Paraffin-embedded Tissue Samples Research Articles

Influence of MRP4 levels on clinical outcomes in biliary tract cancer.

624 Background: Biliary tract cancer (BTC) are rare tumors with a poor prognosis.Multidrug resistance protein 4 (MRP4) is a transmembrane efflux transporter that influences on cellular growth and differentiation. Recent studies have linked high MRP4 levels to increased aggressiveness in pancreatic cancer and other tumor types. This study aimed to evaluate the role of MRP4 in BTC and its association with clinical outcomes in a real-world cohort from endemic areas in Argentina. Methods: We conducted a retrospective analysis of clinical outcomes and MRP4 expression in paraffin-embedded tissue samples from 78 patients with BTC. Immunohistochemistry staining was performed, and MRP4 expression was scored based on staining intensity and the percentage of positive cells. A score exceeding 100 was classified as high MRP4 levels. Statistical analyses included Chi-square, Kaplan-Meier survival curves, and log-rank tests, with significance set as p <0.05 (two-tailed). Results: High MRP4 levels were observed in 46 out of the 78 samples (59%). Patients with gallbladder cancer (n=64) and elevated MRP4 levels had significantly poorer overall survival (OS) compared to those with low MRP4 levels (median 11.3 and 14.5 months, high and low, p =0.038). In the broader BTC cohort evaluable for survival (n=73; 64 gallbladder, 7 cholangiocarcinoma, 1 ampulla of Vater, and 1 biliary undefined) patients with elevated MRP4 levels had non-significant shorter OS (median 12.4 and 13.5 months, high and low, p =0.17). Poorly differentiated tumors were more likely to express high MRP4 levels ( p =0.06). High MRP4 expression was found in 59% of localized stages (0-II) and in 57% of advanced stages (III, IV and relapsed). Conclusions: High MRP4 expression is common in BTC, particularly gallbladder cancer, and could be a marker of poor prognosis. MRP4 levels appear more elevated in poorly differentiated tumors, and are similar in both, early and advanced stages, suggesting its involvement in early tumor progression. Further prospective studies are needed to confirm MRP4 as a biomarker. If MRP4 is a driver gene, targeting with inhibitors could represent a promising therapeutic strategy for these challenging tumors.

MUC5AC levels as predictors of adjuvant therapy outcomes in pancreatic ductal adenocarcinoma: A study of post-surgical gemcitabine-based regimens.

759 Background: The role of tissue mucin 5 AC (MUC5AC) in PDA is not fully understood. In our previous work we showed that mature MUC5AC (MM) detected in the extracellular (EC) space and in apical region has better prognostic value than perinuclear immature MUC5AC (IM). Our previous research demonstrated that mature MUC5AC (MM), present in the extracellular (EC) space and apical region, offers better prognostic value than perinuclear immature MUC5AC (IM). Preclinical studies suggest that MM may confer chemoresistance to gemcitabine (Gem), commonly used in adjuvant therapy (AT) either alone or with capecitabine (Cap) or nab-paclitaxel (NP) in patients not eligible for FOLFIRINOX. This study investigates MM’s effect on outcomes in PDA patients undergoing post-surgical treatment with Gem-only, Gem-NP, and Gem-Cap. Methods: Formalin-fixed, paraffin-embedded tissue samples from resected PDA cases (January 2010 - June 2021) were sourced from the Ohio State University biorepository. Immunohistochemistry using monoclonal antibodies 45M1 (for MM) and CLH2 (for IM) was employed to examine cellular MM and IM expression, with H-scores calculated. Statistical analysis of progression-free survival (PFS) and overall survival (OS) was done using the log-rank test. Results: We had 49 patients available for analysis. The median age of the group was 66 years (range: 38 to 89) with majority Caucasians (90%) and 53% (n=26) females. AT distribution within this group was, 31 Gem-only, 7 Gem-NP, and 11 Gem and capecitabine (Cap). Median expression levels for MM and IM were both 150, and 82% (n=40) were EC-MM positive. Using the median MM H-score as a threshold (< 150 vs. ≥ 150), outcomes were evaluated within each therapy subgroup (Table). Conclusions: This preliminary study suggests that PDA patients with low MM expression may benefit from Gem-Cap therapy. However, larger, prospective studies are needed for definitive conclusions. Comparing outcomes in the sub-groups. Sub-group (n) OS in days (p-value) PFS in days (p-value) MM H-score < 150 vs. ≥ 150 Gem-only (11 vs. 20) 775 vs. 1006 (0.4) 444 vs. 413 (0.4) Gem NP (4 vs. 3) 1409 vs. 551 (0.8) 391 vs. 418 (0.5) Gem-Cap (4 vs. 7) not evaluable (NE) vs. 766 (0.02) NE vs. 415 (0.01)

Immunohistochemical Analysis of a1-Acid Glycoprotein and Tumor Associated Macrophages in Clear Cell Renal Cell Carcinoma.

