Distinct Genomic Characteristics Research Articles

Age does matter in adolescents and young adults vs. older adults with lung adenocarcinoma: A retrospective analysis comparing clinical characteristics and outcomes in response to systematic treatments.

Adenocarcinoma is the most common histological type of lung cancer in adolescents and young adults (AYAs; ˂50 years of age). However, few clinical trials that have investigated systematic treatments regard AYAs as a special cohort, and the differences in progression-free survival (PFS) and overall survival (OS) between AYAs and older adults is still unclear. The present study compared clinical characteristics, targetable genomic mutations, toxicity, efficacy and prognostic response to systematic treatments in AYAs (n=251) and older adults (n=1,098) who were diagnosed with lung adenocarcinoma between January 2013 and December 2017 at YueBei People's Hospital (Shaoguan, China). Compared with older adults, AYAs with lung adenocarcinoma were more frequently female and non-smokers, with a higher ratio of patients receiving chemotherapy and targeted therapy, and fewer untreated. More AYAs harbored targetable genomic mutations, including epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations, while more older adults harbored KRAS proto-oncogene GTPase mutations. EGFR L858R was significantly more prevalent among older adults, while 19Del was common in AYAs. AYAs showed a higher objective response rate (ORR) and a lower grade 3–4 treatment-related adverse event (TRAE) percentage following systematic chemotherapy, but shared a similar ORR and grade 3–4 TRAE percentage with older adults following targeted therapies. AYAs experienced a shorter progression-free survival time following EGFR-tyrosine kinase inhibitor (TKI) treatment due to the higher number of metastatic organs at the time of the initial cancer diagnosis. However, there was a survival advantage of AYAs over older adults in terms of the response to systemic chemotherapy, and an age of ˂50 years was indicated as one of the positive predictors for OS time. Overall, AYAs with lung adenocarcinoma harbored distinctive clinical and genomic characteristics, and exhibited PFS and OS disadvantages following first-line EGFR-TKIs and advantages following systematic chemotherapy. However, the age-related difference in prognosis existed solely in patients who received systematic chemotherapy.

A thousand metagenome-assembled genomes of Akkermansia reveal phylogroups and geographical and functional variations in the human gut.

Akkermansia muciniphila has long been considered to be the only Akkermansia species in the human gut and has been extensively studied. The present study revealed the genomic architecture of Akkermansia in the human gut by analyzing 1,126 near-complete metagenome-assembled genomes, 84 publicly available genomes, and 1 newly sequenced Akkermansia glycaniphila strain from the human gut. We found that 1) the genomes of Akkermansia were clustered into four phylogroups with distinct interspecies similarity and different genomic characteristics and 2) A. glycaniphila GP37, a strain of Akkermansia, was isolated from the human gut, whereas previously, it had only been found in python. Amuc III was present in the Chinese population, and Amuc IV was mainly distributed in Western populations. A large number of gene functions, pathways, and carbohydrate-active enzymes were specifically associated with phylogroups. Our findings based on over a thousand genomes strengthened our previous knowledge and provided new insights into the population structure and ecology of Akkermansia in the human gut.

Abstract 5402: Urothelial carcinomas in different anatomical positions exhibit distinct genomic and immunological characteristics

