Search Results (596)

Malignant pleural mesothelioma (MPM) is an aggressive pleural tumor associated with asbestos exposure. Poor clinical outcome of MPM is often driven by late-stage diagnosis due to non-specific clinical presentation, similarity to pleural lesions (e.g., inflammatory changes, metastatic adenocarcinoma), and limitations of current diagnostic methods. We employed Fourier transform infrared (FTIR) spectroscopy combined with convolutional neural networks (CNNs) to analyze formalin-fixed paraffin-embedded (FFPE) pleural tissue samples from patients with MPM, metastatic adenocarcinoma, pleural inflammation, and normal (healthy) pleura. Glycan analysis of FFPE normal pleura and MPM was performed using ultra-high-performance liquid chromatography (UPLC) and mass spectrometry (MS). Our FTIR-spectral analysis uncovered a strong spectral fingerprint of MPM that was especially apparent in the region typical for C-O and C-C stretches as well as local symmetry region typical for deformation vibrations of CH₂ and C-OH groups, all appearing in carbohydrates. Our orthogonal validation of these findings through a targeted glycomics approach using UPLC confirmed that the MPM N-glycome exhibits a distinct fingerprint that distinguishes it from normal pleural tissue. Through utilization of MS for identifying the exact structures of differentially expressed N-glycan peaks, we also identified two high-mannose N-glycan structures that show a specific biomarker potential for MPM and need to be examined in future studies. Full article

Background: Tight junctions are major components of apical junction complexes and are crucial for the maintenance of cell polarity, healthy tissue architecture, adhesion, and permeability. These junctions include the claudin family of transmembrane proteins, which act as paracellular barriers to regulate selective permeability. Abnormal claudin expression disturbs cell adhesions and is associated with cancer through promoting cell invasion, migration, and metastasis. Claudin 6 (CLDN6) overexpression, in particular, is linked to several types of cancer with malignant phenotypes. The present study aimed to investigate the association between CLDN6 protein expression and its copy number variations (CNVs) with clinicopathological features and survival outcomes of ovarian cancer (OC) patients. Methods: A total of 114 formalin-fixed paraffin-embedded blocks from primary OC patients were used to construct tissue microarray slides. Automated immunostaining was used to assess CLDN6 protein expression levels, and next-generation knowledge discovery platforms were used to further evaluate CLDN6 CNV levels using The Cancer Genome Atlas open-source data. The relationships between CLDN6 CNVs and tumor stage, overall survival, disease-specific survival (DSS), and disease-free survival (DFS) were investigated. Results: This study demonstrated that CLDN6 had a mixed membranous-cytoplasmic expression pattern. The cytoplasmic expression of CLDN6 was significantly associated with tumor stage (p = 0.05), tumor size (p = 0.04), and recurrence (p = 0.05). In Univariate analysis, Kaplan–Meier analysis demonstrated that CLDN6 expression was significantly correlated with DFS (p = 0.01). OC patients with lower cytoplasmic CLDN6 expression levels lived longer and had lower recurrence rates. These findings were further confirmed through CLDN6 CNVs analysis, where OC with lower CLDN6 cytoplasmic expression positively correlated with longer DFS and DSS. No independent prognosticator was found when using Cox-regression multivariate analysis (p > 0.05). Conclusions: These results suggest CLDN6 as an interesting prognosticator to identify OC patients at a higher risk of recurrence in order to provide personalized management, alleviate the burden of this disease on women’s health, and improve their survival outcomes. Full article

Syphilis remains one of the most prevalent sexually transmitted infections. Serology is the diagnostic gold standard, but skin biopsies aid in ambiguous cases. Treponemas can be detected in tissue by immunohistochemistry (IHC) and polymerase chain reaction (PCR); however, their comparative performance across disease stages has not been extensively studied. This retrospective study of 48 formalin-fixed, paraffin-embedded (FFPE) skin biopsies from syphilis cases compared Treponema pallidum IHC and PCR. Of 48 biopsies, 42 (88%) were positive by T. pallidum–specific PCR, whereas IHC identified 45 (94%; p = 0.04). Organisms were denser in primary syphilis (p = 0.03) and predominantly involved the lower third of the epidermis. In 7 of 45 biopsies (15%), treponemas were only detected in the dermis (all secondary syphilis). Therefore, Treponema pallidum IHC demonstrated a slight advantage over PCR, but low Treponema density in about 40% and epidermal absence in nearly 20% of secondary syphilis biopsies highlight a substantial risk of overlooking treponemas on IHC. Systematic assessment including endothelium and perivascular areas is essential. Full article

