Wild-type Group Research Articles

Reaction Pathway Differentiation Enabled Fingerprinting Signal for Single Nucleotide Variant Detection.

Accurate identification of single-nucleotide variants (SNVs) is paramount for disease diagnosis. Despite the facile designof DNA hybridization probes, their limited specificity poses challenges in clinical applications. Here, a differential reaction pathway probe (DRPP) based on a dynamic DNA reaction network is presented. DRPP leverages differences in reaction intermediate concentrations between SNV and WT groups, directing them into distinct reaction pathways. This generates a strong pulse-like signal for SNV and a weak unidirectional increase signal for wild-type (WT). Through the application of machine learning to fluorescence kinetic data analysis, the classification of SNV and WT signals is automated with an accuracy of 99.6%, significantly exceeding the 80.7% accuracy of conventional methods. Additionally, sensitivity for variant allele frequency (VAF) is enhanced down to 0.1%, representing a ten-fold improvement over conventionalapproaches. DRPP accurately identified D614G and N501Y SNVs in the S gene of SARS-CoV-2 variants in patient swab samples with accuracy over 99% (n = 82). It determined the VAF of ovarian cancer-related mutations KRAS-G12R, NRAS-G12C, and BRAF-V600E in both tissue and blood samples (n = 77), discriminating cancer patients and healthy individuals with significant difference (p <0.001). The potential integration of DRPP into clinical diagnostics, along with rapid amplification techniques, holds promise for early disease diagnostics and personalized diagnostics.

Preliminary analysis of the role of small hepatitis B surface proteins mutations in the pathogenesis of occult hepatitis B infection via the endoplasmic reticulum stress-induced UPR-ERAD pathway

Abstract A growing body of evidence has shown that hepatitis B surface antigen (HBsAg) mutations can influence the occurrence of occult hepatitis B infection (OBI), particularly amino acid substitutions in small hepatitis B surface proteins (SHBs). The mechanistic basis for these results, however, remains unclear. This study was designed to explore the potential impact and mechanisms of OBI-related SHBs mutations on serum HBsAg. Huh7 and HepG2 cells were transfected with plasmids encoding wild-type (WT) or OBI-related SHB mutation-containing sequences, after which a chemiluminescence approach was used to detect HBsAg levels in cell culture supernatants. Western blotting was further used to assess HBsAg and endoplasmic reticulum stress (ERS)-related protein levels in lysates prepared from these cells, while the localization of HBsAg within cells was assessed via immunofluorescent staining. Cells transfected with OBI-related SHB mutation-encoding plasmids exhibited lower supernatant HBsAg levels than cells transfected with WT plasmids. Intracellular and extracellular HBsAg levels in these mutant plasmid-transfected cells were lower relative to those for WT plasmid-transfected cells, and HBsAg accumulation within the ER was detected via immunofluorescent staining in cells transfected with OBI-related SHB mutation-encoding plasmids, ERS-related protein content was also significantly increased in mutant plasmid-transfected cells as compared to those in the WT group. These results suggest that proteins harboring OBI-related mutations may tend to accumulate in the ER, thereby triggering an ERS response and impairing the transcription and translation of HBsAg via the activation of the unfolded protein response and ER-associated protein degradation pathway. These effects ultimately reduce the overall assembly of HBV virions in the ER and their associated secretion.

Effect of RAS mutations and related immune characteristics on the prognosis of patients with MSI-H/dMMR colorectal cancer

PurposeMicrosatellite high instability/deficient mismatch repair (MSI-H/dMMR) colorectal cancer (CRC) has an active tumor microenvironment, rendering it more sensitive to immune checkpoint inhibitors. Given that studies involving patients with MSI-H colorectal cancer with RAS mutations are scarce, we explored the effect of RAS mutations on the TME in patients with MSI-H/dMMR cancer and identified potential prognostic factors.MethodsSeventy-five patients diagnosed with MSI-H/dMMR colorectal cancer were retrospectively enrolled and divided into RAS-mutant and -wild-type groups. The expression levels of CD11c+ dendritic cells, CD4+ T cells, CD8+ T cells, and regulatory T cell (Treg) markers were detected, and prognostic factors were analyzed.ResultsRAS-mutant MSI-H colorectal patients were more likely to have: (1) higher platelet values; (2) shorter disease-free survival (DFS); (3) lower infiltrated numbers of CD11c+ dendritic cells, CD4+ T lymphocytes, and CD8+ T lymphocytes, and higher infiltrated numbers of Foxp3+ Treg cells. In MSI-H/dMMR CRC patients: (1) the high CD11c + , CD4 +, and CD8 + cells infiltration group had longer DFS than the low-infiltration group, and Foxp3 + cells infiltration was not significantly correlated with DFS; (2) the RAS mutation status, number of CD11c+ cells infiltrated, and carbohydrate antigen 19–9 (CA19–9) level were the potential prognostic factors.ConclusionRAS mutations in patients with MSI-H/dMMR CRC may reduce the infiltration of CD11c+ dendritic cells, CD4+ T cells, and CD8+ T cells, and increase the infiltration of Foxp3+ Treg cells to affect the tumor microenvironment of patients. RAS gene status, CD11c + cells infiltration, and CA19–9 level were potential prognostic factors for MSI-H/dMMR CRC.

