Search Results (41)

Backgrounds: Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is a critical predictive biomarker for immune checkpoint inhibitor (ICI) therapy in triple-negative breast cancer (TNBC). However, prolonged storage of formalin-fixed paraffin-embedded (FFPE) tissue may reduce antigenicity, potentially leading to false-negative results. False-negative results may lead to the inappropriate selection of ICI therapy. We investigated the effect of FFPE storage duration on PD-L1 immunoreactivity. Methods: We retrospectively analyzed 63 TNBC cases with PD-L1 testing using the 22C3 pharmDx assay at diagnosis and repeated IHC on the same FFPE blocks after varying storage durations (<1, 1–2, 2–3, ≥3 years). PD-L1 positivity was defined as Combined Positive Score (CPS) ≥ 10. Associations with clinicopathologic features, pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC), and survival were evaluated. Results: At diagnosis, 41 patients (65.1%) were PD-L1–positive. In the PD-L1–positive group, decreased staining was observed in 0%, 11%, 13%, and 50% of cases for <1, 1–2, 2–3, and ≥3 years of storage, respectively (p = 0.015). PD-L1 positivity correlated with higher Ki67 and nuclear grade. pCR was achieved in 33% of PD-L1–positive vs. 0% of PD-L1–negative NAC patients (p = 0.0527). Survival analysis showed a non-significant trend toward shorter recurrence-free and overall survival in PD-L1–positive patients. Conclusions: Prolonged FFPE storage, particularly beyond three years, significantly reduces PD-L1 immunoreactivity. Testing on recent specimens is recommended to avoid false-negative results that may impact ICI eligibility. Full article

Background/Objectives: Obtaining reliable DNA profiles from archival tissue preserved as formalin-fixed, paraffin-embedded (FFPE) samples remains a major challenge in both forensic and medical evaluations. The quality of DNA isolated from FFPE material is frequently compromised due to formalin-induced fragmentation and chemical modifications. These limitations are particularly relevant in cases of suspected medical malpractice related to cancer diagnosis or treatment, where retrospective molecular analyses may provide critical evidence. The aim of this study was to evaluate the performance of the Maxwell^® RSC Xcelerate DNA FFPE Kit (Promega) in generating DNA profiles from archival FFPE tissue blocks of endometrial cancer and to identify the limitations associated with this approach. Methods: Archival FFPE blocks of endometrial cancer were analyzed using the Maxwell^® RSC Xcelerate DNA FFPE Kit. DNA yield, purity, and degradation indices were assessed using standard real-time PCR-based quantification methods. Short tandem repeat (STR) profiling was performed with forensic genotyping kits, and the completeness, allele balance, and reliability of obtained profiles were evaluated. The obtained results were compared with reference quality thresholds commonly used in forensic practice. Results: The Maxwell^® RSC Xcelerate Kit allowed for recovery of relatively high DNA yields with consistently low degradation indices, confirming good extraction efficiency from FFPE samples. Nevertheless, despite favorable quantitative values, the generation of complete STR profiles was often unsuccessful. Partial or incomplete profiles were frequent, characterized by allele dropout and imbalance, which substantially reduced their evidentiary value. These findings suggest that DNA fragmentation and fixation-related artifacts impair amplification efficiency and limit the usefulness of STR analysis. Conclusions: This study emphasizes the persistent challenges of DNA profiling from FFPE tissue in forensic-medical contexts. Although the Maxwell^® RSC Xcelerate Kit demonstrated effective DNA recovery, the ability to generate complete and interpretable STR profiles remained limited. Further refinement of extraction protocols, as well as improved interpretative strategies, are required to enhance the reliability and evidentiary significance of molecular analyses based on archival FFPE material. Full article

Cervical cancer screening plays a crucial role in preventing invasive disease through early detection of high-grade lesions. However, traditional cytology and histology often fail to reliably differentiate between transient HPV infections and those likely to progress. This study investigates the feasibility of integrating molecular HPV testing into histopathological workflows using FFPE tissue samples to improve diagnostic precision. A retrospective analysis was conducted on 55 FFPE cervical specimens from patients undergoing colposcopy with biopsy or conization. The workflow included automated DNA extraction and real-time PCR-based HPV genotyping with the Seegene Anyplex II HPV28 assay. HPV DNA was detected in 56.4% of samples, with 21 genotypes, including multiple high-risk types. High viral loads correlated with high-grade lesions, supporting the clinical value of HPV quantification. Compared to histology, molecular analysis reduced potential overdiagnosis by confirming HPV absence in morphologically suspicious but HPV-negative lesions. Integrating viral load and genotyping improved risk stratification, optimizing colposcopy referrals and reducing unnecessary follow-ups. This study introduces a novel, fully automated molecular workflow applicable to FFPE samples, enhancing cervical cancer screening beyond traditional methods. Although based on a limited sample, the findings support the method’s potential for broader implementation and further validation in multicenter settings. Full article