α1-Acid glycoprotein (AGP), also known as orosomucoid, is an acute-phase protein that has been found increased in plasma of cancer patients. This study investigates the role of AGP expression in clear cell renal cell carcinoma (ccRCC) and its association with clinical outcomes. We investigated the correlation between AGP levels and the prognosis of ccRCC through an analysis of The Cancer Genome Atlas (TCGA) database. To examine AGP expression and its clinicopathological associations, immunostaining was performed on paraffin-embedded tissue samples of 92 ccRCC cases. AGP expression was found to be higher in RCC cell lines compared to normal renal epithelial cells. Analysis of the TCGA dataset showed that patients with AGP gene expression had significantly worse overall survival. However, AGP expression was not correlated with age, sex, or cancer stage. A mouse monoclonal antibody against AGP was generated. This antibody reacted with human and mouse hepatocytes, but not in AGP-deficient mice. From 92 examined ccRCC cases, AGP protein expression was detected in 89 cases, with only 3 being negative. AGP expression levels did not correlate with clinicopathological factors, such as age, tumor size, or nuclear grade. CD14, a receptor of AGP, was found to be expressed in Iba1-positive monocytes and tumor-associated macrophages (TAMs) but not in other cell types like lymphocytes or cancer cells. No significant correlation was found between AGP expression and the number of Iba1-positive cells in ccRCC tissues. Iba1-positive cells were correlated with Fuhrman grade, and patients with ≥30% Iba1-positive cells were, on average, significantly younger and had more aggressive tumor. AGP expression is linked to poorer survival in ccRCC, but its association with immune cell infiltration (via Iba1-positive cells) is unclear.

Molecular diagnosis of infective endocarditis from culture-negative valve samples in a tertiary hospital in Iran.

The aim of this study was to investigate the prevalence of Tropheryma whipplei, Chlamydia psittaci, Chlamydia pneumoniae, Legionella, Brucella, and Francisella tularensis in valve samples from endocarditis patients using the real-time PCR method at a major referral heart hospital in Iran. In this study, 146 paraffin-embedded tissue samples from the heart valves of patients with clinical and pathological evidence of infective endocarditis (IE), who underwent heart valve replacement surgery between 2016 and 2020 at Tehran Heart Center were collected. After DNA extraction from paraffin-embedded valve tissue samples, they were surveyed for the presence of T. whipplei, C. psittaci, C. pneumoniae, Legionella, Brucella, and F. tularensis using quantitative real-time PCR (qPCR). The 16S rRNA gene sequence analysis was used for accurate species identification. Based on the molecular results, T. whipplei, Chlamydia spp., and Legionella spp. were detected in 10 (6.84%), 6 (4.1%), and 3 (2.05%) valve samples, respectively. In addition, one of six positive samples for Chlamydia spp. was identified as C. psittaci. No positive samples for F. tularensis and Brucella were found. In addition, all control valve samples were negative for all investigated pathogens. The findings suggest that specific bacterial species such as T. whipplei, Chlamydia spp., and Legionella spp. are associated with the development of IE. Considering the life-threatening nature of IE, it is critical for healthcare systems to prioritize the identification of its causative agents and develop targeted treatment strategies.IMPORTANCEInfective endocarditis (IE) is a serious and potentially life-threatening condition, and it is associated with significant morbidity, mortality, and complications, making it a major concern in both global and national healthcare systems. Late diagnosis and failure to receive appropriate treatment for patients with culture-negative endocarditis caused by hard-to-grow bacteria can lead to the death of patients. Unfortunately, in Iran, less attention is paid to the role of organisms that are difficult to cultivate in laboratory settings such as Tropheryma whipplei, Chlamydia psittaci, Chlamydia pneumoniae, Legionella, Brucella, and Francisella tularensis in causing culture-negative endocarditis, and these pathogens are overlooked by infectious, cardiologists, and health officials. This study underscores the need for special attention in the diagnosis of the agents of IE.

