Widespread and Functional RNA Circularization in Localized Prostate Cancer.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.

TMBstable: a variant caller controls performance variation across heterogeneous sequencing samples.

In cancer genomics, variant calling has advanced, but traditional mean accuracy evaluations are inadequate for biomarkers like tumor mutation burden, which vary significantly across samples, affecting immunotherapy patient selection and threshold settings. In this study, we introduce TMBstable, an innovative method that dynamically selects optimal variant calling strategies for specific genomic regions using a meta-learning framework, distinguishing it from traditional callers with uniform sample-wide strategies. The process begins with segmenting the sample into windows and extracting meta-features for clustering, followed by using a pre-trained meta-model to select suitable algorithms for each cluster, thereby addressing strategy-sample mismatches, reducing performance fluctuations and ensuring consistent performance across various samples. We evaluated TMBstable using both simulated and real non-small cell lung cancer and nasopharyngeal carcinoma samples, comparing it with advanced callers. The assessment, focusing on stability measures, such as the variance and coefficient of variation in false positive rate, false negative rate, precision and recall, involved 300 simulated and 106 real tumor samples. Benchmark results showed TMBstable's superior stability with the lowest variance and coefficient of variation across performance metrics, highlighting its effectiveness in analyzing the counting-based biomarker. The TMBstable algorithm can be accessed at https://github.com/hello-json/TMBstable for academic usage only.

Clinical value of FAT1 mutations to indicate the immune response in colorectal cancer patients.

Immunotherapy is currently approved for CRC whose tumors have high MSI-H. To find additional biomarkers for immunotherapy in CRC, targeted sequencing was performed on tumor tissues from a discovery cohort of 161 CRC patients. Validation cohorts from the cBioPortal were also used for survival and tumor cell infiltration analyses. The FAT1-mutated CRC group often co-occurred with MSI events and displayed a higher tumor mutational burden compared to the FAT1 wild-type CRC. Overall survival was higher in patients with FAT1 mutations than in patients with wild type FAT1. The altered PI3K-AKT pathway and immune pathways were enriched in the FAT1-mutated CRC. A higher infiltration rate of immune cells including CD4+ T cells, CD8+ T cells, macrophages M1 and regulatory T cells were also observed in the colorectal tumors with FAT1 mutation compared to tumors with wild type FAT1. The results showed that CRC patients with FAT1 mutations exhibited an immunotherapy-favorable profile.

Integrated analysis of metabolome, transcriptome, and bioclimatic factors of Acer truncatum seeds reveals key candidate genes related to unsaturated fatty acid biosynthesis, and potentially optimal production area.

BACKGROUND: Lipids found in plant seeds are essential for controlling seed dormancy, dispersal, and defenses against biotic and abiotic stress. Additionally, these lipids provide nutrition and energy and are therefore important to the human diet as edible oils. Acer truncatum, which belongs to the Aceaceae family, is widely cultivated around the world for its ornamental value. Further because its seed oil is rich in unsaturated fatty acids (UFAs)- i.e. α-linolenic acid (ALA) and nervonic acid (NA)- and because it has been validated as a new food resource in China, the importance of A. truncatum has greatly risen. However, it remains unknown how UFAs are biosynthesized during the growth season, to what extent environmental factors impact their content, and what areas are potentially optimal for their production. RESULTS: In this study, transcriptome and metabolome of A. truncatum seeds at three representative developmental stages was used to find the accumulation patterns of all major FAs. Cumulatively, 966 metabolites and 87,343 unigenes were detected; the differential expressed unigenes and metabolites were compared between stages as follows: stage 1 vs. 2, stage 1 vs. 3, and stage 2 vs. 3 seeds, respectively. Moreover, 13 fatty acid desaturases (FADs) and 20 ß-ketoacyl-CoA synthases (KCSs) were identified, among which the expression level of FAD3 (Cluster-7222.41455) and KCS20 (Cluster-7222.40643) were consistent with the metabolic results of ALA and NA, respectively. Upon analysis of the geographical origin-affected diversity from 17 various locations, we found significant variation in phenotypes and UFA content. Notably, in this study we found that 7 bioclimatic variables showed considerable influence on FAs contents in A. truncatum seeds oil, suggesting their significance as critical environmental parameters. Ultimately, we developed a model for potentially ecological suitable regions in China. CONCLUSION: This study provides a comprehensive understanding of the relationship between metabolome and transcriptome in A. truncatum at various developmental stages of seeds and a new strategy to enhance seed FA content, especially ALA and NA. This is particularly significant in meeting the increasing demands for high-quality edible oil for human consumption. The study offers a scientific basis for A. truncatum's novel utilization as a woody vegetable oil rather than an ornamental plant, potentially expanding its cultivation worldwide.