Synthetic biology has fundamentally advanced cell engineering and helped to develop effective therapeutics such as chimeric antigen receptor (CAR)-T cells. For these applications, the detection, localization, and quantification of heterologous fusion proteins assembled from interchangeable building blocks is of high importance. The V5 tag, a 14-residue epitope tag, offers promising characteristics for these applications but has only rarely been used in this context. Thus, we have systematically evaluated the murine anti-V5 tag antibody mu_SV5-Pk1 as well as its humanized version, hu_SV5-Pk1, to analyze cells expressing V5-tagged receptors in samples from various in vitro and in vivo experiments. We found that the V5 tag signal on cells is affected by certain fixation and detachment reagents. Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse tissue samples was performed to sensitively detect cells in tissue. We improved IHC by applying the hu_SV5-Pk1 monoclonal antibody (mAb) to avoid cross-reactivity within and unspecific background signals arising on fixed mouse tissue. Conversely, the absence of unspecific binding by the mu_SV5-Pk1 mAb was evaluated on 46 human normal or cancer tissues. Our findings present a robust toolbox for utilizing the V5 tag and cognate antibodies in synthetic biology applications. Full article

Background: The fragile X protein family comprises three members: the fragile X syndrome protein (FMRP) and its structural homologs, fragile X syndrome 1 and 2 (FXR1 and FXR2). FMRP has a significant role in controlling the genesis and progression of various forms of human cancer. However, studies on the prognostic significance of FXR2 in cancer are scarce. Thus, this study aimed to investigate the clinicopathological significance of FXR2, a member of the FMRP family, in primary breast cancer (BC). Methods: A total of 100 formalin-fixed paraffin-embedded (FFPE) tissue blocks from invasive BC cases were collected from King Abdulaziz Hospital in Saudi Arabia. Immunohistochemistry (IHC) was used to assess FXR2 protein expression in the BC tissues, and the results were correlated with clinicopathological parameters, such as tumor grade, tumor size and hormone receptor status. Additionally, the association between clinicopathological features and FXR2 mRNA expression was assessed using the BC Gene-Expression Miner v5.0 tool on all publicly available DNA microarray (n = 10,872) and RNA sequence (n = 4421) data to validate the results. Results: FXR2 protein expression was significantly associated with human epidermal growth factor 2 (HER2) negativity (p = 0.010) and low Ki67 (p < 0.001). Both DNA microarray and RNA sequence data showed that HER2 negativity was strongly linked to high levels of FXR2 mRNA. High FXR2 mRNA levels were also correlated with hormone receptor negativity and mutated p53. Conclusions: This study suggests that FXR2 may have indirect clinical significance in BC. However, further studies are warranted to deepen our understanding of the association between FXR2 and other clinicopathological parameters, which could lead to improved diagnostic, treatment, and prognostic strategies for BC patients. Full article

Human papillomavirus (HPV) is a major cause of cervical cancer, a significant health concern worldwide. Despite advances in screening methods, including the Pap test and the HPV DNA test, limitations remain in accurately predicting which HPV infections will progress to cervical intraepithelial neoplasia (CIN) and, eventually, invasive cancer. This study evaluates the usefulness in real life of assessing HPV viral load and the presence of multiple HPV genotypes in enhancing the diagnostic accuracy of triage in cervical cancer screening. A retrospective analysis was performed on 55 formalin-fixed paraffin-embedded cervical samples collected from women who underwent colposcopy with a biopsy or conization at San Martino Hospital, Genova, Italy, between January and June 2021. Histological diagnoses were compared with molecular analyses (HPV genotyping, viral load quantification and co-infection) using a multiplex real-time PCR platform. Of the samples analyzed, 56.4% were HPV DNA positive, while 40% tested negative. The molecular analysis identified more HPV-negative cases than the histological analysis (p < 0.05). Higher viral loads and HPV co-infections were more frequent in high-grade CIN lesions. These markers may help identify patients at an elevated risk for persistent infections and cancer progression. These findings support the potential of integrating HPV viral load and genotype co-infection assessments into routine cervical cancer screening protocols to improve early detection and reduce overtreatment and unnecessary interventions. Full article