Effect of homologous recombination deficiency on survival in patients with metastatic colorectal cancer with KRAS mutations.

252 Background: Metastatic colorectal cancer (mCRC) patients exhibit intertumoral genetic heterogeneity, making it difficult to apply precision medicine. In the molecular genetic profile of mCRC, there are relatively few other gene alterations that significantly impact treatment decisions beyond KRAS and MSI status. We aim to investigate the prognostic impact of Homologous recombination deficiency (HRD)-related genetic alterations in mCRC patients, focusing on how mutation status influences on patient outcomes. Methods: The analysis included 364 mCRC patients who underwent comprehensive genomic profiling at Korea University Anam Hospital from Sep. 2016 to Dec. 2020. HRD was defined by the presence of pathogenic variants in genes including ATM, ATR, BARD1, BRCA1/2, BRIP1, CDK12, CHEK2, FANCA, MRE11A, PALB2, RAD50/51. Overall survival (OS) and progression-free survival (PFS) were evaluated using the Kaplan-Meier method. Multivariate Cox proportional hazards regression analyses were performed to assess the independent impact of HRD status. Results: Of the total cohort, 181 of patients (49.78%) harbored KRAS mutations. HRD gene mutations were identified in 38 of patients (10.4%) of patients, predominantly in ATM, BRCA 1/2, ARID1A genes. KRAS-HRD co-mutations were present in 18 of patients (4.7%). There is no significant difference in HRD status between the KRAS mutant and wild-type groups (p=0.73). While first-line treatment PFS was independent of KRAS mutational status (wt 10.8 vs. mut 9.6 months, p=0.31), it was significantly associated with HRD gene mutation status (16.2 vs. 10.6 months, p=0.001). OS was similar with KRAS mutations (34.7 vs. 32.9 months, p=0.72) and HRD mutations (34.7 vs. 39.1 months, p=0.73). However, patients with both KRAS and HRD mutations had significantly worse OS compared to those with either KRAS and HRD mutation (21.3 vs. 36.7 vs. 65.3 months, p=0.034, &lt;0.001). A multivariate analysis was performed to evaluate factors affecting OS in mCRC patients. Among the variables, metastasectomy, which 44% of patients underwent, had a significantly low HR (=0.28) and would contributed to more favorable outcomes when evaluating OS. Neither KRAS nor HRD mutation alone was a significant prognostic factor, but their co-mutation was significantly associated with poor survival (HR=2.39, p=0.048). Conclusions: Poor OS in patients with KRAS-HRD co-mutations, despite metastasectomy, highlights the need for additional treatment strategies beyond surgery. Future studies with larger sample sizes and analyses stratified by metastasectomy status are necessary to strengthen these findings. Variables HR (95% CI) p-value Metastasis site Liver 2.16 (1.60 – 2.92) &lt; 0.001 Peritoneum 1.52 (1.01 – 2.11) 0.013 Bone 1.69 (0.96 – 2.97) 0.067 Metastasectomy 0.28 (0.47 – 0.74) &lt; 0.001 KRAS 1.03 (0.77 – 1.37) 0.826 HRD 0.75 (0.40 – 1.41) 0.367 Co-mutation 2.39 (1.01 – 5.66) 0.048

Plasma shallow whole-genome sequencing profiling in patients with RAS WT metastatic colorectal cancer (mCRC) undergoing first-line anti-EGFR therapy.

214 Background: Acquired resistance (AR) to first-line (1L) chemotherapy (CT) + anti-EGFR inhibitors is a major challenge in the management of RAS WT mCRC patients (pts). While AR in later lines has been explained with point mutations in the EGFR pathway, its real mechanisms in 1L remain poorly understood. Tumor molecular biology can be dynamically studied through circulating tumor DNA (ctDNA) analysis, a real-time, non-invasive approach to detect AR. In this context, we conducted the multicenter prospective PLATFORM-B study, which enrolled 100 RAS WT mCRC pts receiving 1L CT + cetuximab across 15 Spanish Medical Oncology Departments, alongside 30 RAS MUT pts (control) treated with 1L CT + bevacizumab. Peripheral blood samples have been collected at baseline (BL), week 8, and disease progression (PD). Next-generation sequencing analysis (Ion S5 system) of ctDNA at PD revealed AR mutations in only 10% of pts, suggesting that alterations in the EGFR pathway may only partially explain AR in 1L. Therefore, we conducted shallow whole-genome sequencing (shWGS) to further characterize genomic profiles possibly related with novel mechanisms of AR. Methods: Baseline and PD samples with sufficient remaining plasma were considered for this analysis. shWGS was performed with a coverage ranging 0.5X to 2.0X and analyzed using ichorCNA pipeline to evaluate various genomic characteristics such as copy number alterations (CNA), tumor fraction (TF), subclonal fraction (SF), and ploidy. Molecular results were correlated to clinical-pathological features and outcomes. Results: Overall, 35 mCRC pts (27 RAS WT and 8 RAS MUT ) were included in the study. At BL ( N = 26), median TF (mTF) was 0.19, median ploidy 2, median SC fraction 0.5, median SC genome fraction 0.21, and median subclonal CNA fraction 0.41. At PD ( N = 33), mTF was 0.08, median ploidy 2, median SC fraction 0.5, median SC genome fraction 0.17, and median subclonal CNA fraction 0.38. Of all the features analyzed, only the TF showed potential prognostic value. At BL, pts with TF levels above the median had poorer overall survival (OS) compared to those with lower levels, approaching statistical significance ( P = .055). At PD, higher TF levels were statistically significantly associated with worse OS ( P = .02). No meaningful differences between the RAS WT and RAS MUT groups were observed. Specific variations in CNA, TF, SF, or ploidy under treatment are being correlated to clinical data to seek for predictive value. Conclusions: This preliminary analysis of the PLATFORM-B using shWGS showed the prognostic value of TF. Further analyses are undergoing to better understand the molecular landscape of acquired resistance to 1L anti-EGFR-based therapy in RAS WT mCRC pts.