Canine parvovirus type 2 (CPV-2) remains an important cause of morbidity and mortality in young dogs worldwide. Although the enteric form of the disease is well characterized, myocardial involvement associated with CPV-2 infection remains incompletely understood. The present retrospective study aimed to investigate myocardial lesions in young dogs and to evaluate their association with CPV-2 DNA detection using histopathological examination and PCR-based molecular analysis of formalin-fixed paraffin-embedded (FFPE) cardiac tissue. A total of 27 dogs aged between 3 and 11 months, submitted for diagnostic necropsy between 2019 and 2021, were included in the study. Histopathological evaluation was performed on myocardial tissue sections stained with hematoxylin and eosin, while conventional PCR targeting the VP1/VP2 region was used for the detection of CPV-2 DNA. Associations between PCR positivity and histopathological findings were assessed using Fisher’s exact test. Myocardial abnormalities were identified in all examined cases (27/27, 100%), with myocarditis consistently observed throughout the study population. Cardiomyocyte necrosis and myocardial fibrosis were identified in 9/27 (33.3%) and 14/27 (51.9%) cases, respectively. CPV-2 DNA was detected in 9/27 myocardial tissue samples (33.3%). Cardiomyocyte necrosis was significantly more frequent in CPV-2-positive dogs than in PCR-negative animals (77.8% vs. 11.1%; p ≈ 0.001). Similarly, myocardial fibrosis was identified more frequently in CPV-2-positive cases (100.0% vs. 27.8%; p ≈ 0.001). Myocarditis was present in all examined animals and therefore could not be evaluated statistically according to PCR status. The detection of CPV-2 DNA in myocardial tissues exhibiting histopathological lesions supports a possible association between CPV-2 infection and myocardial injury in young dogs. However, the retrospective design, limited sample size, and the use of conventional PCR do not allow conclusions regarding causality or direct viral involvement in lesion development. Further studies incorporating larger case series and complementary diagnostic techniques are needed to clarify the pathogenesis and clinical significance of CPV-2-associated myocardial lesions. Full article

Medulloblastoma is a heterogeneous solid tumor, and its molecular characteristics are the most important prognostic factors for this neoplasm. Unfortunately, the molecular classification of MB-G3 and MB-G-4 medulloblastoma is very complex because of molecular similarity. Therefore, in this work, through unsupervised machine learning-based gene expression profiling, we identified a low molecular profile associated with four molecular groups of medulloblastoma. We performed medulloblastoma expression microarray data mining via the Partek Genomics Suite and Transcriptome Analysis Console (TAC), and we included a total of 25 fresh medulloblastoma tumors that were obtained and hybridized into HG U133 Plus 2.0 Array microarrays. To identify the molecular groups of the 25 patients, we compared them against classified patients, which were obtained from free repositories, and through data mining based on gene expression, compared the expression profiles of our patients. To do so, we performed an analysis via the least squares method via PCA. The molecular groups MB-WNT and MB-SHH were confirmed via immunohistochemistry via β-catenin, YAP1 and GAB1 antibodies in tissue fixed in formalin and embedded in paraffin, and another tissue section was placed on a Visium Spatial slide to perform spatial RNA-seq via Illumina NextSeq 2000 platform sequencers. The data obtained were analyzed with R. We identified the expression profiles associated with the four molecular groups and formed a reference set. Through unsupervised analysis via the least squares method, we assigned the molecular profiles of 25 patients with medulloblastoma, via the integration of bulk and spatial tumor molecular gene expression profiling analysis and with immunohistochemical findings, this strategy was fast and accurate. We observed correlations in three of the trials carried out and, in part, in one study, a patient who presented two tumor strains and two molecular signatures (SHH and G4), which led us to believe that this patient presented mixed phenotypic characteristics. Multigene expression profile analysis of medulloblastoma represents a significant advance in precision medicine; integrating different layers of transcriptomic information allows us to demonstrate underlying molecular changes in the four molecular groups that are essential for personalized therapy. Full article

