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Wei-wei Tian,1 Qian-qiu Wang,1 Wei-da Liu,2 Jian-ping Shen,1 Hong-sheng Wang11Laboratory of Mycobacterial Disease, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of China; 2Department of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of ChinaPurpose: The aim of the present study was to investigate the immune response induced by Mycobacterium marinum infection in vitro and the potential of M. marinum as an immunotherapy for M. tuberculosis infection.Methods: The potential human immune response to certain bacillus infections was investigated in an immune cell–bacillus coculture system in vitro. As a potential novel immunotherapy, M. marinum was studied and compared with two other bacilli, Bacillus Calmette-Guérin (BCG) and live attenuated M. tuberculosis. We examined the changes in both the bacilli and immune cells, especially the time course of the viability of mycobacteria in the coculture system and host immune responses including multinuclear giant cell formation by Wright–Giemsa modified staining, macrophage polarization by cell surface antigen expression, and cytokines/chemokine production by both mRNA expression and protein secretion.Results: The M. marinum stimulated coculture group showed more expression of CD209, CD68, CD80, and CD86 than the BCG and M. tuberculosis (an attenuated strain, H37Ra) groups, although the differences were not statistically significant. Moreover, the M. marinum group expressed more interleukin (IL)-1B and IL-12p40 on day 3 (IL-1B: P = 0.003 and 0.004, respectively; IL-12p40: P = 0.001 and 0.011, respectively), a higher level of CXCL10 on day 1 (P = 0.006 and 0.026, respectively), and higher levels of chemokine (C-X-C motif) ligand (CXCL) 8 and chemokine (C motif) ligand (XCL) 1 on day 3 (CXCL8: P = 0.012 and 0.014, respectively; XCL1: P = 0.000 and 0.000, respectively). The M. marinum stimulated coculture group also secreted more tumor necrosis factor (TNF)-α, IL-1ß, and IL-10 on day 1 (TNF-α: P = 0.000 and 0.000, respectively; IL-1ß: P = 0.000 and 0.000, respectively; IL-10: P = 0.002 and 0.019, respectively) and day 3 (TNF-α: P = 0.000 and 0.000, respectively; IL-1ß: P = 0.000 and 0.001, respectively; IL-10: P = 0.000 and 0.000, respectively). In addition, the colony-forming units (an index of viability) of M. marinum in the M. marinum stimulated coculture group was significantly less than that of BCG and H37Ra in their corresponding bacillus stimulated groups (P = 0.037 and 0.013, respectively).Conclusion: Our results indicated that M. marinum could be a potentially safe and effective immunotherapy.Keywords: immunotherapy, Mycobacterium marinum, bacilli, immunity

Meiwen Yu,1,* Kan Wu,1,* Bing Pei,2 Degang Yang,2 Qiulin Wang,1 Hongsheng Wang,1 Jianping Shen,1 Liangbing Yan,1 Guochen Zhang1 1Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, National Center for STD and Leprosy Control, China CDC, Nanjing, People's Republic of China; 2Department of Infectious Diseases, Shanghai Skin Disease Hospital, Shanghai, People's Republic of China *These authors contributed equally to this work Abstract: The reported number of registered leprosy patients worldwide declined with the introduction of multidrug therapy. However, the emergence of rifampicin resistance in leprosy patients engenders difficulties for an individual patient, and its dissemination could pose a threat to leprosy control. This study reports an elderly patient who was diagnosed with rifampicin-resistant lepromatous leprosy. This case indicates that inadequate treatment and poor compliance may eventually result in rifampicin resistance in Mycobacterium leprae and clinical relapse. Keywords: leprosy, rifampicin, drug resistance, elderly patient

Cancers are driven by alterations in diverse genes, creating dependencies that can be therapeutically targeted. However, many genetic dependencies have proven inconsistent across tumors. Here we describe SCHEMATIC, a strategy to identify a core network of highly penetrant, actionable genetic interactions. First, fundamental cellular processes are perturbed by systematic combinatorial knockouts across tumor lineages, identifying 1,805 synthetic lethal interactions (95% unreported). Interactions are then analyzed by hierarchical pooling, revealing that half segregate reliably by tissue type or biomarker status (51%) and a substantial minority are penetrant across lineages (34%). Interactions converge on 49 multigene systems, including MAPK signaling and BAF transcriptional regulatory complexes, which become essential on disruption of polymerases. Some 266 interactions translate to robust biomarkers of drug sensitivity, including frequent genetic alterations in the KDM5C/6A histone demethylases, which sensitize to inhibition of TIPARP (PARP7). SCHEMATIC offers a context-aware, data-driven approach to match genetic alterations to targeted therapies., Competing Interests: Competing interests J.H.H. and M.A.W. are former or current employees of Ideaya Biosciences and have an equity interest. T.I. is a member of the Ideaya Biosciences scientific advisory board and has an equity interest. T.I. is also a co-founder and member of the advisory board and has an equity interest in Data4Cure and Serinus Biosciences. The terms of these arrangements have been reviewed and approved by the UCSD in accordance with its conflict-of-interest policies. B.M.K. is an employee of Vividion Therapeutics and has equity interests in Pfizer and Vividion Therapeutics. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Importance: Disparity in overall survival (OS) and differences in the frequency of driver gene variants by race and ethnicity have been separately observed in patients with colorectal cancer; however, how these differences contribute to survival disparity is unknown., Objective: To quantify the association of molecular, socioeconomic, and clinical covariates with racial and ethnic disparities in overall survival among patients with colorectal cancer., Design, Setting, and Participants: This single-center cohort study was conducted at a tertiary-level cancer center using relevant data on all patients diagnosed with colorectal cancer from January 1, 1973, to March 1, 2023. The relative contribution of variables to the disparity was determined using mediation analysis with sequential multivariate Cox regression models., Main Outcome: OS, from diagnosis date and from start of first-line chemotherapy., Results: The study population of 47 178 patients (median [IQR] age, 57.0 [49-66] years; 20 465 [43.4%] females and 26 713 [56.6%] males; 3.0% Asian, 8.7% Black, 8.8% Hispanic, and 79.4% White individuals) had a median (IQR) follow-up from initial diagnosis of 124 (174) months and OS of 55 (145) months. Compared with White patients, Black patients had worse OS (hazard ratio [HR], 1.16; 95% CI, 1.09-1.24; P <.001), whereas Asian and Hispanic patients had better OS (HR, 0.66; 95% CI, 0.59-0.74; P <.001; and 0.86; 95% CI, 0.81-0.92; P <.001, respectively). When restricted to patients with metastatic disease, the greatest disparity was between Black patients compared with White patients (HR, 1.2; 95% CI, 1.06-1.37; P <.001). Evaluating changes in OS disparity over 20 years showed disparity decreasing among Asian, Hispanic, and White patients, but increasing between Black patients and White patients (HRs, 1.18; 95% CI, 1.07-1.31 for 2008-2012; 1.24, 95% CI, 1.08-1.42 for 2013-2017; and 1.50; 95% CI, 1.20-1.87 for 2018-2023). Survival outcomes for first-line chemotherapy were worse for Black patients compared with White patients (median OS, 18 vs 26 months; HR, 1.30; 95% CI, 1.01-1.70). Among 7628 patients who had clinical molecular testing, APC, KRAS, and PIK3CA showed higher variant frequency in Black patients (false discovery rate [FDR], 0.01; < 0.001; and 0.01, respectively), whereas BRAF and KIT were higher among White patients (FDR, 0.001 and 0.01). Mediation analysis identified neighborhood socioeconomic status as the greatest contributor to OS disparity (29%), followed by molecular characteristics (microsatellite instability status, KRAS variation and BRAF variation, 10%), and tumor sidedness (9%)., Conclusions: This single-center cohort study identified substantial OS disparity and differing frequencies of driver gene variations by race and ethnicity. Socioeconomic status had the largest contribution but accounted for less than one-third of the disparity, with substantial contribution from tumor molecular features. Further study of the associations of genetic ancestry and the molecular pathogenesis of colorectal cancer with chemotherapy response is needed.