UGT1A1 mutation rates in a minority-rich comprehensive cancer center: A retrospective pharmacogenomic study.

294 Background: Irinotecan is a widely used and effective treatment for various gastrointestinal malignancies. Its toxic metabolite, SN-38, is inactivated by UDP glucuronosyltransferase-1A1 (UGT1A1). However, variability in individual clearance of irinotecan and SN-38 can cause unpredictable toxicities. Mutations in the UGT1A1 gene reduces enzyme activity, resulting in dose-limiting neutropenia and diarrhea, hospitalizations, treatment delays, and dose reductions. The best characterized germline variant is a polymorphism in the UGT1A1*28 allele, but data in underrepresented groups remains particularly lacking. Methods: We conducted a retrospective cohort study of patients who were treated at our minority-rich academic medical center. A total of 63 patients with gastrointestinal malignancies were tested for the UGT1A1*28 polymorphism. Data for irinotecan treatment course, related toxicities, and dose reductions was available for 41 patients. Analysis was performed using Fisher’s exact test, one-way ANOVA, and Welch’s two-sample test. Results: 73% (n = 46) of patients were identified with the UGT1A1*28 polymorphism, including 63% (n = 29) with heterozygous genotype and 36.9% (n = 17) with homozygous genotype. There were significantly more patients of UGT1A1*28 polymorphisms carriers in the African American and Hispanic population as compared to the white population (p = 0.001, 0.01 respectively). The polymorphism frequency was 0.66 in the African American group [95% CI (0.49 – 0.81)], 0.48 in the Hispanic group [95% CI (0.34 – 0.62)], and 0.06 in the white group [95% CI (0.001 – 0.30)]. The frequency was significantly lower in the white population when compared to all other races (p-value = 0.0002), to African American (p- value &lt; 0.001), and to Hispanic (p-value = 0.003) populations. There were higher rates of overall toxicities of any grade in the heterozygotes (76%) and homozygotes (64%) when compared to the wild-type group (56%). 55% (n = 6) of the homozygous and 48% (n = 10) of the heterozygous patients developed G1-G4 neutropenia, as compared to the 22% (n = 2) of the wild-type patients. Conclusions: Ethnic differences are known to exist in UGT1A1*28 polymorphism, with the prevalence being significantly higher in African and Caucasian populations. Historical data based on large-scale genotyping report allele frequencies of 0.42 in African/African American, 0.37 in Hispanic American, and 0.29 in Europeans. Our local population has a higher rate overall in both the African American and Hispanic populations with significantly higher rates than the white population. Patients with the UGT1A1*28 polymorphism experienced more toxicities and neutropenia of any grade. UGT1A1 genotyping is not routinely performed in patients initiating irinotecan treatment, however it may prove to be beneficial in certain demographics with higher rates of UGT1A1*28 polymorphisms.

P-290. Clinical and microbiological characteristics of patients infected with ceftazidime/avibactam-resistant Klebsiella pneumoniae carbapenemase-producing K. pneumoniae strains