Immunohistochemical expression of parathyroid hormone-related protein and ezrin in invasive breast carcinoma of no special type: a retrospective analysis

BackgroundGlobally, breast cancer ranks among the most common malignancies and has a high mortality rate. Invasive breast carcinoma of no special type (IBC-NST) presents a heterogeneous group with variable prognosis. Identifying reliable biomarkers is crucial for improving treatment strategies and predicting outcomes. This study investigates the immunohistochemical expression of parathyroid hormone-related protein (PTHrP) and ezrin in IBC-NST and their correlation with clinicopathological features and overall survival.MethodsThis retrospective study analyzed 160 paraffin-embedded tissue samples, including 123 IBC-NST and 37 normal breast tissues, collected from patients treated at Menoufia University Hospital during the period from January 2018 to January 2022. Immunohistochemical staining for PTHrP and ezrin was performed, and expression levels were quantified using the H score.ResultsPTHrP expression was significantly higher in IBC-NST than in adjacent DCIS and normal tissues (p < 0.001). High PTHrP percent of expression was associated with metastasis (p = 0.009), bone metastasis (p = 0.012), and lymphovascular invasion (p = 0.037). Ezrin expression was also significantly elevated in IBC-NST, with higher H score values correlating with high tumor grade (p = 0.002), high N stage (p = 0.045), advanced AJCC stage grouping (p = 0.0043) and metastasis (p = 0.001). A significant positive correlation was observed between PTHrP and ezrin expression (rs = 0.341, p < 0.001). Kaplan-Meier analysis showed that high ezrin expression, in terms of intensity (p = 0.007) and H score (p = 0.002), was linked to poorer survival.ConclusionThe study highlights the significant roles of PTHrP and ezrin in breast cancer progression. Elevated levels of these proteins are associated with more aggressive disease, suggesting their capability as prognostic indicators and treatment targets in breast cancer. Additional studies are required to investigate their interaction and collective influence on breast cancer metastasis and treatment.

Immunohistochemical Expression of VEGF and Microvessel Density (CD 34) in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: Original Research.

Angiogenesis, the formation of new blood vessels from preexisting ones via capillary sprouting, is a crucial process in tumor growth and metastasis. As a tumor's angiogenic capacity increases, its microvasculature, measured by micro vessel density (MVD), also increases. This study aims to evaluate the expression of Vascular Endothelial Growth Factor (VEGF) and CD34 in oral epithelial dysplasia and oral squamous cell carcinoma through immunohistochemical methods. The study analyzed a total of 40 formalin-fixed, paraffin-embedded tissue samples. These included 10 cases of normal buccal mucosa, 15 cases of oral epithelial dysplasia, and 15 cases of oral squamous cell carcinoma. Immunohistochemical staining was performed using monoclonal anti-VEGF and anti-CD34 antibodies. The intensity and area of staining for VEGF were assessed, and the mean MVD was calculated. Statistical analysis was conducted using Pearson's chi-square test and one-way ANOVA. The expression of VEGF and MVD (indicated by CD34 staining) were significantly higher in oral squamous cell carcinoma compared to oral epithelial dysplasia and normal buccal mucosa. As tumors grow, angiogenesis increases proportionally with tumor volume and disease progression, contributing to tumorigenesis. VEGF serves as a critical mitogen for tumor vascularization, and MVD can be a useful indicator of disease progression.

Predictive role of ABC transporters in the efficacy of enfortumab vedotin for urothelial carcinoma.

To evaluate the correlation between ATP-binding cassette (ABC) transporter expression and therapeutic efficacy of enfortumab vedotin (EV), an antibody-drug conjugate targeting Nectin-4, in urothelial cancer, as only a few studies have been conducted on this topic. This retrospective study included 20 patients with metastatic urothelial carcinoma (mUC), including bladder and upper urinary tract cancers, who were treated with EV at Dokkyo Medical University Hospital between 2022 and 2024. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue samples. Progression-free survival (PFS) was estimated using the Kaplan-Meier method, and differences between subgroups (e.g., Nectin-4 and ABC transporter expression) were compared using the log-rank test. Immunohistochemical analysis indicated that tumours with high ABC transporter expression exhibited shorter PFS time and poorer response to EV. Furthermore, a decrease in Nectin-4 expression and an increase in ABC transporter expression were observed as the disease progressed from non-muscle-invasive to muscle-invasive and metastatic. Patients with Nectin-4-positive and ABC transporter-negative tumours had the longest PFS, underscoring the prognostic significance of these biomarkers. To our knowledge, this study is the first to show a correlation between ABC transporter expression and EV efficacy in urothelial carcinoma. Future research should focus on optimizing treatment strategies based on Nectin-4 and ABC transporter expression to improve outcomes.