Abstract Background Human bladder cancers were recently suggested to be grouped into different molecular subtypes. However, little is known about the difference in gene landscape between upper tract urothelial carcinoma (UTUC) and lower tract urothelial carcinoma (LTUC) of Chinese patients. Herein, we analyzed the genomic characteristics of primary UTUC and LTUC in China. Methods The formalin-fixed paraffin-embedded specimens of 250 LTUC and 121 UTUC patients have been consecutively collected and have underwent next-generation sequencing (NGS) from 2018 to 2021 in 3DMed Clinical Laboratory Inc. Gene mutation and TMB level were analyzed by NGS, detected PD-L1 expression by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. Results NGS analysis showed that, in the samples of UTUC, the top ten gene mutations were CDKN2A/2B 6.6% (8/121), TP53 5.8% (7/121), FGF19 5.8% (7/121), TERT 3.3% (4/121), PREX2 3.3% (4/121), FRS2 3.3% (4/121), KDM6A 2.5% (3/121), PIK3CA 2.5% (3/121), EGFR 2.5% (3/121), MYCL 2.5% (3/121); while in the LTUC, the top gene mutations were TP53 44.4% (111/250), TERT 31.6% (79/250), CDKN2A/2B 25.7% (64/250), FGF3 18% (45/250), KDM6A 17.6% (44/250), PIK3CA 16.4% (41/250), RB1 14.0%(35/250), DGFR3 13.6% (34/250), ARID1A 12.4% (31/250). Furthermore, TMB level and PD-L1 expression in the UTUC and LTUC were also analyzed, and results showed that although the gene mutation was totally different, but no significant difference was found on TMB level (p>0.05). However, among these patients, higher PD-L1 expression level were found in UTUC than LTUC (p<0.05). Conclusion UTUC and LTUC in Chinese patients hold different gene landscape and PD-L1 expression, this suggested these patients may have different prognosis and response differently to targetable therapy and immunotherapy. Citation Format: Ruiqian Li, Hushan Zhang, Qilin Wang, Hong Yang, Yu Bai, Chen Hu, Hongyi Wu, Chongjian Zhang, Ziyong Wang, Qiwei Wang, Pengjie Xu, Jun Li. Urothelial carcinomas in different anatomical positions exhibit distinct genomic and immunological characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5402.

Abstract 3099: Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models

Abstract INTRODUCTION AND OBJECTIVES: Prostate cancer is a highly heterogeneous cancer type with distinct genomic and phenotypic characteristics that drive tumorigenesis and the differential response to drug therapies. A limit number of prostate cancer cell lines and patient-derived xenograft (PDX) models hinders research to improve disease outcome. Some currently available PDX models were derived from the primary tumor samples are insufficient to recapitulate the clinical response at more advanced stages. In this study, we developed patient-derived models from patients with advanced disease and evaluated a novel AKR1C3 inhibitor in these models. METHODS: Samples received from our Pathology Biorepository Shared Resource were divided into four groups and subjected to pathological staining, RNA extraction, xenografting in NSG mice via renal capsule and subcutaneous implantation in SCID mice and conditional reprogramed cultures (CRCs) or organoid culturing. The AKR1C3 inhibitor PB was modified from celecoxib. Androgen receptor (AR), AR-V7 and AKR1C3 expression were determined by western blot. The effects of the AKR1C3 inhibitor on enzalutamide sensitivity were characterized by growth assay and colony formation assay. RESULTS: Eight PDX models have been developed from prostate cancer patients with high Gleason score and/or at the castration-resistant stages. Among the PDX models, one spontaneous indefinite cell line PS1172 was established. Early passage CRCs showed the epithelial morphology with AR positive expression. Through serially passaging PS1172 PDX with castration in SCID mice, the castration resistant cell line 1172CR was re-cultured from castration-resistant PS1172 PDX tumors. 1172CR cells were resistant to enzalutamide treatment and expressed high level of AKR1C3 and AR-V7. A novel AKR1C3 inhibitor (PB) which displayed superior potential to inhibit AKR1C3 activity and suppress enzalutamide resistant prostate cancer cell growth was tested in these models. At the same dose, PB significantly suppressed 1172CR cell growth and colony formation compared to indomethacin and enzalutamide. PB also significantly suppressed AR/AR-V7 protein expression compared to indomethacin in 1172CR cells. CONCLUSION: PS1172 and castration-resistant 1172CR cells are novel models with significant characteristics such as AR-V7 and AKR1C3. These novel prostate cancer models are ideal for small molecule testing and resistant mechanism investigating. Citation Format: Joy C. Yang, Shu Ning, Hans Adomat, Martin Gleave, Allen Gao, Christopher P. Evans, Chengfei Liu. Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3099.