Abstract Background Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae with ceftazidime/avibactam (CZA) resistance emerged after the widely use of CZA. The most common resistance mechanism was mutations in blaKPC. We aimed to study the clinical and microbiological characteristics in patients infected with CZA-resistant KPC-producing K. pneumoniae, and focus on the comparison between the strains with or without KPC variants (wild-type KPC). Methods A retrospective study was conducted in Taipei Veterans General Hospital from February 2019 to July 2023. Hospitalized unique adult patients infected with CZA-resistant KPC-producing K. pneumoniae were included. Clinical characteristics and outcomes were collected, and genes encoding carbapenemases were detected by polymerase chain reaction followed by Sanger sequencing. Results A total of 44 cases with CZA-resistant KPC-producing K. pneumoniae infections were identified, and most of them were critically ill with the median APACHE II score 23.0 (IQR, 19.3-29.8). Pneumonia was the most common infection (n=18). Only 15 patients (34.1%) received appropriate treatment regimens, and tigecycline (n=8) and meropenem-based therapy (n=5) were the common regimens. Twenty-seven strains (61.4%) harbored KPC variants, and a total of 16 different types were identified. KPC-33 (n=10) was the most common, followed by KPC-35 (n=3). The overall 28-day mortality was similar between patients infected with and without KPC variants (22.2% vs 23.5%, p=1.000). A previous isolation of carbapenem-resistant K. pneumoniae and exposure to CZA were more common in KPC variants group compared with wild-type KPC group (92.6% vs 58.8%, p=0.017, 92.6% vs 41.2%, p&amp;lt; 0.001, respectively). KPC variants-producing strains also had a higher proportion of CZA MIC &amp;gt; 16 mg/L (85.2% vs 41.2%, p=0.002) and restored meropenem susceptibility (MIC≤ 4 mg/L) (70.4% vs 5.9%, p&amp;lt; 0.001) than that in wild-type KPC strain. Conclusion CZA-resistant KPC variants-producing K. pneumoniae has the tendency of restored meropenem susceptibility and higher level of CZA resistance compared with the wild type KPC-producing strain. The optimal therapy in these patients needs further study. Disclosures All Authors: No reported disclosures

Assessment of the quality of life in metastatic colorectal cancer patients with KRAS gene mutant: a case-control study

BackgroundThe mutation of the KRAS (Kirsten rat sarcoma virus) gene is a prevalent genetic alteration in metastatic colorectal cancer (mCRC). According to previous research, this mutation significantly affects clinical outcomes and quality of life (QOL). This research investigated the association between KRAS mutant status and various aspects of QOL in mCRC patients.MethodsThis case-control study involved 90 admitted patients with mCRC. The patients were either mCRC with positive KRAS mutants (case group) or those without the mutation (control group). The KRAS mutation status of each patient was determined using standard molecular testing. The QOL was evaluated through validated questionnaires from the European Organization for Research and Treatment of Cancer (EORTC), including QLQ-C30 and the QLQ-CR29 questionnaire for colorectal cancer patients. Differences in QOL between the groups and the association of QOL with the odds of KRAS mutation were analyzed using appropriate statistical tests.ResultsPatients in the wild-type KRAS group had significantly higher scores on the global health status (GHS) of the QLQ-C30 scale compared to those with a KRAS mutation [(64.26 ± 4.63 vs. 49.63 ± 4, crude; p = 0.019, adjusted; p = 0.024). The mean score of the social functioning scale of the KRAS mutation group was significantly higher than the wild-type [(40 ± 5.84 vs. 24.81 ± 4.39, crude; p = 0.040, adjusted; p = 0.021)]. Based on the QLQ-CR29 questionnaire, average QOL scores were suboptimal for both groups but insignificant. Further, both crude and adjusted analyses showed that KRAS mutation odds were significantly linked to improved social functioning [(crude; OR = 1.013; P = 0.044), (adjusted; OR = 1.017; P = 0.019)] and negatively associated with GHS [(crude; OR = 0.983; P = 0.022), (adjusted; OR = 0.982; P = 0.022)].ConclusionThe study revealed a low QOL in mCRC, a notable difference in social functioning, and the GHS among patients with and without mutations. Further research is needed to develop targeted interventions to enhance QOL in patients with KRAS mutation.

Low-dose quinine targets KCNH6 to potentiate glucose-induced insulin secretion.

Insulin secretion is mainly regulated by two electrophysiological events, depolarization initiated by the closure of ATP-sensitive K+ (KATP) channels and repolarization mediated by K+ efflux. Quinine, a natural component commonly used for the treatment of malaria, has been reported to directly stimulate insulin release and lead to hypoglycemia in patients during treatment through inhibiting KATP channels. In this study, we verified the insulinotropic effect of quinine on the isolated mouse pancreatic islets. We also revealed that low-dose quinine (<20 µM) did not directly provoke Ca2+ spikes or insulin secretion under low-glucose conditions but potentiated Ca2+ influx and insulin secretion induced by high glucose, which cannot be explained by KATP inhibition. KCNH6 (hERG2) is a voltage-dependent K+ (Kv) channel that plays a critical role in the repolarization of pancreatic β cells. Patch clamp experiments showed that quinine inhibited hERG channels at low micromolar concentrations. However, whether quinine can target KCNH6 to potentiate glucose-induced insulin secretion remains unclear. Here, we showed that in vivo administration of low-dose quinine (25mg/kg) improved glucose tolerance and increased glucose-induced insulin release in wild-type control mice but not in Kcnh6-β-cell-specific knockout (βKO) mice. Consistently, in vitro treatment of primary islet β cells with low-dose quinine (10 µM) prolonged action potential duration and augmented glucose-induced Ca2+ influx in the wild-type control group but not in the Kcnh6-βKO group. Our results demonstrate that KCNH6 plays an important role in low-dose quinine-potentiated insulin secretion and provide new insights into KCNH6-targeted drug development.