Background: We developed a whole transcriptome sequencing (WTS)-based Consensus Molecular Subtypes (CMS) classifier using FFPE tissue and investigated its prognostic and predictive utility in a large clinico-genomic database of CRC patients (n = 24,939)., Methods: The classifier was trained against the original CMS datasets using an SVM model and validated in an independent blinded TCGA dataset (88.0% accuracy). Kaplan-Meier estimates of overall survival (OS) and time-on-treatment (TOT) were calculated for each CMS (p < 0.05 considered significant)., Results: CMS2 tumors were enriched on left-side of colon and conferred the longest median OS. In RAS-wildtype mCRC, left-sided tumors and CMS2 classification were associated with longer TOT with anti-EGFR antibodies (cetuximab and panitumumab). When restricting to only CMS2, there was no significant difference in TOT between right- versus left-sided tumors. CMS1 tumors were associated with a longer median TOT with pembrolizumab relative to other CMS groups, even when analyzing only microsatellite stable (MSS) tumors., Discussion: A WTS-based CMS classifier allowed investigation of a large multi-institutional clinico-genomic mCRC cohort, suggesting anti-EGFR therapy benefit for right-sided RAS-WT CMS2 tumors and immune checkpoint inhibitor benefit for MSS CMS1. Routine CMS classification of CRC provides important treatment associations that should be further investigated., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

During the recent times, environmental antibiotic resistance genes (ARGs) and their potential transfer to other bacterial hosts of pathogenic importance are of serious concern. However, the dissemination strategies of such ARGs are largely unknown. We tested that saprotrophic soil fungi differentially enriched antibiotic resistant bacteria (ARBs) and subsequently contributed in spatial distribution of selective ARGs. Wafergen qPCR analysis of 295 different ARGs was conducted for manure treated pre-sterilized soil incubated or not with selected bacterial-fungal consortia. The qPCR assay detected unique ARGs specifically found in the mycosphere of ascomycetous and basidiomycetous fungi. Both fungi exerted potentially different selection pressures on ARBs, resulting in different patterns of ARGs dissemination (to distant places) along their respective growing fungal highways. The relative abundance of mobile genetic elements (MGEs) was significantly decreased along fungal highways compared to the respective inoculation points. Moreover, the decrease in MGEs and ARGs (along fungal highways) was more prominent over time which depicts the continuous selection pressure of growing fungi on ARBs for enrichment of particular ARGs in mycosphere. Such data also indicate the potential role of saprotrophic soil fungi to facilitate horizontal gene transfer within mycospheric environmental settings. Our study, therefore, advocates to emphasize the future investigations for such (bacteria-fungal) interactive microbial consortia for potential (spatial) dissemination of resistance determinants which may ultimately increase the exposure risks of ARGs., Competing Interests: Declaration of competing interest The authors declare ‘no conflict of interest’ with any lab or association and have no competing interests with anyone for the conduct/publication of such research work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Acinar cell carcinoma (ACC) accounts for about 1% of pancreatic cancers. The molecular and clinical features of ACC are less characterized than those of pancreatic ductal adenocarcinoma. Methods : We retrospectively evaluated the clinical and molecular features of ACC patients who underwent germline and/or somatic molecular testing at The University of Texas MD Anderson Cancer Center from 2008 to 2022 and two cases from 2023-2024 who underwent RNA and TME analysis by Boston Gene. Patient information was extracted from our institutional database with the approval of the Institutional Review Board. Results: We identified 16 patients with available molecular testing results. Fourteen patients had metastatic disease, one had borderline resectable disease, and one had localized resectable disease at diagnosis. Fifteen patients were wild type for KRAS (one patient had unknown KRAS status). Somatic/germline mutations of DNA damage repair genes ( BRCA1/2 , PALB2 , and ATM ) were present in 5 of 12 patients tested for these genes. One patient was found to have RET fusion and responded favorably to selpercatinib for over 42 months. The median overall survival (OS) was 24 months for patients with metastatic disease. One of the additional two cases who underwent BostonGene testing was found to have NTRK1 fusion. RNA and TME analysis by Boston Gene of the two cases reported immune desert features and relatively lower RNA levels of CEACAM5, CD47, CD74, and MMP1 and higher RNA levels of CDH6 compared with PDAC.

Background: The study aimed to investigate the effect of Glucagon-like peptide-2 (GLP-2) on muscle aging in vivo and in vitro., Methods: Six-week-old C57BL/6J mice were administered with D-galactose (200 mg/kg/day, intraperitoneally) for 8weeks, followed by daily subcutaneous injections of GLP-2 (300 or 600 μg/kg/day) for 4weeks. Skeletal muscle function and mass were evaluated using relative grip strength and muscle weight. The sizes and types of muscle fibers and apoptosis were assessed through histological analysis, immunofluorescence staining, and TUNEL staining, respectively. C2C12 myotubes were treated with D-galactose (40 mg/mL) and GLP-2. Protein expression of differentiation-related myogenic differentiation factor D (MyoD), myogenin (MyoG), and myosin heavy chain (Myhc), degradation-related Muscle RING finger 1 (MuRF-1), and muscle atrophy F-box (MAFbx)/Atrogin-1, and apoptosis-related B-cell leukemia/lymphoma 2 (Bcl-2) and Bax, were assessed using western blots. The Pi3k inhibitor LY294002 was applied to investigate whether GLP-2 regulated myogenesis and myotube aging via IGF-1/Pi3k/Akt/FoxO3a signaling pathway., Results: The results demonstrated that GLP-2 significantly reversed the decline in muscles weight, relative grip strength, diameter, and cross-sectional area of muscle fibers induced by D-galactose in mice. Apart from suppressing the expressions of MuRF-1 and Atrogin-1 in the muscles and C2C12 myotubes, GLP-2 significantly increased the expressions of MyoD, MyoG, and Myhc compared to the D-galactose. GLP-2 significantly suppressed cell apoptosis. Western blot analysis indicated that the regulation of GLP-2 may be attributed to the activation of theIGF-1/Pi3k/Akt/FoxO3a phosphorylation pathway., Conclusions: This study suggested that GLP-2 ameliorated D-galactose induced muscle aging by IGF-1/Pi3k/Akt/FoxO3a pathway., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Microorganisms produce extracellular enzymes to meet elemental requirements and cope with stoichiometric imbalances of resources. To gain insights into the cycling of C, N, and P, the activities of the C∶N∶P acquisition enzymes have been extensively investigated. To detect the effects of long-term fertilization practices on soil nutrient balance and characteristics of soil enzymatic stoichiometry in black soil, four different fertilization treatments were selected： no fertilization （CK）, nitrogen fertilizer （N）, phosphorus fertilizer （P）, and combination of nitrogen and phosphorus fertilizers （NP）. Soil samples were collected in both April 2021 and April 2022 to determine soil enzyme activities and their stoichiometric characteristics. The results showed that soil acid phosphatase and β -D-glucosidase activities were significantly higher in the N and NP treatments than in CK by 68%-158% and 26%-222%, respectively. Soil β -N-acetylaminoglucosidase activities were significantly higher in the P and NP treatments, with the highest around 75.48 nmol·（g·h） -1 and 106.81 nmol·（g·h） -1 , respectively. Two-way ANOVA analysis showed that N and P inputs had a great impact on soil enzyme activities. Redundancy analysis showed that the main factors controlling enzyme activities were soil pH, microbial biomass phosphorus, and soil available P content. It was found that N inputs significantly increased enzyme vector length, which was ranged from 1.32 to 1.52, and the enzyme vector angles were all larger than 45°, suggesting C and P co-limited in the black soils. These findings suggest that 40 years of fertilization have had a great impact on soil enzymes and the related resource use strategy, which provides great implications for assessing soil nutrients balance and soil sustainability.