No Bacterial Biomass Detected in Tissue From Patients With Ovarian Cancer and Serous Tubal Intraepithelial Carcinomas Using 16S rDNA Sequencing.

The ovarian oncobiome is subject to increasing scientific focus, but a potential link between bacterial dysbiosis and ovarian carcinogenesis remains controversial. Our primary aim was to characterize the bacterial microbiota in epithelial ovarian cancer samples. Secondarily, we aimed to compare results from the bacterial microbiota in formalin-fixed, paraffin-embedded ovarian tissue samples from 194 patients with epithelial ovarian cancer, fallopian tube tissue samples from 16 patients with serous tubal intraepithelial carcinomas and in benign fallopian tube tissue samples from 25 patients. Bacterial abundance was investigated by means of 16S rDNA Next Generation Sequencing. The 16S rDNA sequencing run produced a very low number of bacterial reads. Only seven samples displayed bacterial reads above 5000. Validation of detected bacterial reads by qPCR was negative and indicative of results being background amplification. Our results indicate no amount of bacterial biomass in the ovarian cancer, benign fallopian tube and in the samples with serous tubal intraepithelial carcinomas. The findings underline the importance of validating results from bacterial microbiota studies with additional technical assays before any conclusion may be drawn.

Assessment of Exhaled Breath Condensate for ALK, RET, ROS1, and NTRK1 Fusion Transcript Detection in NSCLC: Comparison With Tissue and Liquid Biopsy Samples.

Lung cancer continues to be the primary cause of cancer-related deaths globally, with the majority of cases identified at advanced stages. Genetic alterations, including mutations and gene fusions, are central to its molecular pathogenesis. The discovery of therapeutically targetable gene fusions, such as ALK, RET, ROS1, and NTRK1, has significantly advanced lung cancer management. Conventional methods, such as tissue biopsies, are invasive and unsuitable for continuous molecular monitoring. Consequently, noninvasive approaches, such as liquid biopsies and exhaled breath condensate (EBC), offer promising options for real-time molecular surveillance. This study evaluates the feasibility of identifying fusion transcripts in 30 patients with lung adenocarcinoma by using next-generation sequencing (NGS) on formalin-fixed paraffin-embedded (FFPE) tissue, plasma, and EBC samples. Clinically significant fusion transcripts, including KIF5B-ALK, KIF5B-RET, and SQSTM1-ALK, were detected across different sample types. EBC samples showed strong concordance with tissue biopsy results, particularly in detecting ALK, ROS1, and RET fusions, and demonstrated greater sensitivity than plasma in detecting NTRK1 fusions. Additionally, 30 fusion transcripts of uncertain clinical significance were identified, highlighting the need for further research into their role in lung cancer pathogenesis. In conclusion, EBC samples provide a valuable, noninvasive medium for detecting clinically relevant and previously uncharacterized fusion transcripts in non-small cell lung cancer (NSCLC). The high concordance between EBC and tissue biopsies suggests that EBC could complement tissue biopsy for effective diagnosis and monitoring of NSCLC. These findings underscore the importance of comprehensive molecular profiling using multiple sample types to enhance diagnostic precision and optimize therapeutic outcomes in lung cancer management.

Genotipificación del virus del papiloma humano en pacientes colombianos con carcinoma de células escamosas de esófago y carcinoma gástrico

Introduction: Human papillomavirus (HPV) is one of the most important oncogenic viru-ses detected in different types of cancer, which includes esophageal squamous cell carcino-ma (ESCC) and gastric cancer (GC). However, Colombia does not have data on the associa-tion of HPV with GC. Objetive: The aim of this study was to determine the presence HPV genotypes in patients with ESCC and GC, diagnosed between 2007 and 2014 at the Hospital Central de la Policía Nacional of Bogotá-Colombia (HOCEN).Methods: A total of 33 paraffin embedded tissue samples were examinate by polymerase chain reaction for the L1 gene of HPV. The extracted DNA was amplified using the universal primers MY09 and MY11. Then, the positive samples had high risk HPV genotyping perfor-med on them. The samples were analyzed by Roche Cobas 4800 system.Results: Five (15%) samples identified as positive to HPVDNA [four men (80%) and one woman (20%)], one (20%) was positive for HPV-16. The other four, whose genotypes had not detected by COBAS 4800, samples could correspond to low-risk HPV, or match to some of the high-risk genotypes that had not included in the detection panel of this test. Also, four (80%) HPVDNA positives had identified as adenocarcinoma intestinal type, and one (20%) as ESCC. Conclusion: Our study proves the existance of HPV and p16 in Colombian patients with ESCC and GC, that could be a risk factor for the development of these pathologies in this population.