Background/Objectives: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease frequently requiring liver transplantation (LTx). The gut–liver axis, host genetics, and microbial dysbiosis are thought to contribute to disease progression and post-transplant outcomes. The FUT2 rs601338 polymorphism influences mucosal fucosylation, host–microbial interactions, and susceptibility to infection. This study aimed to investigate the association between FUT2 genotype, colonic mucosal microbiome composition, post-transplant bacteremia, and all-cause mortality in a retrospective single-center PSC cohort. Methods: This retrospective cohort study included PSC patients who underwent LTx at Erasmus MC University Medical Center (Rotterdam, The Netherlands) between 1987 and 2015. Pre-transplant archival formalin-fixed paraffin-embedded (FFPE) colonic biopsy specimens were available for microbiome analysis. Of 169 transplanted patients, FFPE tissue was available for 98 individuals, and FUT2 rs601338 genotyping was successfully performed in 87 patients. Patients were classified as FUT2 non-secretors (AA, n = 28) and secretors (GA/GG, n = 59). Post-transplant bacteremia was assessed based on clinically indicated blood cultures during follow-up. Colonic mucosal microbiome composition was analyzed using 16S rRNA gene sequencing. Results: FUT2 non-secretors showed a distinct colonic mucosal microbiome profile compared with secretors, characterized by differential abundance of selected taxa within Proteobacteria, Firmicutes, and Bacteroidetes. Post-transplant bacteremia occurred in 30 patients and was more frequent among non-secretors (43%) compared with secretors (15%). Both FUT2 non-secretor status and post-transplant bacteremia were associated with reduced all-cause post-transplant survival in Kaplan–Meier analysis and remained associated with mortality in multivariable regression models. Specific microbial taxa were also showed associations with bacteremia, mortality, and established prognostic scores, including the Amsterdam–Oxford Model and Mayo Risk Score. Conclusions: FUT2 genotype is associated with alterations in colonic mucosal microbiome composition, post-transplant bacteremia, and all-cause mortality in PSC patients undergoing liver transplantation. These findings suggest a potential interplay between host genetics, intestinal microbiota, and infectious complications after transplantation. Given the retrospective design, limited sample size, and use of archival FFPE tissue, all findings should be interpreted as exploratory and hypothesis-generating. Prospective multicenter studies using standardized sampling and high-resolution metagenomic approaches are warranted for validation. Full article

Therapeutic agents targeting the PD1–PDL1 interaction, commonly called PD1 blockade, are of great clinical value; however, predicting which patients will benefit has been inconsistent, in part, due to a lack of reliable biomarkers. Quantifying PD1 saturation by PDL1 in tumor tissue has the potential to serve as a biomarker; unfortunately, few diagnostic technologies are available to reliably quantify PD1 saturation in clinical biospecimens. Here, we report on a novel bioassay based on RNA aptamers, called the PD1 LIRECAP assay, that allows for quantification of the saturation of PD1 by PDL1 in formalin-fixed, paraffin-embedded (FFPE) tumor biospecimens. The assay is technically straightforward, high-throughput capable and reproducible. Results showed that quantification of PD1 saturation determined by PD1 LIRECAP assay correlates closely with PD1-mediated signaling and PD1–PDL1 proximity. Analysis of sarcoma FFPE biospecimens confirmed the assay to be consistent and revealed significant differences between patients as well as considerable intratumoral heterogeneity in PD1 saturation by PDL1. We conclude that this novel PD1 LIRECAP platform is technically feasible, reproducible and has the potential to be a superior predictive biomarker assay to predict the outcome of PD1/PDL1-based therapy. Similar assays based on this platform could be used in other systems and settings to quantify the interaction between two molecules. Full article