Background: The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) is poor. Secondary brain metastasis (Br-M) occurs in less than 1% of patients. Clinical characteristics and molecular alterations have not been characterized in this rare patients' subset., Materials and Methods: The Foundry software platform was used to retrospectively query electronic health records for patients with Br-M secondary to PDAC from 2005 to 2023; clinical, molecular, and overall survival (OS) data were analyzed., Results: Br-M was diagnosed in 44 patients with PDAC. Median follow-up was 78 months; median OS from initial PDAC diagnosis was 47 months. Median duration from PDAC diagnosis to Br-M detection was 24 months; median OS from Br-M diagnosis was 3 months. At Br-M diagnosis, 82% (n = 36) of patients had elevated CA19-9. Lung was the most common preexisting metastatic location (71%) with Br-M, followed by liver (66%). Br-M were most frequently observed in the frontal lobe (34%, n = 15), cerebellar region (23%, n = 10), and leptomeninges (18%, n = 8). KRAS mutations were detected in 94.1% (n = 16) of patients who had molecular data available (n = 17) with KRASG12V being the most frequent subtype 47% (n = 8); KRASG12D in 29% (n = 5); KRASG12R in 18% (n = 3). Patients who underwent Br-M surgical resection (n = 5) had median OS of 8.6 months, while median OS following stereotactic radiosurgery only (n = 11) or whole-brain radiation only (n = 20) was 3.3 and 2.8 months, respectively., Conclusion: Br-M is a late PDAC complication, resulting in an extremely poor prognosis especially in leptomeningeal disease. KRAS was mutated in 94.1% of the patients and the KRASG12V subtype was prevalent., (© The Author(s) 2024. Published by Oxford University Press.)

Intra-tumor heterogeneity is an important driver of tumor evolution and therapy response. Advances in precision cancer treatment will require understanding of mutation clonality and subclonal architecture. Currently the slow computational speed of subclonal reconstruction hinders large cohort studies. To overcome this bottleneck, we developed Clonal structure identification through Pairwise Penalization, or CliPP, which clusters subclonal mutations using a regularized likelihood model. CliPP reliably processed whole-genome and whole-exome sequencing data from over 12,000 tumor samples within 24 hours, thus enabling large-scale downstream association analyses between subclonal structures and clinical outcomes. Through a pan-cancer investigation of 7,827 tumors from 32 cancer types, we found that high subclonal mutational load (sML), a measure of latency time in tumor evolution, was significantly associated with better patient outcomes in 16 cancer types with low to moderate tumor mutation burden (TMB). In a cohort of prostate cancer patients participating in an immunotherapy clinical trial, high sML was indicative of favorable response to immune checkpoint blockade. This comprehensive study using CliPP underscores sML as a key feature of cancer. sML may be essential for linking mutation dynamics with immunotherapy response in the large population of non-high TMB cancers.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

Purpose: Appendiceal adenocarcinoma (AA) remains an orphan disease with limited treatment options for patients unable to undergo surgical resection. Evidence supporting the efficacy of combined VEGF and PD-1 inhibition in other tumor types provided a compelling rationale for investigating this combination in AA, where immune checkpoint inhibitors have not been explored previously., Experimental Design: We conducted a prospective, single-arm phase II study evaluating efficacy and safety of atezolizumab in conjunction with bevacizumab (Atezo+Bev) in advanced, unresectable AA., Results: Patients treated with the Atezo+Bev combination had 100% disease control rate (1 partial response, 15 stable disease) with progression-free survival (PFS) of 18.3 months and overall survival not-yet-reached with median duration of follow-up of 40 months. These survival intervals were significantly longer relative to a clinically and molecularly matched synthetic control cohort treated with cytotoxic chemotherapy designed for colorectal cancer (PFS of 4.4 months, P = 0.041)., Conclusions: In light of recent data demonstrating a lack of efficacy of 5-fluorouracil-based chemotherapy, Atezo+Bev is a promising treatment option for patients with low-grade unresectable AA; further study is warranted., Significance: AA remains an orphan disease with limited systemic therapy options for patients who are not candidates for surgical resection. These data suggest activity from combined VEGF and PD-L1 inhibition that warrants further study., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Conventional surveillance methods are poorly sensitive for monitoring appendiceal cancers (AC). This study investigated the utility of circulating tumor DNA (ctDNA) in evaluating systemic therapy response and recurrence after surgery for AC., Methods: Patients from two specialized centers who underwent tumor-informed ctDNA testing (Signatera) were evaluated to determine the association between systemic therapy and ctDNA detection. In addition, the accuracy of ctDNA detection during surveillance for the diagnosis of recurrence after complete cytoreductive surgery (CRS) for grade 2-3 ACs with peritoneal metastases (PM) was investigated., Results: In this cohort of 94 patients with AC, most had grade 2-3 tumors (84.0%) and PM (84.0%). Fifty patients completed the assay in the presence of identifiable disease, among which ctDNA was detected in 4 of 7 (57.1%), 10 of 16 (62.5%), and 19 of 27 (70.4%) patients with grade 1, 2, and 3 diseases, respectively. Patients who had recently received systemic chemotherapy had ctDNA detected less frequently (7 of 16 [43.8%] v 26 of 34 [76.5%]; odds ratio, 0.22 [95% CI, 0.06 to 0.82]; P = .02). Among 36 patients with complete CRS for grade 2-3 AC-PM, 16 (44.4%) developed recurrence (median follow-up, 19.6 months). ctDNA detection was associated with shorter recurrence-free survival (median 11.3 months v not reached; hazard ratio, 14.1 [95% CI, 1.7 to 113.8]; P = .01) and showed high accuracy for the detection of recurrence (sensitivity 93.8%, specificity 85.0%). ctDNA was more sensitive than carcinoembryonic antigen (62.5%), CA19-9 (25.0%), and CA125 (18.8%) and was the only elevated biomarker in four (25%) patients with recurrence., Conclusion: This study revealed a reduced ctDNA detection frequency after systemic therapy and accurate recurrence assessment after CRS. These findings underscore the role of ctDNA as a predictive and prognostic biomarker for grade 2-3 AC-PM management.

Purpose: Dynamic operations platforms allow for cross-platform data extraction, integration, and analysis, although application of these platforms to large-scale oncology enterprises has not been described. This study presents a pipeline for automated, high-fidelity extraction, integration, and validation of cross-platform oncology data in patients undergoing treatment for rectal cancer at a single, high-volume institution., Methods: A dynamic operations platform was used to identify patients with rectal cancer treated at MD Anderson Cancer Center between 2016 and 2022 who had magnetic resonance imaging (MRI) imaging and preoperative treatment details available in the electronic health record (EHR). Demographic, clinicopathologic, tumor mutation, radiographic, and treatment data were extracted from the EHR using a methodology adaptable to any disease site. Data accuracy was assessed by manual review. Accuracy before and after implementation of synoptic reporting was determined for MRI data., Results: A total of 516 patients with localized rectal cancer were included. In the era after institutional adoption of synoptic reports, the dynamic operations platform extracted T (tumor) category data from the EHR with 95% accuracy compared with 87% before the use of synoptic reports, and N (lymph node) category with 88% compared with 58%. Correct extraction of pelvic sidewall adenopathy was 94% compared with 78%, and extramural vascular invasion accuracy was 99% compared with 89%. Neoadjuvant chemotherapy and radiation data were 99% accurate for patients who had synoptic data sources., Conclusion: Using dynamic operations platforms enables automated cross-platform integration of multiparameter oncology data with high fidelity in patients undergoing multimodality treatment for rectal cancer. These pipelines can be adapted to other solid tumors and, together with standardized reporting, can increase efficiency in clinical research and the translation of actionable findings toward optimizing patient outcomes.