Circulating exosomal miR-155-5p contributes to severe acute pancreatitis-associated intestinal barrier injury by targeting SOCS1 to activate NLRP3 inflammasome-mediated pyroptosis.

Severe acute pancreatitis (SAP) represents a common and serious disease that can cause intestinal barrier dysfunction. However, the pathogenesis of this barrier dysfunction remains unclear. Exosomes are a new intercellular communication method involved in multiple diseases. Consequently, the present study sought to determine the function of circulating exosomes in barrier dysfunction associated with SAP. A rat model of SAP was established by injecting biliopancreatic duct with 5% sodium taurocholate. Circulating exosomes were purified from SAP (SAP-Exo) and sham operation rats (SO-Exo) using a commercial kit. In vitro, SO-Exo and SAP-Exo were cocultured with rat intestinal epithelial (IEC-6) cells. In vivo, naive rats were treated with SO-Exo and SAP-Exo. We found SAP-Exo-induced pyroptotic cell death and barrier dysfunction in vitro. In addition, miR-155-5p exhibited a remarkable increase in SAP-Exo than SO-Exo, and miR-155-5p inhibitor partially abolished the negative effect of SAP-Exo on IEC-6 cells. Furthermore, miRNA functional experiments revealed that miR-155-5p could induce pyroptosis and barrier loss in IEC-6 cells. Overexpression of suppressor of cytokine signaling 1 (SOCS1), a miR-155-5p target, could partially reverse IEC-6 cells from the harmful impact of miR-155-5p. In vivo, SAP-Exo significantly triggered pyroptosis in intestinal epithelial cells and caused intestinal injury. In addition, blocking exosome release with GW4869 attenuated intestinal injury in SAP rats. In summary, our study demonstrated that miR-155-5p is highly enriched in circulating exosomes from SAP rat plasma and can be transported to intestinal epithelial cells, where it targets SOCS1 to activate NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis leading to intestinal barrier damage.

A Comparative Study on Platelet-Rich Plasma From Elderly Individuals and Young Adults to Treat Pressure Ulcers in Mice.

OBJECTIVE: The aim of the present study was to compare the therapeutic effects of activated platelet-rich plasma (PRP) prepared from elderly individuals and young adults to treat pressure ulcers (PUs), and to accumulate a theoretical basis for allogeneic PRP treatment of PUs in elderly patients. MATERIALS AND METHODS: Whole blood was extracted from elderly individuals aged >65 y and young adult volunteers for PRP preparation, and platelet concentrations in whole blood and PRP were compared. Growth factors released from activated PRP were assayed using the enzyme-linked immunosorbent assay. C57BL/6 mice were divided into three groups: the control saline, elderly-PRP (Group A), and young adult-PRP (Group B). Ischemia-reperfusion injury-induced PUs were established on the backs of mice. PUs were photographed on days 0, 5, and 10 to assess their sizes. Specimens were collected on day 10 and subjected to hematoxylin and eosin and Masson's staining. Immunohistochemical staining for CD31 was conducted to evaluate vascular formation, and cell invasion was assessed using a Transwell assay. The action of PRP on transforming growth factor-beta (TGF-ß)-dependent fibroblast activity and epithelial-mesenchymal transition was analyzed using immunofluorescence and Western blotting in vitro. RESULTS: The platelet concentrations in whole blood and PRP of young adults were significantly higher than that in elderly individuals. The two PRP treatment groups had similar platelet enrichment coefficients of PRP. After activation, PRP from young adults produced significantly higher levels of platelet-derived growth factor, TGF-ß, and vascular endothelial growth factor than PRP from elderly individuals (P < 0.05). The concentrations of platelet-derived growth factor, TGF-ß, and vascular endothelial growth factor were positively correlated with the platelet concentrations in whole blood and PRP. The effects of PRP in regulating the expressions of TGF-ß, α-smooth muscle actin, vimentin, and E-cadherin were observed in vivo and in vitro. The two PRP treatment groups exhibited better wound healing than the control group, as evidenced by more re-epithelialization, higher collagen content, skin fibrosis, and more blood vessel formation over time. Group B exhibited better wound healing than Group A (P < 0.05). CONCLUSION: PRP exhibits potent wound healing ability in PU therapy, and PRP from young adults is seemingly superior to that from elderly individuals because of a higher concentration of platelets and increased production of growth factors.