Background/Objectives: Despite hematoxylin and eosin (H&E) staining remaining the cornerstone of histopathological diagnosis, substantial intra- and inter-laboratory variability persists. This issue is increasingly relevant in Digital Pathology, where staining inconsistency may affect whole-slide image interpretation and the performance of image analysis algorithms. In the present work, we evaluated the diagnostic adequacy and non-inferiority of a novel tabs-based H&E histochemical staining method compared with conventional liquid reagents. Methods: Fifty formalin-fixed paraffin-embedded tissue samples from routine practice were sectioned in duplicate and stained either conventionally or using H&E Stain Tabs. After slide review, 14 representative tissue samples were selected, scanned at 40× magnification, and used to generate 24 matched image pairs at different magnifications. A blind online survey was completed by 13 expert pathologists using high-quality monitors. Participants assessed overall staining preference and rated stromal, epithelial, cytoplasmic, and nuclear staining quality. Non-inferiority was tested using a predefined margin of −0.10, and paired rating differences were analyzed using the Wilcoxon signed-rank test. Results: Across 312 paired evaluations, the tabs-based method was preferred in 120 cases (38.5%), conventional staining in 118 cases (37.8%), and no preference was expressed in 74 cases (23.7%). The tabs-based method met the criterion for non-inferiority compared with standard staining (z = 2.7). Rating-scale analysis showed significantly better stromal evaluation with the tablet-based method (z = 2.638; p = 0.008), whereas no significant differences were observed for epithelial, cytoplasmic, or nuclear staining. All evaluated images were considered diagnostically adequate. Conclusions: The tabs-based H&E stain was non-inferior to the conventional method and showed particularly favorable performance in the assessment of stromal components. These findings support its potential role in improving staining reproducibility and standardization, particularly in Digital Pathology workflows where pre-analytical and analytical consistency is critical. Full article

Understanding the spatial proteomic landscape of human tumors is essential for dissecting cellular heterogeneity and microenvironmental interactions in cancer biology. Traditional bulk proteomic approaches, however, obscure spatial information and average out signals from distinct cell populations. Here, we present a detailed and reproducible micro-quantitative protocol for spatially resolved proteomic analysis of specific cellular subpopulations isolated from immunohistochemistry (IHC)-labeled formalin-fixed paraffin-embedded (FFPE) tissue sections using laser microdissection (LMD). By combining IHC staining to visually define phenotypically distinct cells within preserved tissue architecture and precise LMD capture, approximately 6000 target cells can be isolated per sample for downstream proteomic quantification. Despite the ultra-low input, optimized lysis and digestion steps ensure consistent peptide recovery and highly reproducible label-free LC–MS/MS data across replicates. Integrating immunohistochemistry staining-guided spatial sampling with ultrasensitive quantitative proteomics, this workflow enables reliable cell-type-specific profiling directly within human tumor tissues. The protocol bridges histopathology and proteomics, offering a practical framework for translational research exploring spatial protein signatures and tumor microenvironmental heterogeneity. Full article

Background: Persistent high-risk human papillomavirus (hrHPV) infection causes over 99% of cervical precancers and cancers worldwide, with HPV genotype 16 (HPV16) responsible for 50% of the cases. Latvia ranks among the top EU countries for cervical cancer incidence and mortality. In the general Latvian population, 4.2% of women are hrHPV-infected, mostly with HPV16. However, information on the circulating HPV16 isolates is missing. Objectives: To study the genomic variability of the Latvian HPV16 isolates, compare them with HPV16 in Europe and across the globe, reveal features associated with the severity of cervical disease and uncover eventual sequence changes due to the national HPV vaccination. Methods: DNA was extracted from the formalin-fixed paraffin-embedded cervical tissues of women diagnosed with cervical intraepithelial neoplasia (CIN) stages I-III and squamous cell carcinoma (SCC) grades 1–3, collected between 2012 and 2024. Samples positive for HPV16 were subjected to whole genome sequencing (WGS) on the Illumina platform (n = 16) or Sanger sequencing of the E6/E7 coding region (n = 31). A consensus HPV16 sequence was generated, and single nucleotide polymorphisms (SNPs) and eventual amino acid substitutions (AAS) were analysed. Results: Complete genomes of 16 HPV16 variants were reconstructed, with 13 related to the European sublineage A1 and 3 to the sublineage A2 references. Sequences showed high conservation; still 93 non-redundant variants were identified. The highest variability was observed for the capsid protein L2, and the lowest, for oncoprotein E7. The prevalence of SNPs and AAS in the Latvian HPV16 variants, specifically in capsid protein L1, did not increase with time, showing no effect of HPV vaccination. Associations between HPV16 sequence features and severity of cervical disease were limited to AAS E6:L90V, which was significantly more common in SCC grade 2/3 than in CINII/III cases (p = 0.015). Conclusions: Highly conserved HPV16 genomes circulating in Latvia harbour a series of unique as well as common nonsynonymous SNPs with respective AAS, with one, AAS E6:L90V, associating with disease severity. No HPV vaccine escape variants were detected. Deciphering complete genomes of HPV16 from CIN and SCC cases in Latvia informs public authorities performing HPV vaccination and is useful for the management of HPV-associated cervical diseases. Full article