Cancer stem cells (CSC) play a critical role in metastasis, relapse, and therapy resistance in colorectal cancer. While characterization of the normal lineage of cell development in the intestine has led to the identification of many genes involved in the induction and maintenance of pluripotency, recent studies suggest significant heterogeneity in CSC populations. Moreover, while many canonical colorectal cancer CSC marker genes have been identified, the ability to use these classical markers to annotate stemness at the single-cell level is limited. In this study, we performed single-cell RNA sequencing on a cohort of 6 primary colon, 9 liver metastatic tumors, and 11 normal (nontumor) controls to identify colorectal CSCs at the single-cell level. Finding poor alignment of the 11 genes most used to identify colorectal CSC, we instead extracted a single-cell stemness signature (SCS&#95;sig) that robustly identified "gold-standard" colorectal CSCs that expressed all marker genes. Using this SCS&#95;sig to quantify stemness, we found that while normal epithelial cells show a bimodal distribution, indicating distinct stem and differentiated states, in tumor epithelial cells stemness is a continuum, suggesting greater plasticity in these cells. The SCS&#95;sig score was quite variable between different tumors, reflective of the known transcriptomic heterogeneity of CRC. Notably, patients with higher SCS&#95;sig scores had significantly shorter disease-free survival time after curative intent surgical resection, suggesting stemness is associated with relapse., Implications: This study reveals significant heterogeneity of expression of genes commonly used to identify colorectal CSCs, and identifies a novel stemness signature to identify these cells from scRNA-seq data., (©2023 American Association for Cancer Research.)

Appendiceal adenocarcinoma (AA) is unique from other gastrointestinal malignancies in that it almost exclusively metastasizes to the peritoneal cavity. However, few studies have investigated the molecular interaction of the peritoneal microenvironment and AA. Here, we use a multi-omics approach with orthotopic and flank-implanted patient-derived xenografts (PDX) to study the effect of the peritoneal microenvironment on AA. AA tumors implanted in the peritoneal microenvironment tended to grow faster and displayed greater nuclear expression of Ki-67 relative to the same tumors implanted in the flank. Comparing the tumor-specific transcriptome (excluding stromal transcription), the peritoneal microenvironment relatively upregulated genes related to proliferation, including MKI67 and EXO1. Peritoneal tumors were also enriched for proliferative gene sets, including E2F and Myc Targets. Proteomic studies found a 2.5-fold increased ratio of active-to-inactive phosphoforms of the YAP oncoprotein in peritoneal tumors, indicating downregulation of Hippo signaling., Implications: The peritoneal microenvironment promotes growth of appendiceal tumors and expression of proliferative pathways in PDXs., (©2024 American Association for Cancer Research.)

Ammonia-oxidation process directly contribute to soil nitrous oxide (N 2 O) emissions in agricultural soils. However, taxonomy of the key nitrifiers (within ammonia oxidising bacteria (AOB), archaea (AOA) and complete ammonia oxidisers (comammox Nitrospira)) responsible for substantial N 2 O emissions in agricultural soils is unknown, as is their regulation by soil biotic and abiotic factors. In this study, cumulative N 2 O emissions, nitrification rates, abundance and community structure of nitrifiers were investigated in 16 agricultural soils from major crop production regions of China using microcosm experiments with amended nitrogen (N) supplemented or not with a nitrification inhibitor (nitrapyrin). Key nitrifier groups involved in N 2 O emissions were identified by comparative analyses of the different treatments, combining sequencing and random forest analyses. Soil cumulative N 2 O emissions significantly increased with soil pH in all agricultural soils. However, they decreased with soil organic carbon (SOC) in alkaline soils. Nitrapyrin significantly inhibited soil cumulative N 2 O emissions and AOB growth, with a significant inhibition of the AOB Nitrosospira cluster 3a.2 (D11) abundance. One Nitrosospira multiformis-like OTU phylotype (OTU34), which was classified within the AOB Nitrosospira cluster 3a.2 (D11), had the greatest importance on cumulative N 2 O emissions and its growth significantly depended on soil pH and SOC contents, with higher growth at high pH and low SOC conditions. Collectively, our results demonstrate that alkaline soils with low SOC contents have high N 2 O emissions, which were mainly driven by AOB Nitrosospira cluster 3a.2 (D11). Nitrapyrin can efficiently reduce nitrification-related N 2 O emissions by inhibiting the activity of AOB Nitrosospira cluster 3a.2 (D11). This study advances our understanding of key nitrifiers responsible for high N 2 O emissions in agricultural soils and their controlling factors, and provides vital knowledge for N 2 O emission mitigation in agricultural ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Although V600E accounts for the majority of the BRAF mutations in metastatic colorectal cancer (mCRC), non-V600 BRAF variants have been shown in recent years to represent a distinct molecular subtype. This study provides a comprehensive profile of BRAF variants in mCRC using a large genomic database of circulating tumor DNA (ctDNA) and analyzing clinical outcomes in a cohort of patients with atypical (non-V600) BRAF variants ( aBRAF ; class II, class III, unclassified). Overall, 1733 out of 14,742 mCRC patients in the ctDNA cohort had at least one BRAF variant. Patients with atypical BRAF variants tended to be younger and male. In contrast to BRAF V600E , BRAF class II and III variants and their co-occurrence with KRAS / NRAS mutations were increased at baseline and especially with those patients predicted to have prior anti-EGFR exposure. Our clinical cohort included 38 patients with atypical BRAF mCRC treated at a large academic referral center. While there were no survival differences between atypical BRAF classes, concurrent RAS mutations or liver involvement was associated with poorer prognosis. Notably, patients younger than 50 years of age had extremely poor survival. In these patients, the high-frequency KRAS / NRAS co-mutation and its correlation with poorer prognosis underlines the urgent need for novel therapeutic strategies. This study represents one of the most comprehensive characterizations to date of atypical BRAF variants, utilizing both ctDNA and clinical cohorts.

Importance: Serum tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and cancer antigen 125 (CA125) have been useful in the management of gastrointestinal and gynecological cancers; however, there is limited information regarding their utility in patients with appendiceal adenocarcinoma., Objective: To assess the association of serum tumor markers (CEA, CA19-9, and CA125) with clinical outcomes and pathologic and molecular features in patients with appendiceal adenocarcinoma., Design, Setting, and Participants: This is a retrospective cohort study at a single tertiary care comprehensive cancer center. The median (IQR) follow-up time was 52 (21-101) months. Software was used to query the MD Anderson internal patient database to identify patients with a diagnosis of appendiceal adenocarcinoma and at least 1 tumor marker measured at MD Anderson between March 2016 and May 2023. Data were analyzed from January to December 2023., Main Outcomes and Measures: Association of serum tumor markers with survival in patients with appendiceal adenocarcinoma. Cox proportional hazards regression analyses were also performed to assess associations between clinical factors (serum tumor marker levels, demographics, and patient and disease characteristics) and patient outcomes (overall survival)., Results: A total of 1338 patients with appendiceal adenocarcinoma were included, with a median (range) age at diagnosis of 56.5 (22.3-89.6) years. The majority of the patients had metastatic disease (1080 patients [80.7%]). CEA was elevated in 742 of the patients tested (56%), while CA19-9 and CA125 were elevated in 381 patients (34%) and 312 patients (27%), respectively. Individually, elevation of CEA, CA19-9, or CA125 were associated with worse 5-year survival; elevated vs normal was 81% vs 95% for CEA (hazard ratio [HR], 4.0; 95% CI, 2.9-5.6), 84% vs 92% for CA19-9 (HR, 2.2; 95% CI, 1.4-3.4), and 69% vs 93% for CA125 (HR, 4.6; 95% CI, 2.7-7.8) (P < .001 for all). Quantitative evaluation of tumor markers was associated with outcomes. Patients with highly elevated (top 10th percentile) CEA, CA19-9, or CA125 had markedly worse survival, with 5-year survival rates of 59% for CEA (HR, 9.8; 95% CI, 5.3-18.0), 64% for CA19-9 (HR, 6.0; 95% CI, 3.0-11.7), and 57% for CA125 (HR, 7.6; 95% CI, 3.5-16.5) (P < .001 for all). Although metastatic tumors had higher levels of all tumor markers, when restricting survival analysis to 1080 patients with metastatic disease, elevated CEA, CA19-9, or CA125 were all still associated worse survival (HR for CEA, 3.4; 95% CI, 2.5-4.8; P < .001; HR for CA19-9, 1.8; 95% CI, 1.2-2.7; P = .002; and HR for CA125, 3.9; 95% CI, 2.4-6.4; P < .001). Interestingly, tumor grade was not associated with CEA or CA19-9 level, while CA-125 was slightly higher in high-grade tumors relative to low-grade tumors (mean value, 18.3 vs 15.0; difference, 3.3; 95% CI, 0.9-3.7; P < .001). Multivariable analysis identified an incremental increase in the risk of death with an increase in the number of elevated tumor markers, with an 11-fold increased risk of death in patients with all 3 tumor markers elevated relative to those with none elevated. Somatic mutations in KRAS and GNAS were associated with significantly higher levels of CEA and CA19-9., Conclusions and Relevance: In this retrospective study of serum tumor markers in patients with appendiceal adenocarcinoma, CEA, CA19-9, and CA125 were associated with overall survival in appendiceal adenocarcinoma. Given their value, all 3 biomarkers should be included in the initial workup of patients with a diagnosis of appendiceal adenocarcinoma.