Establishment and characterization of a golden pompano (Trachinotus blochii) fin cell line for applications in marine fish pathogen immunology.

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1ß, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.

p-Smad3 differentially regulates the cytological behavior of osteoclasts before and after osteoblasts maturation.

BACKGROUND: A series of previous investigations have revealed that p-Smad3 plays a facilitative role in the differentiation and maturation of osteoblasts, while also regulating the expression of certain intercellular communication factors. However, the effects of p-Smad3 in osteoblasts before and after maturation on the proliferation, migration, differentiation, apoptosis and other cellular behaviors of osteoclasts have not been reported. METHODS: MC3T3-E1 cells were cultured in osteogenic induction medium for varying durations, After that, the corresponding conditioned medium was collected and the osteoclast lineage cells were treated. To elucidate the regulatory role of p-Smad3 within osteoblasts, we applied the activator TGF-ß1 and inhibitor SIS3 to immature and mature osteoblasts and collected corresponding conditioned media for osteoclast intervention. RESULTS: We observed an elevation of p-Smad3 and Smad3 during the early stage of osteoblast differentiation, followed by a decline in the later stage. we discovered that as osteoblasts mature, their conditioned media inhibit osteoclasts differentiation and the osteoclast-coupled osteogenic effect. However, it promotes apoptosis in osteoclasts and the angiogenesis coupled with osteoclasts. p-Smad3 in immature osteoblasts, through paracrine effects, promotes the migration, differentiation, and osteoclast-coupled osteogenic effects of osteoclast lineage cells. For mature osteoblasts, p-Smad3 facilitates osteoclast apoptosis and the angiogenesis coupled with osteoclasts. CONCLUSIONS: As pre-osteoblasts undergo maturation, p-Smad3 mediated a paracrine effect that transitions osteoclast cellular behaviors from inducing differentiation and stimulating bone formation to promoting apoptosis and coupling angiogenesis.

Recombinant human growth hormone promotes wound angiogenesis in burned mice through the ERK signaling pathway.

Burns are the most severe type of trauma, and the resulting ischemia and hypoxia damage can promote the dysfunction and even failure of tissues and organs throughout the body, endangering patients' life safety. Recombinant human growth hormone (rhGH) has the functions of promoting protein synthesis to reverse negative nitrogen balance, accelerating wound healing, and improving immune function, which is widely used in the treatment of burns. However, the exact mechanism and pathway of rhGH's action is not yet fully understood. In this study, we observed the wound repair effect of recombinant human growth hormone (rhGH) on burned mice and further analyzed the mechanism of action, which can provide more comprehensive reference opinions for clinical practice. First, by establishing a burn mouse model and and intervening with different doses of rhGH, we found that the wound healing capacity of mice was significantly enhanced and the inflammatory and oxidative stress responses were obviously alleviated, confirming the excellent promotion of wound repair and anti-inflammatory and antioxidant effects of rhGH. Subsequently, we found that the expression of p-ERK1/2/ERK1/2, EGF, TGF-ß, and VEGF proteins was elevated in the traumatic tissues of mice after rhGH intervention, suggesting that the pathway of action of rhGH might be related to the activation of ERK pathway to promote the regeneration of traumatic capillaries.