Deciphering genomic characteristics of ERBB family members potentially involved in the recovery of anti-cancer immunity for intrahepatic cholangiocarcinoma patients.

e16003 Background: Previous studies showed that the efficacy of immune checkpoint inhibitor (ICI) is enhanced in hepatobiliary cancers harboring activating gene alterations of ERBB family members. The purpose of this study was to explore genomic characteristics of ERBB family members as well as the relationship of them with the activity of anti-cancer immunity in Chinese intrahepatic cholangiocarcinoma (ICC) patients. Methods: Fifty-four ICC patients which carried at least one gene mutation of ERBB family members were pre-selected from 508 ICC patients included in the China Pan-cancer project of OrigiMed 2020 (https://www.cbioportal.org/study/summary?id=pan_origimed_2020). Targeted sequencing was implemented with cancer-related 450-gene panel. Tumor mutation burden (TMB) and microsatellite status were evaluated. To explore the molecular mechanisms, two human cholangiocarcinoma cell lines, RBE and HuCCT1, were transfected with a lentivirus to obtain the cells with stable S310F mutation and overexpression of ERBB2. Results: The variation frequencies of EGFR, ERBB2, ERBB3 and ERBB4 were 2.8% (14/508), 3.5% (18/508), 3.3% (17/508) and 2.4% (12/508) in Chinese ICC patients, respectively. All the patients harboring ERBB family gene mutations were classified as TMB-H (cutoff = 10 Muts/Mb), when co-mutated with ACVR2A or TGFBR2. Moreover, ACVR2A and TGFBR2 were identified as driver genes in the cohort with oncodriveCLUST. TMB and the proportion of MSI-H patients were significantly elevated when ERBB family members co-mutated with ACVR2A (mutation vs wild: TMB, 38.2 ± 19.1 vs 7.0 ± 8.6 Muts/Mb, p<0.0001; MSI-H, 77.8% vs 4.9%, p<0.0001), or with TGFBR2 (TMB, 33.1 ± 8.7 vs 9.0 ± 14.4 Muts/Mb, p<0.0001; MSI-H, 83.3% vs 9.1%, p<0.001). Besides, ACVR2A and TGFBR2 were enriched in TGF-β pathway. Flow cytometry analysis revealed that ERBB2 S310F mutation and amplification were increased by approximate 3-fold and 3.5-fold in PD-L1 expression of cell line RBE ( p < 0.0001, respectively); by approximate 2-fold and 3-fold upregulation of PD-L1 expression in cell line HUCCT1 ( p < 0.0001, respectively). Western-blot results suggested that ERBB2 mutation/overexpression mediated PD-L1 upregulation through the RAS/ERK/c-JUN pathway. T cell cytotoxicity-mediated killing assay demonstrated that ERBB2 S310F mutation and overexpression induced the recovery of anti-cancer immunity by about 30% ( p < 0.05) both in RBE and HuCCT1 cells. Conclusions: The present study revealed two distinct genomic characteristics potentially involved in the recovery of anti-cancer immunity of intrahepatic cholangiocarcinoma patients: on one hand, co-mutation of ERBB family genes with ACVR2A or TGFBR2 was enriched in TGF-β pathway, contributing to MSI-H/TMB-H; on the other hand, ERBB2 might impact expression of PD-L1 by regulating the RAS/ERK/c-Jun pathways.

Immune-Associated Gene Signatures Serve as a Promising Biomarker of Immunotherapeutic Prognosis for Renal Clear Cell Carcinoma.