The relevance of KRAS mutation alleles to clinical outcome remains inconclusive in pancreatic adenocarcinoma (PDAC). We conducted a retrospective study of 803 patients with PDAC (42% with metastatic disease) at MD Anderson Cancer Center. Overall survival (OS) analysis demonstrated that KRAS mutation status and subtypes were prognostic (p < 0.001). Relative to patients with KRAS wildtype tumors (median OS 38 months), patients with KRAS G12R had a similar OS (median 34 months), while patients with KRAS Q61 and KRAS G12D mutated tumors had shorter OS (median 20 months [HR: 1.9, 95% CI 1.2-3.0, p = 0.006] and 22 months [HR: 1.7, 95% CI 1.3-2.3, p < 0.001], respectively). There was enrichment of KRAS G12D mutation in metastatic tumors (34% vs 24%, OR: 1.7, 95% CI 1.2-2.4, p = 0.001) and enrichment of KRAS G12R in well and moderately differentiated tumors (14% vs 9%, OR: 1.7, 95% CI 1.05-2.99, p = 0.04). Similar findings were observed in the external validation cohort (PanCAN's Know Your Tumor® dataset, n = 408)., (© 2024. The Author(s).)

Oncogenic KRAS (KRAS*) contributes to many cancer hallmarks. In colorectal cancer, KRAS* suppresses antitumor immunity to promote tumor invasion and metastasis. Here, we uncovered that KRAS* transforms the phenotype of carcinoma-associated fibroblasts (CAF) into lipid-laden CAFs, promoting angiogenesis and tumor progression. Mechanistically, KRAS* activates the transcription factor CP2 (TFCP2) that upregulates the expression of the proadipogenic factors BMP4 and WNT5B, triggering the transformation of CAFs into lipid-rich CAFs. These lipid-rich CAFs, in turn, produce VEGFA to spur angiogenesis. In KRAS*-driven colorectal cancer mouse models, genetic or pharmacologic neutralization of TFCP2 reduced lipid-rich CAFs, lessened tumor angiogenesis, and improved overall survival. Correspondingly, in human colorectal cancer, lipid-rich CAF and TFCP2 signatures correlate with worse prognosis. This work unveils a new role for KRAS* in transforming CAFs, driving tumor angiogenesis and disease progression, providing an actionable therapeutic intervention for KRAS*-driven colorectal cancer., Significance: This study identified a molecular mechanism contributing to KRAS*-driven colorectal cancer progression via fibroblast transformation in the tumor microenvironment to produce VEGFA driving tumor angiogenesis. In preclinical models, targeting the KRAS*-TFCP2-VEGFA axis impaired tumor progression, revealing a potential novel therapeutic option for patients with KRAS*-driven colorectal cancer. This article is featured in Selected Articles from This Issue, p. 2489., (©2023 American Association for Cancer Research.)

Appendiceal cancer is a rare, orphan disease with no therapies currently approved by the FDA for its treatment. Given the limited data regarding drug efficacy, these tumors have historically been treated with chemotherapy designed for colon cancer. However, an overwhelming body of molecular data has demonstrated that appendiceal adenocarcinoma is a distinct entity with key molecular differences from colon cancer, notably rare APC mutation. Recognizing that APC loss-of-function is thought to contribute to taxane resistance and that taxanes are effective in the treatment of other gastrointestinal tumors, including gastric, esophageal, and small bowel adenocarcinoma, we completed a single-center retrospective study to assess efficacy. In a cohort of 13 patients with metastatic appendiceal adenocarcinoma, treated with taxane chemotherapy the median overall survival was 8.8 months. Of 10 evaluable patients, we observed 3 responses, 4 patients with stable disease, and 3 with progression (30% response rate, 70% disease control rate). The results of this study showing activity of taxane-based chemotherapy in appendiceal adenocarcinoma support further clinical investigation of taxane therapy in this orphan disease., (© The Author(s) 2023. Published by Oxford University Press.)

Immune checkpoint inhibitors improve survival in patients with mismatch repair deficiency/microsatellite instability-high (MSI-H) colorectal cancer. The recurrence outcomes following discontinuation of immunotherapy after prolonged disease control have not been definitively reported in large series. Records from patients with advanced MSI-H colorectal cancer from The University of Texas - MD Anderson Cancer Center who received immunotherapy between 2014 and 2022 and stopped after prolonged clinical benefit were reviewed. Median progression-free and overall survival were estimated. Associations between the event of recurrence and coexisting mutations (KRAS/NRAS, BRAFV600E), metastatic organ involvement (lung, liver, lymph node, or peritoneum), metastatic timing (synchronous vs. metachronous), prior immunotherapy [anti-PD-(L)1 alone or in combination with anti-CTLA antibodies], etiology of MSI status (sporadic vs. hereditary non-polyposis colorectal cancer), and duration of immunotherapy were assessed. Sixty-four patients with MSI-H colorectal cancer without progression on immunotherapy were reviewed. Of these 48 and 16 received anti-PD(L)1 antibody alone or in combination with anti-CTLA-4 antibody, respectively. Median exposure to immunotherapy was 17.6 months (range, 1.3-51.9). After a median follow-up of 22.6 months (range, 0.3-71.7) after stopping immunotherapy, 56 of 64 patients (88%) remained without disease progression. Lung metastases were associated with recurrence/progression (OR, 6.1; P = 0.04), but coexisting mutation, primary tumor sidedness, and immunotherapy were not. These data provide a retrospective, single-institution analysis that showed that most patients with advanced MSI-H colorectal cancer do not recur after treatment cessation, regardless of the reason for stopping treatment or a variety of patient and disease features, supporting an optimistic prognosis of sustained disease control., Significance: Outcomes for patients with MSI-H colorectal cancer stopping immunotherapy after disease control remain unknown. Sixty-four patients with MSI-H colorectal cancer from our institution stopping treatment for sustained benefit or toxicity were retrospectively assessed. After median follow up of 22 months and median immunotherapy exposure of 18 months, 88% patients remained without progression. All patients who recurred or progressed and were rechallenged with immunotherapy have continued to experience disease control., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Appendiceal adenocarcinomas (AA) are a rare and heterogeneous mix of tumors for which few preclinical models exist. The rarity of AA has made performing prospective clinical trials difficult, which has partly contributed to AA remaining an orphan disease with no chemotherapeutic agents approved by the FDA for its treatment. AA has a unique biology in which it frequently forms diffuse peritoneal metastases but almost never spreads via a hematogenous route and rarely spreads to lymphatics. Given the localization of AA to the peritoneal space, intraperitoneal delivery of chemotherapy could be an effective treatment strategy. Here, we tested the efficacy of paclitaxel given by intraperitoneal administration using three orthotopic patient-derived xenograft (PDX) models of AA established in immunodeficient NSG mice. Weekly intraperitoneal paclitaxel treatment dramatically reduced AA tumor growth in all three PDX models. Comparing the safety and efficacy of intravenous with intraperitoneal administration, intraperitoneal delivery of paclitaxel was more effective, with reduced systemic side effects in mice. Given the established safety record of intraperitoneal paclitaxel in gastric and ovarian cancers, and lack of effective chemotherapeutics for AA, these data showing the activity of intraperitoneal paclitaxel in orthotopic PDX models of mucinous AA support the evaluation of intraperitoneal paclitaxel in a prospective clinical trial., Significance: The activity and safety of intraperitoneal paclitaxel in orthotopic PDX models of mucinous appendiceal adenocarcinoma supports the evaluation of intraperitoneal paclitaxel in a prospective clinical trial of this rare tumor type., (©2023 American Association for Cancer Research.)