Ginsenoside Rc alleviates osteoporosis by the TGF-ß/Smad signaling pathway.

Osteoporosis is a common chronic bone disorder in postmenopausal women. Ginsenosides are primary active components in ginseng and the effects of various ginsenoside variants in osteoporosis treatment have been widely revealed. We planned to explore the impact of ginsenoside Rc on bone resorption in an osteoporosis rat model. We used ovariectomized rats to assess the potential impact of ginsenoside Rc on osteoporosis. µ-CT was implemented for analyzing the microstructure of the distal left femur in rats. H&E staining together with Masson staining were applied for bone histomorphometry evaluation. ELISA kits were implemented to detect serum concentrations of TRACP-5b, OCN, CTX, as well as PINP. Ginsenoside Rc treatment lessened the serum levels of TRACP-5b as well as CTX, while increasing serum levels of OCN, and PINP of OVX rats. Moreover, we found that ginsenoside Rc contributed to the synthesis of type I collagen via increasing Col1a1 and Col1a2 levels in femur tissues of ovariectomized rats. Our findings also revealed that ginsenoside Rc activated the TGF-ß/Smad pathway by increasing TGF-ß as well as phosphorylated Smad2/3 protein levels. Ginsenoside Rc alleviates osteoporosis in rats through promoting the TGF-ß/Smad pathway.

Multidimensional Cell-Free DNA Fragmentomic Assay for Detection of Early-Stage Lung Cancer.

Rationale: Cell-free DNA (cfDNA) analysis holds promise for early detection of lung cancer and benefits patients with higher survival. However, the detection sensitivity of previous cfDNA-based studies was still low to suffice for clinical use, especially for early-stage tumors. Objectives: Establish an accurate and affordable approach for early-stage lung cancer detection by integrating cfDNA fragmentomics and machine learning models. Methods: This study included 350 participants without cancer and 432 participants with cancer. The participants' plasma cfDNA samples were profiled by whole-genome sequencing. Multiple cfDNA features and machine learning models were compared in the training cohort to achieve an optimal model. Model performance was evaluated in three validation cohorts. Measurements and Main Results: A stacked ensemble model integrating five cfDNA features and five machine learning algorithms constructed in the training cohort (cancer: 113; healthy: 113) outperformed all the models built on individual feature-algorithm combinations. This integrated model yielded superior sensitivities of 91.4% at 95.7% specificity for cohort validation I (area under the curve [AUC], 0.984), 84.7% at 98.6% specificity for validation II (AUC, 0.987), and 92.5% at 94.2% specificity for additional validation (AUC, 0.974), respectively. The model's high performance remained consistent when sequencing depth was down to 0.5× (AUC, 0.966-0.971). Furthermore, our model is sensitive to identifying early pathological features (83.2% sensitivity for stage I, 85.0% sensitivity for <1 cm tumor at the 0.66 cutoff). Conclusions: We have established a stacked ensemble model using cfDNA fragmentomics features and achieved superior sensitivity for detecting early-stage lung cancer, which could promote early diagnosis and benefit more patients.

Collaborative Human-Computer Vision Operative Video Analysis Algorithm for Analyzing Surgical Fluency and Surgical Interruptions in Endonasal Endoscopic Pituitary Surgery: Cohort Study.