Pathology is foundational to biomarker-driven and translational oncology research, yet systemic barriers limit full pathology engagement in Canadian cancer clinical trials, compromising the tissue-based questions such trials are designed to answer. This commentary and white paper synthesizes the perspectives of a national pathology working group convened at the 2025 Canadian Cancer Trials Group (CCTG) Annual General Meeting with a descriptive internal audit of the CCTG Tumour Tissue Data Repository (TTDR), in which biospecimen attrition was tabulated at the patient level by disease site; no inferential testing was performed. The TTDR data revealed substantial attrition across disease sites, with no tissue submitted for 32–44% of patients and slides submitted in place of formalin-fixed paraffin-embedded blocks for up to 51% of cases, reflecting persistent misalignment between protocol expectations and laboratory capacity. From these observations, five interrelated gaps were identified—in trial design, funding and resourcing, digital pathology infrastructure, academic recognition, and knowledge translation around consent and ethics governance. Five corresponding strategies are proposed to align research demands with pathology capacity, reduce attrition, and strengthen biomarker-driven trials. As a consensus- and experience-driven analysis rather than a systematic review, these recommendations are intended to frame a national conversation and a starting point for prospective evaluation. Full article

Objective: The aim of this study was to evaluate the expression of PD-L1, PD-1, CD3, CD4, and CD8 in tumor tissues of patients with head and neck squamous cell carcinoma from southern Poland, and to assess their prognostic value in relation to disease-free survival (DFS), taking into account HPV16 status and other clinical, pathological, and demographic factors. Material/Methods: This study included 155 unselected patients with head and neck squamous cell carcinoma (HNSCC) from the southern Poland region, who underwent diagnostic evaluation and surgical treatment. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were obtained from these centers. The patients were treated at the Maria Skłodowska-Curie National Research Institute of Oncology, Kraków Branch, between 1991 and 2014, with treatment approaches including induction therapy (preoperative), adjuvant therapy (postoperative), or definitive chemoradiotherapy with cisplatin. Protein expression was assessed using immunohistochemistry and quantified (TPS, CPS, H-score). Relationships between expression levels and epidemiological, clinical, and histopathological features were analyzed. Results: The results show that PD-L1 overexpression was associated with worse DFS, whereas overexpression of PD-1, CD3, CD4, and CD8 correlated with improved DFS. These associations were statistically significant in the HPV16-negative subgroup, while no such correlations were found in HPV16-positive patients. In multivariate analysis, independent prognostic factors associated with improved DFS included HPV16 infection, absence of PD-L1 overexpression, overexpression of CD4 and CD8, and combined chemoradiotherapy with cisplatin. Conclusions: These findings confirm the prognostic relevance of PD-L1, PD-1, and T-cell markers in HNSCC, particularly in HPV16-negative patients, and support further research into the use of these biomarkers in personalized treatment strategies. Full article

Osteosarcoma is the most common primary bone tumor in children, adolescents, and young adults, and metastasis remains the main determinant for a poor outcome. We conducted an exploratory retrospective cohort study using formalin-fixed, paraffin-embedded tissue from 97 patients with histopathologically confirmed osteosarcoma treated between 2005 and 2019 to evaluate telomere maintenance mechanisms. To assess alternative lengthening of telomeres (ALT), colocalization of telomeric DNA and promyelocytic leukemia protein (TEL–PML) was evaluated as a tissue-based proxy. TEL–PML colocalization was assessed using combined PML immunofluorescence and telomere DNA PNA-FISH. Furthermore, telomerase reverse transcriptase (TERT) expression was evaluated by immunohistochemistry in relation to metastasis and disease progression. Logistic regression models were adjusted for age, sex, and smoking. TEL–PML was evaluable in 45/97 cases, including 10 positive and 35 negative tumors; the remaining samples were non-evaluable because of non-determinable signal or predominant necrosis. TERT immunohistochemistry was scorable in 58/97 cases, of which 33 were positive. TEL–PML evaluability was associated with amputation specimens, whereas TERT positivity was associated with non-osteoblastic histology and was inversely associated with age. Neither TEL–PML nor TERT was significantly associated with metastasis, recurrence, or death. Exploratory time-to-metastasis curves suggested an earlier increase of metastatic events among TEL–PML-positive cases. However, the small number of positive tumors precludes definitive prognostic interpretation. These hypothesis-generating findings indicate that TEL–PML assessment is feasible in osteosarcoma, but it is strongly influenced by tissue adequacy. On the other hand, TERT immunohistochemistry appears to reflect subtype- and age-related heterogeneity rather than providing robust outcome stratification in this cohort. Full article