Towards Reliable Methodology: Microbiome Analysis of Fresh Frozen vs. Formalin-Fixed Paraffin-Embedded Bladder Tissue Samples: A Feasibility Study.

Studies have shown that the human microbiome influences the response to systemic immunotherapy. However, only scarce data exist on the impact of the urinary microbiome on the response rates of bladder cancer (BC) to local Bacillus Calmette-Guérin instillation therapy. We launched the prospective SILENT-EMPIRE study in 2022 to address this question. We report the results of the pilot study of SILENT-EMPIRE, which aimed to compare the microbiome between fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples in the cancerous tissue and adjacent healthy tissue of BC patients. Our results show that alpha diversity is increased in FF samples compared to FFPE (coverage index p = 0.041, core abundance index p = 0.008). No significant differences concerning alpha diversity could be detected between cancerous and non-cancerous tissue in the same BC patients. This study demonstrates that microbiome analysis from both FF and FFPE samples is feasible. Implementing this finding could aid in the translation of research findings into clinical practice.

High frequency of HPV high-risk preventable genotypes in Ecuadorian women with invasive cervical cancer.

To determine the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer samples from Ecuadorian women who attended the Cancer Institute (Sociedad de Lucha Contra el Cáncer - SOLCA). Archived formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples collected during 2017-2021 were deparaffinized, and nucleic acid extraction and purification was performed using silica columns. The obtained nucleic acids were analyzed using INNO-LiPA® HPV Genotyping Extra II per the manufacturer's specifications. Data were retrieved from records, and HPV genotypes were determined from the FFPE samples. The study included samples from 190 women diagnosed with invasive cervical cancer, with a median age of 52.78 years. Squamous cell carcinoma accounted for 78.94% of the cases, while 21.05% had adenocarcinoma. Among the 190 samples, 80.53% tested positive for HPV DNA, while 19.47% were negative. The most common genotypes detected were HPV 16 (64.05%), 18 (16.99%), and 58 (6.54%). HPV infection frequency was higher in samples from patients with elementary level education (p < 0.05). This study provides valuable insights into the distribution of HPV genotypes in invasive cervical cancer samples from Ecuadorian women. The results indicate an elevated presence of HPV 16, HPV 18, and HPV 58, which are vaccine-preventable genotypes.

Prevalence of SPOP and IDH Gene Mutations in Prostate Cancer in a Jordanian Population.

Speckle-type POZ (SPOP) is described as an essential tumor suppressor factor in gastric cancer, colorectal cancer, and prostate cancer (PCa). SPOP gene mutations were reported in primary human PCa. Isocitrate dehydrogenase-1 (IDH1) oncogene mutations were detected in gliomas, acute myeloid leukemia, some benign and malignant cartilaginous tumors, and only 1% of PCa. This study aimed to investigate the prevalence of mutations of SPOP and IDH1 genes in PCa in the Jordanian population. One hundred formalin-fixed paraffin-embedded tissue samples were collected from patients diagnosed with prostate adenocarcinoma. The obtained specimens were subjected to genomic DNA extraction, PCR amplification, and direct sequencing of exons 4, 5, 6, and 7 of the SPOP gene and exon 6 of the IDH1 gene. SPOP gene mutations were found in 17% of PCa cases, while no mutation was detected in the screened exon 6 of the IDH1 gene. Clinicopathological data demonstrated a strong correlation between prostate-specific antigen (PSA) levels and both Gleason score (GS) and the International Society of Urological Pathology (ISUP) grade group (GG). There was no significant correlation between PSA levels and age (p = 0.816) nor there were significant associations for SPOP mutational status with age (p = 0.659), PSA levels (p = 0.395), GS (p = 0.259), and ISUP GG (p = 0.424) in the tested population. The study found a strong correlation between PSA levels and both GS and ISUP GG. It also identified a high frequency (17%) of SPOP gene mutations in Jordanian Arab PCa patients, mainly in exon 7. No IDH1 mutations were detected in exon 6.