BACKGROUND: The endonasal endoscopic approach (EEA) is effective for pituitary adenoma resection. However, manual review of operative videos is time-consuming. The application of a computer vision (CV) algorithm could potentially reduce the time required for operative video review and facilitate the training of surgeons to overcome the learning curve of EEA. OBJECTIVE: This study aimed to evaluate the performance of a CV-based video analysis system, based on OpenCV algorithm, to detect surgical interruptions and analyze surgical fluency in EEA. The accuracy of the CV-based video analysis was investigated, and the time required for operative video review using CV-based analysis was compared to that of manual review. METHODS: The dominant color of each frame in the EEA video was determined using OpenCV. We developed an algorithm to identify events of surgical interruption if the alterations in the dominant color pixels reached certain thresholds. The thresholds were determined by training the current algorithm using EEA videos. The accuracy of the CV analysis was determined by manual review, and the time spent was reported. RESULTS: A total of 46 EEA operative videos were analyzed, with 93.6%, 95.1%, and 93.3% accuracies in the training, test 1, and test 2 data sets, respectively. Compared with manual review, CV-based analysis reduced the time required for operative video review by 86% (manual review: 166.8 and CV analysis: 22.6 minutes; P<.001). The application of a human-computer collaborative strategy increased the overall accuracy to 98.5%, with a 74% reduction in the review time (manual review: 166.8 and human-CV collaboration: 43.4 minutes; P<.001). Analysis of the different surgical phases showed that the sellar phase had the lowest frequency (nasal phase: 14.9, sphenoidal phase: 15.9, and sellar phase: 4.9 interruptions/10 minutes; P<.001) and duration (nasal phase: 67.4, sphenoidal phase: 77.9, and sellar phase: 31.1 seconds/10 minutes; P<.001) of surgical interruptions. A comparison of the early and late EEA videos showed that increased surgical experience was associated with a decreased number (early: 4.9 and late: 2.9 interruptions/10 minutes; P=.03) and duration (early: 41.1 and late: 19.8 seconds/10 minutes; P=.02) of surgical interruptions during the sellar phase. CONCLUSIONS: CV-based analysis had a 93% to 98% accuracy in detecting the number, frequency, and duration of surgical interruptions occurring during EEA. Moreover, CV-based analysis reduced the time required to analyze the surgical fluency in EEA videos compared to manual review. The application of CV can facilitate the training of surgeons to overcome the learning curve of endoscopic skull base surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT06156020; https://clinicaltrials.gov/study/NCT06156020.

Left Atrial Low Voltage Areas Predicts Recurrence of Atrial Fibrillation after Catheter Ablation: A Meta-Analysis.

BACKGROUND: Low voltage areas (LVAs) on left atrial (LA) voltage mapping correlate with atrial fibrosis. However, there is no uniform standard for the definition of LVAs, or mapping techniques and mapping rhythms, so that the predictive value of left atrial LVAs for recurrence of atrial fibrillation (AF) is uncertain. This study aimed to explore the relationship between the presence of pre-ablation left atrial LVAs and the risk of recurrent AF after catheter ablation. METHODS: The databases of PubMed, Embase, Web of science, Cochrane library, Scopus, Wanfang Datebase, China National Knowledge Infrastructure, China Biology Medicine and China Scientific Journal Datebase were searched from inception to 31 July 2023. Relevant studies regarding left atrial LVAs prior to ablation to predict postoperative recurrence of AF were identified and analyzed. The efficacy endpoints were defined as the recurrence of atrial arrhythmia lasting over 30 s. RESULTS: A total of 12 studies with 1070 patients were included. We found the presence of pre-ablation left atrial LVAs correlated with the risk of recurrent AF after ablation (hazard ratio (HR) = 2.87, 95% confidence interval (CI): 2.33-3.52). The presence of pre-ablation left atrial LVAs can predict the risk of recurrent AF after ablation both in the follow-up duration ≤12 months group and follow-up duration >12 months group (follow-up duration ≤12 months: HR = 2.93, 95% CI: 2.20-3.90; follow-up duration >12 months: HR = 2.80, 95% CI: 2.09-3.77). The presence of pre-ablation left atrial LVAs correlated with the risk of recurrent AF after ablation in paroxysmal AF (HR = 2.89, 95% CI: 1.97-4.24). CONCLUSIONS: The presence of pre-ablation left atrial LVAs correlate with the risk of recurrent AF after catheter ablation.