SCN10A gene polymorphism is associated with pain sensitivity and postoperative analgesic effects in patients undergoing gynecological laparoscopy

BackgroundPostoperative pain intensity is influenced by various factors, including genetic variations. The SCN10A gene encodes the Nav1.8 sodium channel protein, which is crucial for pain signal transmission in peripheral sensory neurons.ObjectivesThis study aims to investigate the relationship between genetic mutations in the SCN10A gene (rs6795970) and postoperative analgesic effects following gynecological laparoscopic surgery.MethodsTwo hundred female patients undergoing gynecological laparoscopic surgery under general anesthesia were included. pain sensitivity was evaluated using the catastrophizing scale and pain sensitivity questionnaire (PSQ). Patients received patient-controlled intravenous analgesia with sufentanil and dexmedetomidine for 48 h post-surgery. Postoperative pain indicators, such as visual analog scale (VAS) scores, Ramsay scores, and side effects were recorded. SCN10A rs6795970 mutations were identified using MassARRAY SNP typing technology, and patients were categoried into homozygous mutant (AA), wild type (GG), and heterozygous mutation (GA) groups for analysis.ResultsPatients in the AA group had higher scores on the pain Catastrophizing Scale, PSQ-total, PSQ-minor, and PSQ-moderate compared to GA and GG groups (P < 0.05). VAS scores at 4, 6, and 12 h post-operation were higher in the AA group than the GG group (P < 0.05). Ramsay scores were lower in AA patients at 2 and 4 h post-operation compared to GA and GG groups (P < 0.05). The AA group exhibited more self-control analgesic pump compressions within the first 24 h post-surgery, quicker depletion of analgesics in the pump, and lower patient satisfaction with pain relief compared to GA and GG groups (P < 0.05).ConclusionsFemale patients with homozygous SCN10A mutations may experience higher preoperative pain scores and increased sensitivity to postoperative pain following gynecological laparoscopic surgery with intravenous patient-controlled analgesia.Trial registration: www.chictr.org.cn, registration number: ChiCTR2200062425.

Loss of Myostatin Affects m6A Modification but Not Semen Characteristics in Bull Spermatozoa.

N6-methyladenosine (m6A) modification is a key methylation modification involved in reproductive processes. Myostatin gene editing (MT) in cattle is known to enhance muscle mass and productivity. However, the changes in m6A modification in MT bull sperm remain poorly understood. In the MT and wild-type (WT) groups, we identified 25,542 and 22,253 m6A peaks, respectively, mainly concentrated in the coding sequence (CDS) and 3' untranslated region (UTR) of genes. The MT group showed an increase in gene transcription, but there was no significant difference in the overall m6A peaks pattern. There was also no significant difference in m6A motif and chromosome distribution between MT and WT groups. Most genes had less m6A modification sites. A total of 1120 m6A peaks were significantly different, corresponding to 1053 differentially m6A-methylated genes (DMMGs). These DMMGs are mainly associated with G protein-coupled receptor signaling pathways and the overall composition of the cell membrane. Furthermore, an MCL clustering analysis of 111 differentially m6A-methylated and expressed genes identified seven key genes (RHOA, DAAM1, EXOC4, GNA12, PRICKLE1, SCN1A, and STXBP5L), with the cytoskeleton and migration-related gene, RHOA, being the most important gene located at the center of the gene network. However, the analysis of sperm morphology and motility indicated no significant changes in semen volume, sperm count, sperm viability, plasma membrane integrity, acrosome membrane integrity, or mitochondrial membrane integrity. This study provides a map of m6A methylation in spermatozoa from MT and WT bulls, identifies key differential m6A genes that are affected by the myostatin gene but do not affect sperm morphology and viability in MT bulls, and provides a theoretical basis for the breeding quality of MT bulls.

Kirsten Rat Sarcoma Virus Mutations Effect On Tumor Doubling Time And Prognosis Of Solid Dominant Stage I Lung Adenocarcinoma.