The Prognostic Predictive Value of TP53 mutation Variant Allele Frequency in Diffuse Large B-Cell Lymphoma

To explore the effect of TP53 mutation variant allele frequency(VAF) on the prognosis of diffuse large B-cell lymphoma(DLBCL) patients. This study included 155 patients with DLBCL who were first diagnosed in the People's Hospital of Xinjiang Uygur Autonomous Region from March 2009 to March 2022. Complete clinical data and paraffin-embedded tumor tissue samples were obtained, and DNA was extracted from tumor tissues. The gene mutation profile of DLBCL patients was detected and analyzed by second-generation sequencing technology. Kaplan-Meier method was used to analyze the mutation status of TP53 gene and the relationship between mutation VAF and OS. Cox regression univariate and multivariate analysis was use to analyze the independent factors affecting OS. A nornogram model for predicting 1, 3, and 5 years OS in DLBCL patients were established to evaluated the performance of the model based on C-index and calibration curves. The average value of TP53 mutation VAF in male DLBCL patients was significantly higher than that in female patients (P < 0.05). Patients with TP53 mutantion had shorter OS than those with wild-type patients (P =0.030). The optimal VAF threshold for TP53 mutation based on OS stratification was 33.61% (P < 0.001), and patients with TP53 mutation VAF ≥34% had shorter OS than those with TP53 mutation VAF < 34% and wild-type patients (P < 0.001). Multivariate Cox analysis showed that TP53 mutation VAF≥34% was an independent poor predictor of OS ( HR =4.05, P < 0.001), and IPI score ≥3 was an independent predictor of OS poor ( HR =2.27, P =0.008). In combination with factors with independent prognostic significance obtained from multi-factor analysis, we constructed a nomogram model for predicting 1-year, 3-year, 5-year OS in DLBCL patients. The results showed that the C index of TP53 mutation VAF combined with IPI model was 0.743, which predicted the value of 1-year, 3-year, and 5-year OS in DLBCL patients. Calibration curves show that the model has good agreement between predicted and actual survival of DLBCL patients at 1-year, 3-year, and 5-year. TP53 mutation VAF has prognostic value in DLBCL patients, and TP53 mutation VAF≥34% is an independent risk factor for OS in DLBCL patients. The prognosis model of TP53 mutation VAF combined with IPI nomogram constructed in this study has good predictive performance for DLBCL patients.

Correlations between 14-gene RNA-level assay and clinical and molecular features in resectable non-squamous non-small cell lung cancer: a cross-sectional study.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Accurate risk stratification is essential for optimizing treatment strategies. A 14-gene RNA-level assay of lung cancer, which involves quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples, offers a promising approach. The aim of our study was to assess the relationships between risk stratification, as determined by a 14-gene RNA-level assay, and various clinical and molecular characteristics. We retrospectively collected the preoperative clinical information and molecular testing information from 102 resectable non-squamous NSCLC patients. The 14-gene RNA-level assay was performed by extracting RNA from FFPE samples, followed by reverse transcription and quantification via quantitative polymerase chain reaction (qPCR) to assess the expression levels of 11 cancer-associated genes and three housekeeping genes. These gene expression levels were used to calculate a risk score, enabling patient stratification into distinct risk groups. Based on the 14-gene risk stratification, we analyzed the correlations between the clinical and molecular characteristics across the high-, medium-, and low-risk groups. A total of 102 patients were included in the study. The mean age was 55.19 years, 67 (65.7%) patients were female, and 18 (17.6%) had a smoking history. The 14-gene risk stratification classified patients into low-risk (n=63), intermediate-risk (n=25), and high-risk (n=14) groups. No significant differences were observed in baseline demographics between the three risk groups. High-risk patients had significantly higher mean computed tomography (CT) value (P=0.01) and enhanced CT value (P=0.02) compared to low-risk patients. Genomic profiling of 89 patients revealed specific mutations that were significantly associated with the higher-risk groups. Tumor mutational burden (TMB) was higher in higher-risk groups (P=0.007). In clinically low-risk patients (n=85) as recognized by the NCCN guidelines, the 14-gene risk stratification model reclassified 30 out from the 85 clinically low-risk patients, with 19 placed in the medium-risk group and 11 in the high-risk group, while the remaining samples were still classified as low-risk. Additionally, we found that three patients who were not recommended for adjuvant therapy by the Multiple-gene INdex to Evaluate the Relative benefit of Various Adjuvant therapies (MINERVA) model were classified as high risk and 13 as intermediate risk. Our results indicate that 14-gene RNA-level assay is correlated with specific genetic mutations, including TP53, KRAS, and LRP1B. These insights provide a stronger foundation for integrating molecular risk assessment with clinical and imaging data, offering more comprehensive information to guide more targeted and effective adjuvant therapy strategies in the future management of lung cancer.