As the most common type of renal cell carcinoma (RCC), the renal clear cell carcinoma (ccRCC) is highly malignant and insensitive to chemotherapy or radiotherapy. Although systemic immunotherapies have been successfully applied to ccRCC in recent years, screening for patients who can benefit most from these therapies is still essential and challenging due to immunological heterogeneity of ccRCC patients. To this end, we implemented a series of deep investigation on the expression and clinic data of ccRCC from The Cancer Genome Atlas (TCGA) International Consortium for Cancer Genomics (ICGC). We identified a total of 946 immune-related genes that were differentially expressed. Among them, five independent genes, including SHC1, WNT5A, NRP1, TGFA, and IL4R, were significantly associated with survival and used to construct the immune-related prognostic differential gene signature (IRPDGs). Then the ccRCC patients were categorized into high-risk and low-risk subgroups based on the median risk score of the IRPDGs. IRPDGs subgroups displays distinct genomic and immunological characteristics. Known immunotherapy-related genes show different mutation burden, wherein the mutation rate of VHL was higher than 40% in the two IRPDGs subgroups, and SETD2 and BAP1 mutations differed most between two groups with higher frequency in the high-risk subgroup. Moreover, IRPDGs subgroups had different abundance in tumor-infiltrating immune cells (TIICs) with distinct immunotherapy efficacy. Plasma cells, regulatory cells (Tregs), follicular helper T cells (Tfh), and M0 macrophages were enriched in the high-risk group with a higher tumor immune dysfunction and rejection (TIDE) score. In contrast, the low-risk group had abundant M1 macrophages, mast cell resting and dendritic cell resting infiltrates with lower TIDE score and benefited more from immune checkpoint inhibitors (ICI) treatment. Compared with other biomarkers, such as TIDE and tumor inflammatory signatures (TIS), IRPDGs demonstrated to be a better biomarker for assessing the prognosis of ccRCC and the efficacy of ICI treatment with the promise in screening precise patients for specific immunotherapies.

Tumor Microenvironment Characterization for Assessment of Recurrence and Survival Outcome in Gastric Cancer to Predict Chemotherapy and Immunotherapy Response.

BackgroundThe tumor microenvironment (TME) is crucial for tumor recurrence, prognosis, and therapeutic responses. We comprehensively investigated the TME characterization associated with relapse and survival outcomes of gastric cancer (GC) to predict chemotherapy and immunotherapy response.MethodsA total of 2,456 GC patients with complete gene-expression data and clinical annotations from twelve cohorts were included. The TME characteristics were evaluated using three proposed computational algorithms. We then developed a TME-classifier, a TME-cluster, and a TME-based risk score for the assessment of tumor recurrence and prognosis in patients with GC to predict chemotherapy and immunotherapy response.ResultsPatients with tumor recurrence presented with inactive immunogenicity, namely, high infiltration of tumor-associated stromal cells, low infiltration of tumor-associated immunoactivated lymphocytes, high stromal score, and low immune score. The TME-classifier of 4 subtypes with distinct clinicopathology, genomic, and molecular characteristics was significantly associated with tumor recurrence (P = 0.002), disease-free survival (DFS, P <0.001), and overall survival (OS, P <0.001) adjusted by confounding variables in 1,193 stage I–III GC patients who underwent potential radical surgery. The TME cluster and TME-based risk score can also predict DFS (P <0.001) and OS (P <0.001). More importantly, we found that patients in the TMEclassifier-A, TMEclassifier-C, and TMEclassifier-D groups benefited from adjuvant chemotherapy, and patients in the TMEclassifier-B group without chemotherapy benefit responded best to pembrolizumab treatment (PD-1 inhibitor), followed by patients in the TMEclassifier-A, while patients in the C and D groups of the TMEclassifier responded poorly to immunotherapy.ConclusionWe determined that TME characterization is significantly associated with tumor recurrence and prognosis. The TME-classifier we proposed can guide individualized chemotherapy and immunotherapy decision-making.

Therapy-related myeloid neoplasms with normal karyotype show distinct genomic and clinical characteristics compared to their counterparts with abnormal karyotype.

Therapy-related myeloid neoplasms (t-MNs) are a complication of treatment with cytotoxic chemotherapy and/or radiation therapy. The majority of t-MNs show chromosomal abnormalities associated with myelodysplastic syndrome (MDS) or KMT2A rearrangements and are characterized by poor clinical outcomes. A small but substantial subset of patients have normal karyotype (NK) and their clinical characteristics and mutational profiles are not well studied. We retrospectively studied patients diagnosed with t-MN at three institutions and compared the mutational profile and survival data between t-MNs with NK and t-MNs with abnormal karyotype (AK). A total of 204 patients with t-MN were identified including 158 with AK and 46 with NK. NK t-MNs, compared to AK, were enriched for mutations in TET2 (p<0.0001), NPM1 (p<0.0001), ASXL1 (p=0.0003), SRSF2 (p<0.0001), RUNX1 (p=0.0336) and STAG2 (p=0.0099) and showed a significantly lower frequency of TP53 mutations (p<0.0001). Overall survival (OS) was significantly lower in AK t-MNs as compared to NK cases (p=0.0094). In our study, NK t-MNs showed a significantly better OS, a higher prevalence of MN-associated mutations and a lower frequency of TP53 mutations compared to their AK counterparts. The distinct clinical and mutational profile of NK t-MNs merits a separate classification.