Testing the generalizability of cfDNA fragmentomic features across different studies for cancer early detection.

cfDNA fragmentomic features have been used in cancer detection models; however, the generalizability of the models needs to be tested. We proposed a type of cfDNA fragmentomic feature named chromosomal arm-level fragment size distribution (ARM-FSD), evaluated and compared its performance and generalizability for lung cancer and pan-cancer detection with existing cfDNA fragmentomic features (as reference) by using cohorts from different institutions. The ARM-FSD lung cancer model outperformed the reference model by ∼10% when being tested by two external cohorts (AUC: 0.97 vs. 0.86; 0.87 vs. 0.76). For pan-cancer detection, the performance of the ARM-FSD based model is consistently higher than the reference (AUC: 0.88 vs. 0.75, 0.98 vs. 0.63) in a pan-cancer and a lung cancer external validation cohort, indicating that ARM-FSD model produces stable performance across multiple cohorts. Our study reveals ARM-FSD based models have a higher generalizability, and highlights the necessity of cross-study validation for predictive model development.

Enhancing Built-in Electric Fields via Molecular Symmetry Modulation in Supramolecular Photocatalysts for Highly Efficient Photocatalytic Hydrogen Evolution.

Nature-inspired supramolecular self-assemblies are attractive photocatalysts, but their quantum yields are limited by poor charge separation and transportation. A promising strategy for efficient charge transfer is to enhance the built-in electric field by symmetry breaking. Herein, an unsymmetric protonation, N-heterocyclic π-conjugated anthrazoline-based supramolecular photocatalyst SA-DADK-H+ was developed. The unsymmetric protonation breaks the initial structural symmetry of DADK, resulting in ca. 50-fold increase in the molecular dipole, and facilitates efficient charge separation and transfer within SA-DADK-H+. The protonation process also creates numerous active sites for H2O adsorption, and serves as crucial proton relays, significantly improving the photocatalytic efficiency. Remarkably, SA-DADK-H+ exhibits an outstanding hydrogen evolution rate of 278.2 mmol g-1 h-1 and a remarkable apparent quantum efficiency of 25.1 % at 450 nm, placing it among the state-of-the-art performances in organic semiconductor photocatalysts. Furthermore, the versatility of the unsymmetric protonation approach has been successfully applied to four other photocatalysts, enhancing their photocatalytic performance by 39 to 533 times. These findings highlight the considerable potential of unsymmetric protonation induced symmetry breaking strategy in tailoring supramolecular photocatalysts for efficient solar-to-fuel production.

Molecular features of gastroenteropancreatic neuroendocrine carcinoma: A comparative analysis with lung neuroendocrine carcinoma and digestive adenocarcinomas.

Objective: There is an ongoing debate about whether the management of gastroenteropancreatic (GEP) neuroendocrine carcinoma (NEC) should follow the guidelines of small-cell lung cancer (SCLC). We aim to identify the genetic differences of GEPNEC and its counterpart. Methods: We recruited GEPNEC patients as the main cohort, with lung NEC and digestive adenocarcinomas as comparative cohorts. All patients undergone next-generation sequencing (NGS). Different gene alterations were compared and analyzed between GEPNEC and lung NEC (LNEC), GEPNEC and adenocarcinoma to yield the remarkable genes. Results: We recruited 257 patients, including 99 GEPNEC, 57 LNEC, and 101 digestive adenocarcinomas. Among the mutations, KRAS, RB1, TERT, IL7R, and CTNNB1 were found to have different gene alterations between GEPNEC and LNEC samples. Specific genes for each site were revealed: gastric NEC ( TERT amplification), colorectal NEC ( KRAS mutation), and bile tract NEC ( ARID1A mutation). The gene disparities between small-cell NEC (SCNEC) and large-cell NEC (LCNEC) were KEAP1 and CDH1. Digestive adenocarcinoma was also compared with GEPNEC and suggested RB1, APC, and KRAS as significant genes. The TP53/ RB1 mutation pattern was associated with first-line effectiveness. Putative targetable genes and biomarkers in GEPNEC were identified in 22.2% of the patients, and they had longer progression-free survival (PFS) upon targetable treatment [12.5 months vs. 3.0 months, HR=0.40 (0.21-0.75), P=0.006]. Conclusions: This work demonstrated striking gene distinctions in GEPNEC compared with LNEC and adenocarcinoma and their clinical utility.

Identification of TCR rearrangements specific for genetic alterations in EGFR-mutated non-small cell lung cancer: results from the ADJUVANT-CTONG1104 trial.