Background and Objectives: This study examined factors influencing the onset and progression of colorectal tumors, including patients’ epidemiological data, tumor location (right-sided, left-sided, and rectal), histomorphology, perineural or intraneural invasion, lymph node status, immune reactions, mismatch repair (MMR) status, and commonly observed mutations. Our primary goal was to evaluate their predictive and prognostic value and interactions. Materials and Methods: We analyzed a retrospective cohort of 100 patients with colorectal adenocarcinoma diagnosed between 2020 and 2023, using formalin-fixed paraffin-embedded (FFPE) tumor blocks. The methods included routine H&E microscopy, immunohistochemistry, Next-Generation Sequencing (NGS), and subsequent statistical analysis. Results: The findings showed a median diagnosis age of 70 years, with no gender-specific tumor localization. Right-sided tumors were prevalent, especially among patients with a defective MMR (dMMR), which represented 89% of dMMR cases. MMR status significantly correlated with tumor localization. We observed significant relationships between tumor grade, lymphovascular invasion, and overall tumor stage. Higher tumor grades and stages correlated with increased lymphovascular invasion and lymph node involvement. Interestingly, tumor budding did not correlate with lymph node metastasis but was significantly associated with higher tumor grades. Most BRAF mutations were found in right-sided tumors, indicating a significant correlation with this localization. Conclusions: This study focuses on the diversity of colorectal cancer (CRC) by examining how genetic and histological characteristics vary based on tumor location or other tumor variables. Full article

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. Full article

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids’ quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6–8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6–8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond. Full article

Background and Objectives: The most common mutation in malignant melanoma (MM) is the single-point mutation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) oncogene. Our study aims to evaluate BRAF V600E mutation, highlighting its frequency differences in primary versus metastatic MM. Materials and Methods: The study group comprised 133 patients diagnosed with MM in several county hospitals of the north-eastern region of Romania who have been assigned for investigation into BRAF V600E mutation in the private medical system. The material consisted of archived formalin-fixed paraffin-embedded (FFPE) blocks. BRAF V600E mutation was identified using the fully automated Idylla^TM BRAF mutation test system. Results: Out of the total of 133 cases, 78 cases were primary tumors, while 55 cases were metastatic MMs. Genetic analysis revealed the presence of BRAF V600E mutation in 66 cases (49.62%) and the wild-type genotype in 67 cases (50.37%). We found a statistically significant difference of the mutation frequency according to age (p = 0.0072). The mutated genotype was found in 45 cases out of 78 primary MMs (57.69%) and in 21 cases out of 55 secondary MMs (38.18%), with a statistically significant difference in favor of primary tumors (p = 0.0413). The correlations between the histopathological types, Clark’s level, Breslow index, ulceration, and lymphovascular invasion, respectively, and the mutated genotype were not statistically significant. BRAF V600E mutation was identified in 15 out of 40 secondary tumors with lymph node location (37.5%) and in 6 out of 15 secondary tumors with another location (40%) without statistically significant differences between the mutation frequency and the location of the secondary tumors. Conclusions: Our results support MM high genetic heterogeneity, pointing out the relationship between BRAF V600E mutation and several clinicopathological characteristics, in primary and metastatic MMs, stressing the importance of BRAF testing implementation in Romania. Full article

The expression level of the progesterone receptor (PGR) plays a crucial role in determining the biological characteristics of serous ovarian carcinoma. Low PGR expression is associated with chemoresistance and a poorer outcome. In this study, our objective was to explore the relationship between tumor progesterone receptor levels and RNA profiles (miRNAs, piwiRNAs, and mRNAs) to understand their biological characteristics and behavior. To achieve this, we employed next-generation sequencing of small non-coding RNAs, quantitative RT-PCR, and immunohistochemistry to analyze both FFPE and frozen tumor samples, as well as blood plasma from patients with benign cystadenoma (BSC), serous borderline tumor (SBT), low-grade serous ovarian carcinoma (LGSOC), and high-grade serous ovarian carcinoma (HGSOC). Our findings revealed significant upregulation of MMP7 and MUC16, along with downregulation of PGR, in LGSOC and HGSOC compared to BSC. We observed significant correlations of PGR expression levels in tumor tissue with the contents of miR-199a-5p, miR-214-3p, miR-424-3p, miR-424-5p, and miR-125b-5p, which potentially target MUC16, MMP7, and MMP9, as well as with the tissue content of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p, which are associated with the epithelial–mesenchymal transition (EMT) of cells. The levels of EMT-associated miRNAs were significantly correlated with the content of hsa_piR_022437, hsa_piR_009295, hsa_piR_020813, hsa_piR_004307, and hsa_piR_019914 in tumor tissues. We developed two optimal logistic regression models using the quantitation of hsa_piR_020813, miR-16-5p, and hsa_piR_022437 or hsa_piR_004307, hsa_piR_019914, and miR-93-5p in the tumor tissue, which exhibited a significant ability to diagnose the PGR-negative tumor phenotype with 93% sensitivity. Of particular interest, the blood plasma levels of miR-16-5p and hsa_piR_022437 could be used to diagnose the PGR-negative tumor phenotype with 86% sensitivity even before surgery and chemotherapy. This knowledge can help in choosing the most effective treatment strategy for this aggressive type of ovarian cancer, such as neoadjuvant chemotherapy followed by cytoreduction in combination with hyperthermic intraperitoneal chemotherapy and targeted therapy, thus enhancing the treatment’s effectiveness and the patient’s longevity. Full article