Rare but critical bleeding events in primary immune thrombocytopenia (ITP) present life-threatening complications in patients with ITP, which severely affect their prognosis, quality of life, and treatment decisions. Although several studies have investigated the risk factors related to critical bleeding in ITP, large sample size data, consistent definitions, large-scale multicenter findings, and prediction models for critical bleeding events in patients with ITP are unavailable. For the first time, in this study, we applied the newly proposed critical ITP bleeding criteria by the International Society on Thrombosis and Hemostasis for large sample size data and developed the first machine learning (ML)-based online application for predict critical ITP bleeding. In this research, we developed and externally tested an ML-based model for determining the risk of critical bleeding events in patients with ITP using large multicenter data across China. Retrospective data from 8 medical centers across the country were obtained for model development and prospectively tested in 39 medical centers across the country over a year. This system exhibited good predictive capabilities for training, validation, and test datasets. This convenient web-based tool based on a novel algorithm can rapidly identify the bleeding risk profile of patients with ITP and facilitate clinical decision-making and reduce the occurrence of adversities., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Science China Press. Published by Elsevier B.V. All rights reserved.)

Objective: To observe the clinical characteristics and impact on mortality of carbapenem-resistant Pseudomonas aeruginosa (CRPA) colonized or infected patients with hematological disorders in order to provide evidence for the prevention and treatment of CRPA., Methods: The patients who were colonized or infected with CRPA in the Department of Hematology of The First Affiliated Hospital of Zhejiang Chinese Medical University from January 2020 to March 2021 were selected as the research subjects, the clinical data such as hospitalization time, primary disease treatment regimen, granulocyte count, previous infection and antibiotic regimen of these patients were analyzed, meanwhile, antibiotic regimen and efficacy during CRPA infection, 30-day and long-term survival were also analyzed., Results: A total of 59 patients were included in this study, and divided into CRPA infection group (43 cases) and CRPA colonization group (16 cases). Univariate logistic regression analysis showed that ECOG score ( P =0.003), agranulocytosis ( P <0.001), and exposure to upper than 3 rd generations of cephalosporins and tigecycline within 30 days ( P =0.035, P =0.017) were the high-risk factors for CRPA infection. Multivariate logistic regression analysis showed that ECOG score of 3/4 ( OR =10.815, 95% CI : 1.260-92.820, P =0.030) and agranulocytosis ( OR =13.82, 95% CI : 2.243-85.176, P =0.005) were independent risk factors for CRPA infection. There was a statistically significant difference in cumulative survival rate between CRPA colonization group and CRPA infection group ( χ 2 =14.134, P < 0.001). Kaplan-Meier survival analysis showed that the influencing factors of 30-day survival in patients with CRPA infection were agranulocytosis ( P =0.022), soft tissue infection ( P =0.03), and time of hospitalization before CRPA infection ( P =0.041). Cox regression analysis showed that agranulocytosis was an independent risk factor affecting 30-day survival of patients with CRPA infection ( HR =3.229, 95% CI :1.093-3.548, P =0.034)., Conclusions: Patients with hematological disorders have high mortality and poor prognosis after CRPA infection. Bloodstream infection and soft tissue infection are the main causes of death. Patients with high suspicion of CRPA infection and high-risk should be treated as soon as possible.

As a dietary supplement, hyaluronic acid (HA) has exhibited appreciable immunomodulatory activity and an ameliorative effect on rodent colitis. However, its high viscosity is not only refractory to absorb through the gut, but also causes flatulence. In contrast to HA, hyaluronic acid oligosaccharides (o-HAs) can overcome the above-mentioned constraints, but their treatment effect still remains ill-defined contemporarily. Herein, the current study intends to compare the modulatory effects of HA and o-HA on colitis and assess the underlying molecular mechanism. We first showed that o-HA had a better preventive effect than HA in alleviating colitis symptoms, as evidenced by lower body weight loss, lower disease activity index scores, a lower inflammatory response (TNF-α, IL-6, IL-1β, p-NF-κB), and more intact colon epithelial integrity in vivo . The best efficiency was observed in the o-HA treated group with a dosage of 30 mg kg -1 . In an in vitro barrier function assay, o-HA exerted a better protective effect on the transepithelial electrical resistance (TEER), FITC permeability, and wound healing and modulated the expression of tight junction (TJ) proteins (ZO-1, occludin) in lipopolysaccharide (LPS)-stimulated Caco-2 cells. In summary, both HA and o-HA showed the potential to reduce inflammation and ameliorate intestinal damage in DSS-induced colitis and LPS-induced inflammation, but o-HA had improved outcomes. The results also provided a glimpse of the latent mechanism by which HA and o-HA enhanced intestinal barrier function via MLCK/p-MLC signaling pathway suppression.

Purpose: Activating mutations in KRAS , NRAS , and BRAF are known to cause resistance to anti-epidermal growth factor receptor (EGFR) therapy; however, only approximately 40% of patients with colorectal cancer (CRC) with RAS WT tumors respond to anti-EGFR treatment. We sought to discover novel biomarkers to predict response to anti-EGFR antibody treatment in CRC and to understand mechanisms of resistance to anti-EGFR therapy., Materials and Methods: Transcriptomic profiles from three clinical and two preclinical cohorts treated with cetuximab were used to assign consensus molecular subtypes (CMS) to each sample and correlated with outcomes., Results: Restricting to RAS WT patients, we observed that CMS2 tumors (canonical subtype) had significantly higher response rates relative to other CMS when treated with cetuximab combination with doublet chemotherapy (Okita et al cohort: 92% disease control rate (DCR) for CMS2, chi-square P = .04; CALGB/SWOG 80405 cohort: 90% objective response rate (ORR) for CMS2, chi-square P < .001) and with single-agent cetuximab (68%, chi-square P = .01). CMS2 tumors showed best response among right-sided (ORR = 80%) and left-sided (ORR = 92%) tumors in the CALGB/SWOG 80405 cohort. CMS2 cells lines were most likely to be sensitive to cetuximab (60%) and CMS2 patient-derived xenograft had the highest DCR (84%). We found Myc, E2F, and mammalian target of rapamycin pathways were consistently upregulated in resistant samples (enrichment score >1, false discovery rate <0.25). Inhibitors of these pathways in resistant cell lines exhibited additive effects with cetuximab., Conclusion: These data suggest that CRC transcriptional profiles, when used to assign CMS, provide additional ability to predict response to anti-EGFR therapy relative to using tumor sidedness alone. Notably both right-sided and left-sided CMS2 tumors had excellent response, suggesting that anti-EGFR therapy be included as a treatment option for right-sided CMS2 tumors.