Tumor response T cells, which have specific T cell receptor (TCR) rearrangements in tumor-infiltrating lymphocytes, determine their ability to interact with the mutation-derived neoantigens presented by antigen-presenting cells. Little is known about the genetic alterations related to specific TCR clones in non-small cell lung cancer (NSCLC) patients who have an epidermal growth factor receptor (EGFR) mutation. In this study, tumor tissues were collected from 101 patients with stage II/III resectable NSCLC with an EGFR mutation (57 patients were treated with gefitinib and 44 were treated with chemotherapy) in the ADJUVANT-CTONG1104 trial for high-throughput TCRß V region and exome sequencing. Ten clonal TCRs were associated with EGFR exon 19 deletion (del), EGFR exon 21 mutation (L858R), RB1 alteration, TP53 exon 4/5 missense mutation, TP53 nonsense mutation, or copy number gains in NKX2-1 and CDK4. Among the TCRs, there was frequent use of Vß20-1Jß2-3 specifically for EGFR exon 19 del or Vß9Jß2-1 specifically for EGFR exon 21 mutation (L858R), and these were significantly associated with favorable overall survival (OS) for NSCLC patients harboring EGFR exon 19 del or exon 21 L858R, particularly in the adjuvant gefitinib setting. Moreover, in comparison with the chemotherapy-preferable (CP) group, higher frequencies of Vß20-1Jß2-3 and Vß9Jß2-1 were found in the highly TKI-preferable (HTP) or TKI-preferable (TP) groups. Altogether, we identified ten TCR rearrangements specific for genetic alterations in NSCLC. Importantly, high abundance Vß20-1Jß2-3 or Vß9Jß2-1 may be an immune biomarker for guiding adjuvant gefitinib decisions for NSCLC patients harboring EGFR exon 19 del or EGFR exon 21 L858R.

Rare mutation-dominant compound EGFR-positive NSCLC is associated with enriched kinase domain-resided variants of uncertain significance and poor clinical outcomes.

BACKGROUND: Compound epidermal growth factor receptor (EGFR) mutations are less responsive to tyrosine kinase inhibitors (TKIs) than single EGFR mutations in non-small cell lung cancer (NSCLC). However, the detailed clinical characteristics and prognosis of various compound EGFR mutations remain to be elucidated. METHODS: We retrospectively studied the next-generation sequencing (NGS) data of treatment-naïve tumors from 1025 NSCLC patients with compound EGFR mutations, which were sub-categorized into different combinations of common mutations (19-Del and EGFR exon 21 p.L858R), rare mutations, and variants of uncertain significance (VUSs). Prognosis and drug resistance to first-line TKIs were analyzed in 174 and 95 patients, respectively. RESULTS: Compound EGFR mutations were enriched with EGFR exon 21 p.L858R and rare mutations, but not 19-Del (P < 0.001). The common + rare and rare + rare subtypes had fewer concurrent mutations in the PI3K pathway (P = 0.032), while the rare + rare and common + VUSs subtypes showed increased association with smoking- and temozolomide-related mutational signatures, respectively (P < 0.001). The rare mutation-dominant subtypes (rare + VUSs and rare + rare) had the worst clinical outcomes to first-line TKIs (P < 0.001), which was further confirmed using an external cohort (P = 0.0066). VUSs in the rare + VUSs subtype selectively reside in the EGFR kinase domain (P < 0.001), implying these tumors might select additional mutations to disrupt the regulation/function of the kinase domain. CONCLUSIONS: Different subtypes of compound EGFR mutations displayed distinct clinical features and genetic architectures, and rare mutation-dominant compound EGFR mutations were associated with enriched kinase domain-resided VUSs and poor clinical outcomes. Our findings help better understand the oncogenesis of compound EGFR mutations and forecast prognostic outcomes of personalized treatments.

Prognostic value of genetic aberrations and tumor immune microenvironment in primary acral melanoma.