Background and Objectives: BRAF mutational status in resected non-small cell lung cancer (NSCLC) in the Korean population is poorly understood. We explored BRAF (particularly BRAF V600E) mutational status among Korean patients with NSCLC. Materials and Methods: This study included 378 patients with resected primary NSCLC who were enrolled from January 2015 to December 2017. The authors obtained formalin-fixed paraffin-embedded (FFPE) tissue blocks and performed peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR) for detecting BRAF V600, real-time PCR for detecting BRAF V600E, and immunohistochemical analyses using the mutation-specific Ventana VE1 monoclonal antibody. For positive cases in any methods mentioned above, direct Sanger sequencing was additionally performed. Results: The PNA-clamping method revealed the BRAF V600 mutation in 5 (1.3%) of the 378 patients. Among these five patients, real-time PCR, direct Sanger sequencing detected BRAF V600E mutations in three (0.8%) patients. Thus, two cases showed differences in their PNA-clamping and the others. Direct Sanger sequencing of PNA-clamping PCR product was performed for two cases showing negative results on direct Sanger sequencing; both contained BRAF mutations other than V600E. All patients harboring BRAF mutations had adenocarcinomas, and all patients with V600E mutation exhibited minor micropapillary components. Conclusions: Despite the low incidence of the BRAF mutation among Korean patients with NSCLC, lung adenocarcinoma patients with micropapillary components should be prioritized in terms of BRAF mutation testing. Immunohistochemical staining using Ventana VE1 antibody may serve as a screening examination for BRAF V600E. Full article

The implementation of next-generation sequencing (NGS) in clinical oncology has enabled the analysis of multiple cancer-associated genes for diagnostics and treatment purposes. The detection of pathogenic and likely pathogenic mutations is crucial to manage the disease. Obtaining the mutational profile may be challenging in samples with low yields of DNA—reflected by the type of biological material, such as formalin-fixed paraffin-embedded tissue (FFPE), needle biopsies, and circulating free/tumor DNA, as well as a sparse tumor content. Moreover, standardized strict procedures for the extraction of DNA in a clinical setting might contribute to lower amounts of DNA per µL. The detection of variants in low-yield DNA samples remains a challenge in clinical diagnostics, where molecular analyses such as NGS are needed. Here, we performed vacuum centrifugation on DNA extracted from five FFPE tissue blocks, with concentrations below 0.2 ng/µL. Through NGS analysis, we found that low-yield DNA samples could be concentrated to sufficient levels, without compromising the mutational profile. Full article

Background: Evolving targeted therapy on Janus Associated Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, especially pertaining to STAT-3 protein in non-Hodgkin lymphoma (NHL), provides new treatment strategies. STAT-3 protein also relates to the prognostication of NHL. Hence, we aimed to evaluate the expression of STAT-3 protein in NHL cases diagnosed in Hospital Universiti Sains Malaysia (USM). Methods: A retrospective cross sectional study using formalin fixed paraffin embedded (FFPE) tissue blocks of 95 NHL cases were obtained. STAT-3 immunostaining was performed and evaluated. The proportion and association between the expression of STAT-3 protein with subtypes of NHL were statistically analyzed. Results: The majority of the cases (78.9%) had positive STAT-3 protein expression. 64.2% were among aggressive B cell NHL, whilst 20.0% of them were diffuse large B cell lymphoma, a non-germinal center B subtype (DLBCL-NGCB). There is also an association between STAT-3 protein expression with DLBCL subtypes (p = 0.046). Conclusion: Our study demonstrated a remarkable expression of STAT-3 protein in NHL, in which DLBCL subtypes had significant association. A larger scale study with a combination of JAK protein evaluation should be undertaken in the future. Full article

Heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV) was first described in farmed Atlantic salmon in Chile in 2011. However, as PRV induces long-lasting infections, it is not known when Chilean farmed salmon may have started to show PRV positivity. This study aimed to evaluate the presence/absence of PRV-1 in formalin-fixed, paraffin-embedded Atlantic salmon heart tissues (FFPE) cultured in Chile during 1992 and 1999. The most frequent histopathological findings in the 42 FFPE blocks were mild focal cardiomyocyte degeneration (57.1%) and a mild focal mononuclear inflammatory infiltrate (21.4%) in the ventricular stratum spongiosum of the heart. One of the 42 heart samples analyzed by RT-qPCR was positive for PRV-1 (2.4%). All samples were negative for other viral and bacterial pathogens that can induce similar histological changes in the heart. Taken together, our results show that PRV-1 has been present in Chile—as a low-virulence genogroup—since at least 1994, 17 years before the first HSMI outbreak in 2011. Finally, archaeovirology can be a valid alternative to contribute to the understanding of the epidemiology of diseases in aquaculture. Full article