Root-associated fungi play a vital role in maintaining nutrient absorption and health of host plants. To compare the responses of root-associated fungal community structures to nitrogen (N) and/or phosphorus (P) additions across differential mycorrhizal types, we collected roots of nine plant species belonging to three mycorrhizal types (arbuscular mycorrhiza, ectomycorrhiza, and ericoid mycorrhiza) under control and N and/or P addition treatments from a subtropical forest, and detected the diversity and community composition of fungi inhabiting roots through the high-throughput sequencing technique. The results showed that root-associated fungal communities of all nine plant species were mainly composed of Basidiomycota and Ascomycota. The relative abundance of Ascomycota and Basidiomycota was significantly lower and higher under the P addition than that under control, respectively. The relative abundance of Ascomycota of ericoid mycorrhizal trees was significantly higher than those of arbuscular mycorrhizal and ectomycorrhizal trees, while the relative abundance of Basidiomycota was significantly lower than the other two mycorrhizal types. Compared with the control, P addition significantly reduced the α-diversity and changed community composition of root-associated fungi across different mycorrhizal plant types, while no effect of N addition or mycorrhizal type was observed. Compared with the control and N addition treatments, NP addition caused root-associated fungal communities of all plants becoming integrally divergent. In addition, the fungal communities of ectomycorrhizal mycorrhizal trees became apparently convergent in comparison with those of arbuscular and ericoid mycorrhizal trees under the NP addition. Collectively, our results highlighted that P was a critical factor influencing community structures of tree root-associated fungi in subtropical forest soils. This study would enhance our understanding of the responses and maintenance mechanisms of plant root-associated fungal diversity under global environmental changes in the subtropical region.

Importance: Appendiceal adenocarcinoma is a rare tumor, and given the inherent difficulties in performing prospective trials in such a rare disease, there are currently minimal high-quality data to guide treatment decisions, highlighting the need for more preclinical and clinical investigation for this disease., Objective: To prospectively evaluate the effectiveness of fluoropyrimidine-based systemic chemotherapy in patients with inoperable low-grade mucinous appendiceal adenocarcinoma., Design, Setting, and Participants: This open-label randomized crossover trial recruited patients at a single tertiary care comprehensive cancer center from September 2013 to January 2021. The data collection cutoff was May 2022. Enrollment of up to 30 patients was planned. Eligible patients had histological evidence of a metastatic low-grade mucinous appendiceal adenocarcinoma, with radiographic imaging demonstrating the presence of mucinous peritoneal carcinomatosis and were not considered candidates for complete cytoreductive surgery. Key exclusion criteria were concurrent or recent investigational therapy, evidence of bowel obstruction, and use of total parenteral nutrition. Data were analyzed from November 2021 to May 2022., Interventions: Patients were randomized to either 6 months observation followed by 6 months of chemotherapy, or initial chemotherapy followed by observation., Main Outcomes and Measures: The primary end point was the percentage difference in tumor growth in treatment and observation groups. Key secondary end points included patient-reported outcomes in the chemotherapy and observation periods, objective response rate, rate of bowel complications, and differences in overall survival (OS)., Results: A total of 24 patients were enrolled, with median (range) age of 63 (38 to 82) years, and equal proportion of men and women (eg, 12 men [50%]); all patients had ECOG performance status of 0 or 1. A total of 11 patients were randomized to receive chemotherapy first, and 13 patients were randomized to receive observation first. Most patients (15 patients [63%]) were treated with either fluorouracil or capecitabine as single agent; 3 patients (13%) received doublet chemotherapy (leucovorin calcium [folinic acid], fluorouracil, and oxaliplatin or folinic acid, fluorouracil, and irinotecan hydrochloride), and bevacizumab was added to cytotoxic chemotherapy for 5 patients (21%). Fifteen patients were available to evaluate the primary end point of difference in tumor growth during treatment and observation periods. Tumor growth while receiving chemotherapy increased 8.4% (95% CI, 1.5% to 15.3%) from baseline but was not significantly different than tumor growth during observation (4.0%; 95% CI, -0.1% to 8.0%; P = .26). Of 18 patients who received any chemotherapy, none had an objective response (14 patients [77.8%] had stable disease; 4 patients [22.2%] had progressive disease). Median (range) OS was 53.2 (8.1 to 95.5) months, and there was no significant difference in OS between the observation-first group (76.0 [8.6 to 95.5] months) and the treatment-first group (53.2 [8.1 to 64.1] months; hazard ratio, 0.64; 95% CI, 0.16-2.55; P = .48). Patient-reported quality-of-life metrics identified that during treatment, patients experienced significantly worse fatigue (mean [SD] score, 18.5 [18.6] vs 28.9 [21.3]; P = .02), peripheral neuropathy (mean [SD] score, 6.67 [12.28] vs 38.89 [34.88]; P = .01), and financial difficulty (mean [SD] score, 8.9 [15.2] vs 28.9 [33.0]; P = .001) compared with during observation., Conclusions and Relevance: In this prospective randomized crossover trial of systemic chemotherapy in patients with low-grade mucinous appendiceal adenocarcinoma, patients did not derive clinical benefit from fluorouracil-based chemotherapy, given there were no objective responses, no difference in OS when treatment was delayed 6 months, and no difference in the rate of tumor growth while receiving chemotherapy., Trial Registration: ClinicalTrials.gov Identifier: NCT01946854.

Soil-borne fungal pathogens pose a major threat to global agricultural production and food security. Pathogen-suppressive bacteria and plant beneficial protists are important components of soil microbiomes and essential to plant health and performance, but it remains largely unknown regarding how agricultural management practices influence the relative importance of protists and bacteria in plant disease suppression. Here, we characterized soil microbiomes (including fungi, protists, and bacteria) in bulk and sorghum rhizosphere soils with various long-term inorganic and organic fertilization regimes, and linked the changes in fungal plant pathogens with the protistan and bacterial communities. We found that the relative abundances of fungal pathogens were significantly decreased by organic fertilization regimes, and there was a significant difference in the community composition of fungal pathogens between inorganic and organic fertilization regimes. Organic fertilization significantly enhanced predatory protists but reduced the proportions of protistan phototrophs. Co-occurrence network analysis revealed more intensive connections between fungal plant pathogens with protists, especially predatory protists, than with bacterial taxa, which was further supported by stronger associations between the community structure of fungal pathogens and predatory protists. We identified more protist consumer taxa than bacterial taxa as predictors of fungal plant pathogens, and structural equation modelling revealed a more important impact of protist consumers than bacteria on fungal pathogens. Altogether, we provide new evidence that the disease inhibitory effects of long-term organic fertilization regimes could be best explained by the potential predation pressure of protists. Our findings advance the mechanistic understanding of the role of predator-prey interactions in controlling fungal diseases, and have implications for novel biocontrol strategies to mitigate the consequences of fungal infections for plant performance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We conducted a nitrogen (N) and phosphorus (P) addition experiment in Qianjiangyuan National Park in 2015, to investigate the response of ammonia-oxidizing microorganisms and denitrifying microorganisms. There were four treatments, including N addition (N), P addition (P), NP, and control (CK). Soil samples were collected in April (wet season) and November (dry season) of 2021. The abundance of amoA gene of ammonia-oxidizing microorganisms ( i.e ., ammonia-oxidizing archaea, AOA; ammonia-oxidizing bacteria, AOB; comammox) and denitrifying microbial genes ( i.e ., nirS , nirK, and nosZ ) were determined using quantitative PCR approach. The results showed that soil pH was significantly decreased by long-term N addition, while soil ammonium and nitrate contents were significantly increased. Soil available P and total P contents were significantly increased with the long-term P addition. The addition of N (N and NP treatments) significantly increased the abundance of AOB- amoA gene in both seasons, and reached the highest in the N treatment around 8.30×10 7 copies·g -1 dry soil. The abundance of AOA- amoA gene was significantly higher in the NP treatment than that in CK, with the highest value around 1.17×10 9 copies·g -1 dry soil. There was no significant difference in N-related gene abundances between two seasons except for the abundance of comammox- amoA . Nitrogen addition exerted significant effect on the abundance of AOB- amoA , nirK and nosZ genes, especially in wet season. Phosphorus addition exerted significant effect on the abundance of AOA- amoA and AOB- amoA genes in both seasons, but did not affect denitrifying gene abundances. Soil pH, ammonium, nitrate, available P, and soil water contents were the main factors affecting the abundance of soil N-related functional genes. In summary, the response of soil ammonia-oxidizing microorganisms and denitrifying microorganisms was more sensitive to N addition than to P addition. These findings shed new light for evaluating soil nutrient availability as well as their response mechanism to global change in subtropical forests.