To analyze the impact of Kirsten-Rat-Sarcoma Virus (KRAS) mutations on tumor-growth as estimated by tumor-doubling-time (TDT) among solid-dominant clinical-stage I lung adenocarcinoma. Moreover, to evaluate the prognostic role of KRAS mutations, TDT and their combination in completely-resected pathologic-stage I adenocarcinomas. In this single-center retrospective analysis, completely resected clinical-stage I adenocarcinomas presenting as solid-dominant nodules (consolidation-to-tumor ratio > 0.5) in at least 2 preoperative computed-tomography scans were enrolled. Nodules' growth was scored as fast (TDT < 400 days) or slow (TDT > 400 days). KRAS-mutated adenocarcinomas were identified with next-generation sequencing. Logistic- and Cox-regressions were used to identify predictors of fast-growth and disease-free survival (DFS), respectively. Among 151 patients, 83 (55%) had fast-growing nodules and 64 (42.4%) were KRAS-mutated. Fast-growing nodules outnumbered in the KRAS-mutated group (n = 45; 70.3%), median TDT 95-days (interquartile range, IQR 43.5-151.5) compared to the KRAS wild-type group (38, 43.7%), median TDT 138-days (IQR 70.3-278.5). KRAS-mutations predicted faster-growth at multivariable analysis (P = .009). In a subgroup analysis including 108 pathologic-stage I adenocarcinomas, neither KRAS-mutations (P = .081) nor fast-growing pattern (P = .146) affected DFS. Nevertheless, the association of KRAS-mutations and fast-growing pattern identified a subgroup of patients with worse DFS (P = .02). The combination of fast-growing and KRAS-mutations (hazard-ratio 2.97 [95%CI 1.22-7.25]; P = .017) and average nodule diameter at diagnosis (hazard-ratio 1.08 [95%CI 1.03-1.14]; P = .004) were independent predictors of worse DFS. KRAS mutations are associated to faster growth, in clinical-stage I adenocarcinoma presenting at diagnosis as solid-dominant nodules undergoing complete resection. Moreover, faster-growth identifies a subgroup of pathologic-stage I KRAS-mutated adenocarcinomas with higher recurrences.

Diverse clinical outcomes for the EGFR‑mutated and ALK‑rearranged advanced non‑squamous non‑small cell lung cancer.

EGFR and ALK are key driver mutations in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors are recommended as the first-line treatment for advanced NSCLC with driving oncogenes because they have fewer side effects and provide better disease control than chemotherapy. The present retrospective analysis aimed to investigate how altered driver genes impact cancer outcomes and clinical presentation. A total of 628 patients with advanced-stage NSCLC and documented EGFR and ALK mutations were enrolled at Changhua Christian Hospital in Taiwan between August 2011 and January 2021. EGFR mutations were identified by PCR. ALK rearrangements were identified by immunostaining. Patients without EGFR or ALK mutations were labeled as wild-type (WT). EGFR mutation was detected in 446 (71.02%) patients, ALK rearrangement in 36 (5.73%) patients and WT in 146 (23.25%) patients. EGFR mutations resulted in higher frequency of lung, brain and multiple extrapulmonary metastases than ALK rearrangement. The ALK group exhibited the longest median overall survival (OS), followed by EGFR and WT groups (ALK: 51.60±13.32, EGFR: 24.03±1.22 and WT: 19.63±2.43 months, respectively; P=0.011). In patients with brain metastases, ALK group had a longer median OS than the EGFR group. Because there were few recruited patients with ALK rearrangement, the results were not significant. According to the results of Cox regression model analysis, driver mutations with EGFR and ALK, lower smoking pack-years, younger age, better performance status, no pleural metastasis and fewer extrapulmonary metastases were key prognostic factors. In conclusion, diverse clinical outcomes are driven by different driver mutations. EGFR mutation leads to more extrapulmonary metastases. Median OS was superior in ALK-rearranged NSCLC than EGFR-mutated NSCLC regardless of brain metastases.

Nb-FAR-1: A key developmental protein affects lipid droplet accumulation and cuticle formation in Nippostrongylus brasiliensis.

Fatty acid and retinol binding proteins (FARs) are lipid-binding protein that may be associated with modulating nematode pathogenicity to their hosts. However, the functional mechanism of FARs remains elusive. We attempt to study the function of a certain FAR that may be important in the development of Nippostrongylus brasiliensis. Nb-FAR-1 was highly expressed throughout developmental stages by RNA-seq data and qPCR analyses, and Nb-FAR-1 was a secretory protein and abundant in the excretory-secretory products. Nb-FAR-1 could bind fatty acids and retinol. Fatty acid pattern of parasitic adults was more similar to rat intestine than to free-living L3s, indicating that N. brasiliensis may be dependent on the host to obtain fatty acids. Lentivirus-mediated RNAi was performed on L3s, resulting in a reduction in the expression of Nb-far-1 gene. Furthermore, these RNAi effects could be maintained in several generations. The offspring L3s in Nb-far-1 RNAi group had a reduction in lipid droplets within the subcuticle and the swelling of the perioral epidermis, accompanied with down-regulated expression of enzymes in amino acid and glycerolipid metabolism and glycometabolism for growth by RNA-seq data. Adults in Nb-far-1 RNAi group had the crumpled epidermis loosely attached to the basal membrane of body surface and the breakage of mouth epidermis, accompanied with a decrease in adult egg-shedding and an appearance of abnormal eggs. In vitro culture of eggs showed decreased efficiency of egg hatchability and larval development in the Nb-far-1 RNAi group. Transcriptomic analysis showed that interference with Nb-far-1 expression induced downregulated expression of major sperm protein and serpin for reproduction, and collagen for epidermis formation in adults, most of which were relatively high expression in adults but low expression in L3s in the WT group. Thus, Nb-FAR-1 may affect the reproduction, growth, and development of N. brasiliensis by regulating the level of lipids.

43 G>T polymorphism in the sucrase-isomaltase gene in the Chinese population prevents the glucose-lowering effect of acarbose.