DNA Methylation Panels for the Differentiation of Lung and Gastric Adenocarcinomas from Other Common Primary Adenocarcinomas.

Background/Objectives: Gastric and lung adenocarcinomas are among the most common adenocarcinomas worldwide. Our research aimed to validate methylation biomarkers that differentiate gastric and lung adenocarcinomas from hepatocellular carcinoma, cholangiocarcinoma, colorectal carcinoma, pancreatic adenocarcinoma and paired healthy tissues. Methods: The study analyzed 178 formalin-fixed, paraffin-embedded tissue samples, including 14 gastric adenocarcinomas, 15 lung adenocarcinomas, 15 hepatocellular carcinomas, 15 cholangiocarcinomas, 15 colorectal carcinomas, 15 pancreatic adenocarcinomas and their paired healthy tissues. Methylation status was determined experimentally by methylation-sensitive high resolution melting. The diagnostic panels were validated on bioinformatics datasets from The Cancer Genome Atlas and Gene Expression Omnibus, comprising 1981 and 773 samples, respectively. Sensitivity, specificity, diagnostic accuracy and predictive values for each cancer type were calculated for the experimental, Gene Expression Omnibus and The Cancer Genome Atlas datasets. Results: The gastric cancer-specific panel showed a sensitivity of 78.6-83.9%, a specificity of 86.6-94.6% and a diagnostic accuracy of 89.9-96.1% to differentiate between all tumors, and a sensitivity of 78.6-83.9%, a specificity of 89.2-96.4% and a diagnostic accuracy of 88-96.1% to differentiate between all tumors and healthy tissues. The lung adenocarcinoma-specific panel showed a sensitivity of 61.1-93.3%, a specificity of 70.3-90.8% and a diagnostic accuracy of 74.2-90.6% to differentiate between all tumors, and a sensitivity of 61.1-93.3%, a specificity of 77.9-93.4% and a diagnostic accuracy of 79.2% to 93.1% to differentiate between all tumors and healthy tissues. Conclusions: This study demonstrates the potential of using diagnostic methylation panels to differentiate gastric and lung adenocarcinomas from other common adenocarcinomas and paired healthy tissues.

Deep learning-based classifier for carcinoma of unknown primary using methylation quantitative trait loci.

Cancer of unknown primary (CUP) constitutes between 2% and 5% of human malignancies and is among the most common causes of cancer death in the United States. Brain metastases are often the first clinical presentation of CUP; despite extensive pathological and imaging studies, 20%-45% of CUP are never assigned a primary site. DNA methylation array profiling is a reliable method for tumor classification but tumor-type-specific classifier development requires many reference samples. This is difficult to accomplish for CUP as many cases are never assigned a specific diagnosis. Recent studies identified subsets of methylation quantitative trait loci (mQTLs) unique to specific organs, which could help increase classifier accuracy while requiring fewer samples. We performed a retrospective genome-wide methylation analysis of 759 carcinoma samples from formalin-fixed paraffin-embedded tissue samples using Illumina EPIC array. Utilizing mQTL specific for breast, lung, ovarian/gynecologic, colon, kidney, or testis (BLOCKT) (185k total probes), we developed a deep learning-based methylation classifier that achieved 93.12% average accuracy and 93.04% average F1-score across a 10-fold validation for BLOCKT organs. Our findings indicate that our organ-based DNA methylation classifier can assist pathologists in identifying the site of origin, providing oncologists insight on a diagnosis to administer appropriate therapy, improving patient outcomes.

Low Expression of Mitochondrial Ribosomal Protein S5 is Associated With Poor Prognosis in Patients With Clear Cell Renal Cell Carcinoma.