BACKGROUND: Acral melanoma (AM) is the most common subtype in Chinese melanoma patients with a very poor prognosis. However, our understanding of the disease pathogenesis and molecular landscape is limited by the few studies that have been conducted. Here, we profiled the clinical characteristics, mutational landscapes and tumor immune microenvironment of AM patients to gain insights into disease characteristics and potential treatment strategies. METHODS: A total of 90 AM patients were enrolled and their tissue samples were subjected to next-generation sequencing and multiplexed immunohistochemistry tests. Kaplan-Meier curves and log-rank tests were used to analyze the prognostic potential of various genetic aberrations and immune cell compositions in AM. RESULTS: The median disease-free survival was 21.3 months and estimated median overall survival (OS) was 60 months. More advanced stages, older ages and thickness of greater than 4 mm were associated with worse prognosis in AM patients (HR = 2.57, 95% CI 1.25-5.29, p = 0.01; HR = 2.77, 95% CI 1.22-6.28, p = 0.02; HR = 3.43, 95% CI 1.51-7.82, p < 0.01, respectively), while patients who received post-surgical treatments had better survival (HR = 0.36, 95% CI 0.17-0.76, p = 0.01). The most frequently altered genes included BRAF (14.5%), KIT (16.9%), NRAS (12%), NF1 (10.8%), APC (7.2%), and ARID2 (6%). Copy number variations (CNV) were commonly found in CCND1 (19.3%), CDK4 (19.3%), MDM2 (14.5%) and FGF19 (12%). CDK4 amplifications was independently associated with shorter OS in AM patients (HR = 3.61, 95% CI 1.38-9.46, p = 0.01). CD8 + T cells (p < 0.001) and M1 macrophages (p = 0.05) were more highly enriched in the invasive margin than in the tumor center. Patients with higher levels of M1 macrophage infiltration in the invasive margin derived markedly longer OS (HR = 0.43, 95% CI 0.20-0.95, p = 0.03). Interestingly, in CDK4-amplified patients, there tended to be a low level of M1 macrophage infiltration in the invasive margin (p = 0.06), which likely explains the poor prognosis in such patients. CONCLUSIONS: Our study provided a comprehensive portrait of the clinicopathological features, genetic aberrations and tumor microenvironment profiles in AM patients and identified candidate prognostic factors, which may facilitate development of additional therapeutic options and better inform clinical management of AM patients. Based on these prognostic factors, further studies should focus on enhancing the infiltration of M1 macrophages, especially in CDK4-amplified AM patients.

Utility of Circulating Free DNA Fragmentomics in the Prediction of Pathological Response after Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

BACKGROUND: A "Watch and Wait" (W&W) approach has become an alternative to surgery for locally advanced rectal cancer (LARC) after neoadjuvant chemoradiotherapy (nCRT). Precise prediction of pathological complete response (pCR) will improve patient selection for W&W. We investigated the utility of cell-free DNA (cfDNA) fragmentomics in predicting pCR. METHODS: We recruited 119 LARC patients and evaluated nCRT response by pCR status and pathological or MRI tumor regression grade (mrTRG). Plasma samples before, during, and after nCRT were applied to deep targeted-panel sequencing, with 103 patients having complete samples. cfDNA fragment and 5'-end motif profiles were used to construct elastic-net logistic regression models to predict non-pCR. Predictive performance was measured by area under the receiver operator characteristic curve (AUC), sensitivity, and specificity. RESULTS: In the training cohort, the model based on 5'-end motif profile plus mrTRG achieved the highest cross-validation AUC (0.92, 95% CI, 0.91-0.93). The AUC in a testing cohort was 0.96 (95% CI, 0.90-1.00). The models based on 5'-end motif profile alone or in combination with mrTRG both maintained good predictive ability for patients without detectable circulating tumor DNA (AUC 0.94, 95% CI, 0.93-0.95; AUC 0.95, 95% CI, 0.94-0.96). In an external validation cohort, the model trained with a local 5'-end motif profile obtained an AUC of 0.878 (95% CI, 0.801-0.956) in discriminating colorectal cancer from healthy subjects. CONCLUSIONS: The combination of a 5'-end motif profile with mrTRG has the potential to predict the response to nCRT, and therefore may improve the patient selection for a W&W approach.