Acarbose is an α-glucosidase inhibitor widely used clinically for its significant hypoglycemic effect, albeit with inter-individual variations in response. The sucrase-isomaltase (SI) enzyme is the primary target of acarbose. This study aims to investigate the impact of genetic polymorphisms in the SI gene on the pharmacodynamics of acarbose. The Illumina sequencing platform and variation-related databases were employed to analyze probable gene polymorphism sites of SI. Based on the SI polymorphism sites, Chinese subjects (n=66) were categorized into the wild-type homozygous group (Group A) and the heterozygous variant group (Group B, SI 43 G>T). The validated hexokinase method was utilized to determine glucose concentrations in participants' serum samples. The differences in blood glucose concentration reduction and pharmacodynamic parameters peak concentration (Cmax) and area under the curve (AUC0-2h) after administering the same dose of acarbose were analyzed between the two groups of subjects. Our results showed that the mean changes in glucose Cmax, AUC0-2h, maximum increase, and maximum decrease in Group B were each lower by 67.66%, 63.05%, 53.17%, and 50% compared to Group A (all p<0.05). These data suggested that genetic polymorphism of the SI gene can significantly influence the hypoglycemic efficacy of acarbose, and the polymorphism of SI is associated with individual differences in clinical treatment outcomes.

Immunohistochemical markers of potential utility in identifying POLE-mutant endometrial carcinomas: An assessment of autocrine motility factor (AMF) and autocrine motility factor receptor (AMFR).

POLE status determination is necessary for the molecular classification of endometrial carcinomas (EC). However, this determination is only achievable by molecular techniques, which are not available in many practice settings. A previously published study reported elevated AMF/GPI and AMFR/gp78 levels in POLE-mutant EC. We examined the relationship between POLE status and AMF and AMFR expression. Our study included 55 molecularly classified EC, assessed for AMF and AMFR immunohistochemically. Staining intensity was scored 0 (negative), 1 (weak), 2 (medium), 3 (strong), extent was scored 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%), with those parameters summed for the final score for each case. The molecular subtypes POLE mutant, mismatch repair-deficient, no specific molecular profile, p53 abnormal had mean AMF scores of 5.909, 4.643, 5.000, 4.667, respectively. The POLE-mutant subtype had a significantly higher average AMF score than POLE wild-type (POLEwt) group (p=0.003). Using POLE mutant status as an end-point, ROC analysis showed that an AMF immunohistochemical score of 6 and above had an 81.8% sensitivity, 61.4% specificity, AUC of 0.708 (95% CI, 0.565-0.851). POLE-mutant subtype had higher prevalence of a score of 6 and above than the POLEwt group (9/11 vs 17/44 cases, p=0.010). An AMF score 6 and above increased the likelihood of being POLE-mutant by a factor of 10.496 (95% CI, 1.592-69.212). Similarly, the POLE-mutant subtype had higher prevalence of AMFR scores of 5 and above than the POLEwt group (p=0.023). AMF may offer some promise in identifying POLE-mutant EC with relatively high sensitivity, but suboptimal specificity indicates that it cannot be applied alone in practice. Additional studies are required to determine whether AMF can be combined with other markers to more optimally identify POLE mutant EC.

Development and Validation of a Prognostic Molecular Phenotype and Clinical Characterization in Grade III Diffuse Gliomas Treatment with Radio-Chemotherapy.

The relationship between molecular phenotype and prognosis in high-grade gliomas (WHO III and IV, HGG) treated with radiotherapy and chemotherapy is not fully understood and needs further exploration. The HGG patients following surgery and treatment with radiotherapy and chemotherapy. Univariate and multivariate Cox analyses were used to assess the independent prognostic factors. The nomogram model was established, and its accuracy was determined via the calibration plots. A total of 215 and 88 patients had grade III glioma and grade IV glioma, respectively. Grade III oligodendroglioma (OG-G3) patients had the longest mPFS and mOS than other grade III pathology, while grade III astrocytoma (AA-G3) patients were close to IDH-1 wildtype glioblastoma (GBM) and had a poor prognosis. The IDH-1 mutant group had a better mPFS and mOS than the IDH-1 wildtype group in all grade III patients, OG-G3 and AA-G3 patients. Furthermore, 1p/19q co-deletion group had a longer mPFS and mOS than 1p/19q non-deletion group in all grade III patients. IDH-1 mutation and 1p/19q co-deletion patients had the best prognosis than other molecular types. Also, the MGMT methylation and IDH-1 mutation or 1p/19q co-deletion group had a longer mPFS and mOS than the MGMT unmethylation and IDH-1 wildtype or 1p/19q non-codeletion of grade III patients. In addition, the low Ki-67 expression group had a better prognosis than high Ki-67 expression group in grade III patients. Univariate and multivariate COX showed that 1p/19q co-deletion and MGMT methylation were the independent prognostic factors for mPFS and mOS. The calibration curve showed that the established nomogram could well predict the survival based on these covariates. The AA-G3 with IDH-1 wildtype, MGMT unmethylation or 1p/19q non-codeletion patients was resistant to radiotherapy and chemotherapy, has a poor prognosis and needs a more active treatment.

