incidence and ranking as the fifth leading cause of cancer-related deaths. Understanding the sociodemographic, clinicopathological, and molecular characteristics of CRC patients is crucial for prognosis, effective management, and treatment. Several molecular alterations and clinicopathological variables have been associated with the prognosis of the disease in different populations; However, we do not know if there are differentially expressed genes specific to Colombian population that are associated with CRC prognosis. Objective: Perform differential expression analysis using RNA-seq data obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples from Colombian patients with CRC. Methodology: For this preliminary analysis were included 5 patients diagnosed with CRC between 2010-2014 at Instituto Nacional de Cancerologia from Colombia. From each patient, formalin-fixed and paraffin-embedded (FFPE) samples of tumor tissue and non-tumor tissue from the surgical remnant were obtained. Extracted RNA was used to perform whole-genome RNA sequencing (RNA-seq) at a depth of 100X. Bioinformatic analysis was performed using Fastqc, Cutadapt, Ribodetector, and STAR on a Linux server. The DESeq2 package in RStudio was used to perform the differential expression analysis between tumor samples vs. non-tumorous. A significant differential expression was considered using an FDR-adjusted p ≤0.05 and a log fold change of 2. Results and discussion: A list of 18 differentially expressed genes was obtained which 5 underexpressed stand out: B3GNT6 (LFC:-10.66; padj=0.0093), ITLN1 (LFC:-6.23; padj=0.0231), NR3C2 (LFC:-3.34; padj=0.0231), NLGN1 (LFC:- 0.46; padj=0.0301), CEACAM7 (LFC:-5.91; padj= 0.0301); and one overexpressed gene: RPL9 (LFC:5.44; padj=0.0267). These genes have been previously characterized and associated with the progression and prognosis of this disease in other populations. Conclusions: 6 differentially expressed genes were found using RNA-seq in FFPE samples. These genes have been associated with CRC progression and prognosis in previous studies, but have not been evaluated in the Colombian population. From this perspective, we propose to continue with the analysis by increasing the number of samples and evaluating their association with the different clinicopathological variables and their impact on the prognosis of the disease. Citation Format: Wendy J. Montero Ovalle, Silvia J Serrano Gómez, Diego F Ballen Lozano. Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C094.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

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.

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.

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.

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.

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence in situ Hybridization.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Mutation-Selected Amplification droplet digital PCR: A new single nucleotide variant detection assay for [formula omitted] mutant in tumor and plasma samples

The early detection of gene mutations in physiological and pathological processes is a powerful approach to guide decisions in precision medicine. However, detecting low-copy mutant DNA from clinical samples poses a challenge due to the enrichment of wild-type DNA backgrounds. In this study, we devised a novel strategy, named Mutation-Selected Amplification droplet digital PCR (MSA-ddPCR), to quantitatively analyze single nucleotide variants (SNVs) at low variant allele frequencies (VAFs). Using TP53R249S (a hotspot mutation associated with hepatocellular carcinoma) as a model, we optimized the concentration ratio of primers, the annealing temperature and nucleic acid amplification modifiers. Subsequently, we evaluated the linear range and precision of MSA-ddPCR by detecting TP53R249S and TP53wild-type (TP53WT) plasmid DNA, respectively. MSA-ddPCR demonstrated superior ability to discriminate between mutant DNA and wild-type DNA compared to traditional TaqMan-MGB PCR. We further applied MSA-ddPCR to analyze the TP53R249S mutation in 20 plasma samples and 15 formalin-fixed paraffin-embedded (FFPE) tissue samples, and assessed the agreement rates between MSA-ddPCR and amplicon high-throughput sequencing. The results showed that the limit of blanks of MSA-ddPCR are 0.449 copies μL−1 in the FAM channel and 0.452 copies μL−1 in the VIC channel. MSA-ddPCR could accurately quantify VAFs as low as 0.01 %, surpassing existing PCR and next-generation sequencing (NGS) methods. In the detection of clinical samples, a high correlation was found between MSA-ddPCR and amplicon high-throughput sequencing. Additionally, MSA-ddPCR outperformed sequencing methods in terms of detection time and simplicity of data analysis. MSA-ddPCR can be easily implemented into clinical practice and serve as a robust tool for detecting mutant genes due to its high sensitivity and accuracy.