Background/Objectives: Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cancer, characterized by frequent metastasis and poor prognosis. Epithelial–mesenchymal transition (EMT) plays a pivotal role in tumor progression. Protocadherin 9 (PCDH9) has emerged as a potential tumor suppressor, but its relationship with EMT markers in ccRCC remains unclear. This study aimed to investigate the expression patterns and prognostic significance of PCDH9, β-catenin (CTNNB1), Snail (SNAI1), and Vimentin (VIM) in ccRCC. Methods: Immunofluorescence analysis was performed on formalin-fixed paraffin-embedded tissue sections from 48 ccRCC patients (31 low-grade, 17 high-grade) and adjacent normal renal cortex. Findings were validated using The Cancer Genome Atlas (TCGA-KIRC) dataset via GEPIA2/GEPIA3 platforms, including differential expression, correlation, and survival analyses. Results:PCDH9 mRNA was significantly downregulated in ccRCC tumors (TCGA-KIRC), while VIM was upregulated at the transcriptomic level. Tissue-level immunofluorescence quantification revealed discordant patterns, highlighting the influence of cellular heterogeneity on bulk protein assessment. The strong positive correlation between PCDH9 and CDH1 observed in normal kidney was completely lost in tumor tissue. Unexpectedly, PCDH9 showed positive correlations with EMT transcription factors (ZEB1, SNAI1) in tumors. In univariate survival analysis, high PCDH9 and CTNNB1 expression were associated with improved overall survival. Multivariate Cox regression revealed endpoint-specific prognostic signatures: VIM independently predicted disease progression, while SNAI1 predicted overall mortality. CTNNB1 was consistently protective across both endpoints. Conclusions: Our findings support a tumor-suppressive role for PCDH9 in ccRCC and reveal disruption of epithelial adhesion molecule co-regulation during tumorigenesis. The identification of endpoint-specific prognostic signatures has implications for patient stratification and suggests that ccRCC exhibits a partial EMT phenotype rather than classical EMT. Full article

Background/Objectives: Formalin-fixed paraffin-embedded tissue is widely used in pathology and molecular diagnostics, yet its variable quality can critically influence the accuracy of sequencing-based analyses. This study investigated pre-analytical and analytical factors that can affect exome sequencing performance in a Brazilian multicenter cohort of patients with breast cancer and prostate adenocarcinoma. Methods: Tumor samples were reviewed for diagnostic confirmation, and those with a minimum cellularity of 20% underwent DNA extraction, fluorometric quantification, fragmentation analysis, and exome sequencing. Pre-analytical parameters, including tumor content, DNA yield, and fragmentation profile, were recorded and correlated with sequencing results. Results: Only 36.7% of all analyzed samples (163/444) generated valid whole-exome sequencing data, corresponding to 55.6% of those that proceeded to sequencing (163/293). Although 94.5% of specimens met the minimum ≥20% cellularity threshold and 66.0% advanced to sequencing, a substantial proportion failed to yield usable exome data. Successful sequencing was associated with shorter storage durations (p < 0.001) and superior analytical parameters (higher autosomal coverage, longer read lengths, lower duplication rates, and higher target coverage at ≥500× and ≥100×; p < 0.001). Detailed fixation-related variables (e.g., formalin type, fixation time, ischemia time) were not consistently available across centers, representing a major limitation for causal interpretation of pre-analytical effects. Conclusions: Our study identified a high failure rate in sequencing archival tissue, highlighting the need to prioritize more recently collected specimens and refine standardized sample handling protocols to maintain DNA integrity. These improvements are essential for optimizing sequencing workflow performance and feasibility in real-world settings. Full article