Introduction: Appendiceal neoplasms have a propensity for peritoneal dissemination. The standard of care for select individuals is CRS/HIPEC. In the current 8 th AJCC Staging system, a finding of only intraperitoneal acellular mucin (M1a) is classified as Stage IVa. There is concern that the current AJCC system may over-stage patients., Methods: This was a single-institution retrospective review of 164 cases of mucinous appendiceal neoplasm. Patients undergoing CRS/HIPEC with M1a disease were compared to patients with peritoneal deposits containing tumor cells (well-differentiated adenocarcinoma; low-grade mucinous carcinoma peritonei-M1b,G1). Overall and recurrence-free survival were assessed., Results: Median age was 51 years, 70% were female, and 75% White. Sixty-four patients had M1a disease and 100 M1b,G1 disease. M1a disease had a lower median PCI score (11 vs. 20, p = .0001) and a higher rate of complete CRS (62% vs. 50%, p = .021). Median follow-up was 7.6 years (IQR 5.6-10.5 years). For M1a disease, there were no recurrences and only one patient died during the study interval. In comparison, for M1b disease, 66/100 (66%) recurred with a 5-year RFS of 40.5% (HR 8.0, 95% CI 4.9-15.1, p < .0001), and 31/100 (31%) died with a 5-year OS of 84.8% (HR 4.5, 95% CI 2.2-9.2, p < .0001)., Conclusions: Acellular mucin (M1a disease) after CRS/HIPEC for appendiceal neoplasm is associated with longer OS and RFS compared to M1b, G1 disease. Current AJCC staging does not accurately reflect the differing outcomes of these two patient populations. The presence of acellular mucin in the peritoneal cavity should not be perceived as a metastatic equivalent., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Over the past two decades of successive clinical trials in metastatic colorectal cancer (CRC), the median overall survival of both control and experimental arms has steadily improved. However, the incremental change in survival for metastatic CRC patients not treated on trial has not yet been quantified. We performed a retrospective review of 1420 patients with de novo metastatic CRC who received their primary treatment at the University of Texas M.D. Anderson Cancer Center (UTMDACC) from 2004 through 2019. Median OS was roughly stable for patients diagnosed between 2004 and 2012 (22.6 months) but since has steadily improved for those diagnosed in 2013 to 2015 (28.8 months), and 2016 to 2019 (32.4 months). Likewise, 5-year survival rate has increased from 15.7% for patients diagnosed from 2004 to 2006 to 26% for those diagnosed from 2013 to 2015. Notably, survival improved for patients with BRAF V600E mutant as well as microsatellite unstable (MSI-H) tumors. Multivariate regression analysis identified surgical resection of liver metastasis (HR = 0.26, 95% CI, 0.19-0.37), use of immunotherapy (HR = 0.44, 95% CI, 0.29-0.67) and use of third line chemotherapy (regorafenib or trifluridine/tipiracil, HR = 0.74, 95% CI, 0.58-0.95), but not year of diagnosis (HR = 0.99, 95% CI, 0.98-1), as associated with better survival, suggesting that increased use of these therapies are the drivers of the observed improvement in survival., (© 2023. The Author(s).)

Purpose: Acquired resistance to anti-epidermal growth factor receptor (EGFR) inhibitor (EGFRi) therapy in colorectal cancer (CRC) has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR , among other MAPK-pathway members. However, this was primarily on the basis of single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients., Methods: We analyzed paired plasma samples from patients with RAS/BRAF/EGFR wild-type metastatic CRC enrolled in three large randomized trials evaluating EGFRi in the first line in combination with chemotherapy and as a single agent in third line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, infusional fluorouracil, leucovorin, and oxaliplatin, and SN38 were developed, and transcriptional changes profiled., Results: Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared with those treated in combination with cytotoxic chemotherapy (9%). Furthermore, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition. By contrast, commonly acquired resistance alterations in the MAPK pathway do not affect sensitivity to cytotoxic chemotherapy., Conclusion: These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

Introduction: Senile osteoporosis is one of the most common age-related diseases worldwide. Glucagon like peptide-2 (GLP-2), a naturally occurring gastrointestinal peptide, possesses therapeutic effects on bone loss in postmenopausal women and ovariectomized rats. However, the role of GLP-2 in senile osteoporosis and underlying mechanisms has not been explored., Methods: GLP-2 was subcutaneously injected into the 6-month-old male senile osteoporosis model of senescence-accelerated mouse prone 6 (SAMP6) mice for 6 weeks. SAMP6 subjected to normal saline and senescence-accelerated mouse resistant 1 served as control groups. Micro-computed tomography was performed to evaluate the bone mass and microarchitecture of the mice. Osteoblastic and osteoclastic activities were determined by biochemical, quantitative real-time PCR, histological, and histomorphometric analyses combined with hematoxylin-eosin, toluidine blue, and tartrate-resistant acid phosphatase staining. We also examined the proteins and structure of intestinal tight junction using immunohistochemical assay as well as a transmission electron microscope. Serum inflammation marker levels were measured using ELISA. Additionally, anti-oxidative enzymes GPX-4 and SOD-2 and receptors of GLP-2 and vitamin D expression in the ileum and colon were detected under immunofluorescence staining., Results: Six-week GLP-2 treatment attenuated bone loss in SAMP6 mice, as evidenced by increased bone mineral density, improved microarchitecture in femora, and enhanced osteogenic activities. In contrast, the activity of osteoclastic activity was not obviously inhibited. Moreover, GLP-2 ameliorated tight junction structure and protein expression in the intestinal barrier, which was accompanied by the reduction of TNF-α level. The expression of receptors of intestinal GLP-2 and vitamin D in the ileum was elevated. Furthermore, the oxidative stress in the intestines was improved by increasing the GPX-4 and SOD-2 signaling., Conclusion: Our findings suggest that GLP-2 could ameliorate age-associated bone loss, tight junction structure, and improved antioxidant enzyme activity in the gut in SAMP6 mice. Amelioration of gut barrier dysfunction may potentially contribute to improving bone formation and provide evidence for targeting the entero-bone axis in the treatment of senile osteoporosis., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Background: Pigment disorder dermatoses are common diseases with complex mechanisms. There are various methods for the clinical treatment of pigmentation diseases, but these have a poor curative effect and many adverse reactions. Currently, looking for safe and effective whitening agents is a popular research topic. Stromal vascular fractions (SVFs) are a compound cell component of adipose-derived stem cells (ADSCs) that can promote tissue regeneration, healing, and vascularization. The purpose of this experiment was to investigate the inhibitory effect of SVFs on pigmentation in guinea pigs., Methods: After guinea pig subcutaneous fat was digested and centrifuged, SVFs were isolated and quantified. SVF was injected into the pigmentation area of the prepared guinea pig pigmentation model. The amount of inducible nitric oxide synthase (iNOS) was determined using immunohistochemical analysis, histopathological staining, and the Fontana-Masson (F-M) method for measuring melanin formation., Results: The skin of the guinea pigs obtained stable and homogenous coloration following three treatments with narrow-band ultraviolet B (NB-UVB). Hematoxylin-eosin (HE) staining revealed that compared to the control group, the cuticle, granular layer, and spinous layer were thicker and the number of epidermal melanocytes and melanin granules increased. While the quantity of pigment granules in the treated group dramatically decreased, it did not significantly change in the blank control group. F-M staining revealed that melanin granules greatly expanded following ultraviolet irradiation and were continuously distributed in basal cells and spinous layers. The entire epidermis was evenly covered in melanin granules. The level of melanin dramatically decreased following therapy. According to immunohistochemical labeling, epidermal cells' cytoplasm and membranes are where iNOS is primarily found. In the epidermis of the irradiated group, iNOS expression was much higher than in the control group, and following treatment, it decreased in the experimental group., Conclusions: SVFs have a reliable treatment effect on ultraviolet B (UVB)-induced pigmentation in guinea pig skin. SVFs can significantly inhibit pigmentation, effectively shorten the fading time of pigmentation, and play a role in skin whitening, providing a new breakthrough for the treatment of pigmentation diseases., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5433/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes., (© 2022. The Author(s).)