Mitochondrial ribosomal protein S5 (MRPS5) is abnormally expressed in various tumor tissues and may be a key molecule for the regulation of tumors. Our aim is to investigate the relationship between the expression of MRPS5 in clear cell renal cell carcinoma (ccRCC) and the prognosis of patients. MRPS5 expression in fresh tumoral tissues and peritumoral tissues of patients with ccRCC was examined by quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemical staining, respectively. MRPS5 expression level in paraffin-embedded tumoral tissue samples with ccRCC was evaluated by immunohistochemical scoring criteria. The relationship between the expression of MRPS5 and the clinicopathological parameters and prognosis of patients with ccRCC was analyzed statistically. The expression of MRPS5 mRNA and protein in fresh tumoral tissues was lower than that in peritumoral tissues. Among 160 paraffin-embedded tumoral tissue samples, 99 cases (61.9%) showed high expression and 61 cases (38.1%) showed low expression of MRPS5. The expression level of MRPS5 was significantly correlated with T classification, TNM stage, and Fuhrman grade. Kaplan-Meier method and log-rank test indicated that patients with low MRPS5 expression had significantly poorer overall survival and recurrence-free survival than high MRPS5 expression. Multivariate analysis revealed that MRPS5 expression was an independent predictor of overall survival and recurrence-free survival, respectively. MRPS5 low expression was a risk factor for the prognosis of patients. The expression level of MRPS5 is significantly correlated with the postoperative survival status, which has the potential to be used as a novel prognostic biomarker for patients with ccRCC.

Development of a High-Throughput Multiplex Immunofluorescence Pipeline in Classic Hodgkin Lymphoma Whole Slide Formalin-Fixed Paraffin-Embedded Tissue Samples

Development of a High-Throughput Multiplex Immunofluorescence Pipeline in Classic Hodgkin Lymphoma Whole Slide Formalin-Fixed Paraffin-Embedded Tissue Samples

DNA methylation biomarker panels for differentiating various liver adenocarcinomas, including hepatocellular carcinoma, cholangiocarcinoma, colorectal liver metastases and pancreatic adenocarcinoma liver metastases

BackgroundDNA methylation biomarkers are one of the most promising tools for the diagnosis and differentiation of adenocarcinomas of the liver, which are among the most common malignancies worldwide. Their differentiation is important because of the different prognoses and treatment options. This study aimed to validate previously identified DNA methylation biomarkers that successfully differentiate between liver adenocarcinomas, including the two most common primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as two common metastatic liver cancers, colorectal liver metastases (CRLM) and pancreatic ductal adenocarcinoma liver metastases (PCLM), and translate them to the methylation-sensitive high-resolution melting (MS-HRM) and digital PCR (dPCR) platforms.MethodsOur study included a cohort of 149 formalin-fixed, paraffin-embedded tissue samples, including 19 CRLMs, 10 PCLMs, 15 HCCs, 15 CCAs, 15 colorectal adenocarcinomas (CRCs), 15 pancreatic ductal adenocarcinomas (PDACs) and their paired normal tissue samples. The methylation status of the samples was experimentally determined by MS-HRM and methylation-specific dPCR. Previously determined methylation threshold were adjusted according to dPCR data and applied to the same DNA methylation array datasets (provided by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)) used to originally identify the biomarkers for the included cancer types and additional CRLM projects. The sensitivities, specificities and diagnostic accuracies of the panels for individual cancer types were calculated.ResultsIn the dPCR experiment, the DNA methylation panels identified HCC, CCA, CRC, PDAC, CRLM and PCLM with sensitivities of 100%, 66.7%, 100%, 86.7%, 94.7% and 80%, respectively. The panels differentiate between HCC, CCA, CRLM, PCLM and healthy liver tissue with specificities of 100%, 100%, 97.1% and 94.9% and with diagnostic accuracies of 100%, 94%, 97% and 93%, respectively. Reevaluation of the same bioinformatic data with new additional CRLM projects demonstrated that the lower dPCR methylation threshold still effectively differentiates between the included cancer types. The bioinformatic data achieved sensitivities for HCC, CCA, CRC, PDAC, CRLM and PCLM of 88%, 64%, 97.4%, 75.5%, 80% and 84.6%, respectively. Specificities between HCC, CCA, CRLM, PCLM and healthy liver tissue were 98%, 93%, 86.6% and 98.2% and the diagnostic accuracies were 94%, 91%, 86% and 98%, respectively. Moreover, we confirmed that the methylation of the investigated promoters is preserved from primary CRC and PDAC to their liver metastases.ConclusionsThe cancer-specific methylation biomarker panels exhibit high sensitivities, specificities and diagnostic accuracies and enable differentiation between primary and metastatic adenocarcinomas of the liver using methylation-specific dPCR. High concordance was achieved between MS-HRM, dPCR and bioinformatic data, demonstrating the successful translation of bioinformatically identified methylation biomarkers from the Illumina Infinium HumanMethylation450 BeadChip (HM450) and lllumina MethylationEPIC BeadChip (EPIC) platforms to the simpler MS-HRM and dPCR platforms.