Pathological Features and Differential Efficacy of Cisplatin-Based Adjuvant Chemotherapy in Lung Cancer Harboring Epidermal Growth Factor Receptor Mutations

We aimed to elucidate the efficacy of conventional cisplatin-based adjuvant chemotherapy for patients with lung cancers harboring epidermal growth factor receptor (EGFR) mutation. This retrospective cohort study included 110 patients (EGFR mutation group: n = 51; EGFR wild-type group: n = 59) receiving cisplatin-based adjuvant chemotherapy following complete resection of non-small-cell non-squamous-cell lung cancer (2010-2021). Clinicopathological characteristics, recurrence-free survival (RFS), and overall survival (OS) were investigated. The pStage distribution was not statistically different. The EGFR mutation group was characterized by more advanced pN, papillary predominance, and presence of micropapillary components, whereas the EGFR wild-type group exhibited more advanced pT and solid predominant patterns. The median RFS was significantly worse in the EGFR mutation group (23.0 vs. 76.1 months, p = 0.017). Nevertheless, the median OS was not significantly different (85.6 months vs. not reached, p = 0.151). Multivariable analysis demonstrated that EGFR mutation and lymphatic invasion were significant risk factors in RFS; however, no independent factors were identified in OS. Cisplatin-based adjuvant chemotherapy might be less effective in patients with EGFR-mutated lung cancer. The style of progression and histological pattern related with EGFR mutation may be associated with the efficacy of adjuvant chemotherapy and poor RFS.

Assessment of MGMT and TERT Subtypes and Prognosis of Glioblastoma by Whole Tumor Apparent Diffusion Coefficient Histogram Analysis.

Adult glioblastomas (GBMs) are associated with high recurrence and mortality. Personalized treatment based on molecular markers may help improve the prognosis. We aimed to evaluate whether apparent diffusion coefficient (ADC) histogram analysis can better predict MGMT and TERT molecular characteristics and to determine the prognostic relevance of genetic profile in patients with GBM. MRI, clinical, and pathological data of 79 patients with GBM were retrospectively collected. The ADC values based on histogram analysis were described using 10th percentile (p10), 90th percentile (p90), mean, median, minimum, maximum, skewness, kurtosis, and entropy. The independent-sample t test, linear correlation analysis, receiver operating characteristics (ROC) curve analysis, Kaplan-Meier analysis, and Cox proportional hazard regression were performed. MGMT promoter methylation and TERT promoter mutation were detected in 53.2% and 44.3% of GBM patients, respectively. The ADCp10 in MGMT promoter unmethylated group was significantly lower than that in the MGMT promoter methylated group (p=0.005). There were significant differences in ADCmin, ADCp10, ADCmean, and entropy between TERT promoter mutant and wild-type groups. Entropy showed the best diagnostic performance in differentiating between positive and negative TERT groups (AUC=0.722, p=0.001). Overall survival (OS) showed a positive correlation with ADCmin. The TERT promoter mutation was the only independent prognostic factor for GBM. ADC histogram analysis may be a potential noninvasive biomarker for differentiating MGMT and TERT molecular markers and providing prognostic information for GBM patients.

Enhanced Immunogenicity of Foot-and-Mouth Disease Virus-like Particles Using a Water-in-Oil-in-Water Adjuvant.

Foot-and-mouth disease (FMD) causes significant economic losses, prompting vaccination as a primary control strategy. Virus-like particles (VLPs) have emerged as promising candidates for FMD vaccines but require adjuvants to enhance their immunogenicity. In this study, we evaluated the immunogenicity of a VLP-based vaccine with a water-in-oil-in-water (W/O/W) emulsion adjuvant, named WT. The WT adjuvant was mixed with FMD VLPs to form the VLPs+WT vaccine. The size and stability of the vaccine were analyzed. BALB/c mice were immunized with the VLPs+WT vaccine, and immunological responses were assessed through antibody measurements, cytokine profiling, and gene expression analysis. In addition, splenic lymphocyte proliferation and signaling pathways were examined. The VLPs+WT vaccine exhibited a homogeneous size of 324.60 ± 2.30 nm and a viscosity of 8.76 mPa·s, indicating good stability. Immunized mice showed steady weight gain and no organ abnormalities. Compared to the VLPs group, the VLPs+WT group induced significantly higher levels of specific antibodies that persisted for 12 weeks, similar to the commercial VLPs+ISA201 vaccine. The VLPs+WT vaccine also enhanced the secretion of Th1-related (IgG2a, IFN-γ) and Th2-related (IgG1, IL-4) molecules. WT stimulated splenic lymphocyte proliferation and differentiation, primarily activating B-cell receptor signaling and phagosome pathways. It also upregulated genes associated with MHC and interferon stimulation while promoting the expression of MyD88, PI3K, AKT, p65, and p-p65 proteins. These findings suggest that WT is an effective adjuvant for FMD VLP-based vaccines, with potential for improving vaccine efficacy.