Single Cell Deconvolution of Myeloblasts Reveals Depletion of HSCs and Expansion of Inflammatory Granulocyte-Monocyte Progenitors (GMPs) Associated with Adverse Outcomes in Chronic Myelomonocytic Leukemia (CMML)

Single Cell Deconvolution of Myeloblasts Reveals Depletion of HSCs and Expansion of Inflammatory Granulocyte-Monocyte Progenitors (GMPs) Associated with Adverse Outcomes in Chronic Myelomonocytic Leukemia (CMML)

PL03.2.A Radiotherapy is associated with a deletion signature that contributes to poor outcomes in glioma patients

Abstract BACKGROUND Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain largely unknown. MATERIAL AND METHODS We analyzed the mutational spectra following treatment with RT in whole genome or exome sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 3693 post-treatment metastatic tumors from the Hartwig Medical Foundation (HMF). RESULTS We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors (P = 3e-03, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and HMF, P = 1.5e-04 and P = 6e-16, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P &amp;lt; 2.2e-16, Kolmogorov-Smirnov test) and a lack of microhomology at breakpoints (P = 6.6e-05, paired Wilcoxon signed-rank test). Furthermore, mutational signature analysis confirmed the distinct genomic characteristics of RT-associated deletions when compared to deletions arising via homologous recombination deficiency or microsatellite instability. These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, RT resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions in IDHmut glioma (P= 1.9e-05, Fisher’s exact test). Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and HMF, P = 3.4e-02 and P &amp;lt; 1e-04, respectively; log-rank test). CONCLUSION Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Furthermore, CDKN2A homozygous deletion at recurrence may be leveraged as a promising clinical biomarker of RT-resistance in IDHmut glioma. Taken together, the identified genomic scars as a result of RT reflect a more aggressive tumor with increased levels of resistance to follow up treatments.

Activity of Wnt/PCP Regulation Pathway Classifies Patients of Low-Grade Glioma Into Molecularly Distinct Subgroups With Prognostic Difference.

Wingless/Int-1 (Wnt) signaling is one of the most well-known oncogenic pathways. Numerous studies have uncovered an aberrant expression of Wnt in cancer and its association with multiple oncogenic processes, such as cell proliferation, epithelial–mesenchymal transition (EMT), and invasiveness. Most previous studies mainly focused on the canonical branch of Wnt signaling pathway, i.e., Wnt/β-catenin signaling. The Wnt/planar cell polarity (PCP) signaling pathway, as the most recently described branch of Wnt signaling, was much less investigated in oncology research. In this study, we thoroughly characterized the activity of the Wnt/PCP regulation pathway in low-grade glioma (LGG) patients. Subtyping based on the expression pattern of the Wnt/PCP regulation pathway revealed three (C1–C3) subgroups with significant survival differences. Each group displayed distinct genomic characteristics. For instance, C1 was enriched with capicua transcriptional repressor (CIC) truncating mutations and 1p19q codel. C2 was characterized with tumor protein p53 (TP53) and ATRX chromatin remodeler (ATRX) inactivating mutations but depletion of telomerase reverse transcriptase (TERT) promoter mutations. C3 showed elevated malignancy reflected from several oncogenic characteristics, such as tumor heterogeneity and cell stemness, and demonstrated the worst survival outcome. In addition, C3 showed elevated macrophage segregation via induction of cytokines that are able to enhance the permeability of the brain–blood barrier (BBB). Lastly, we developed a prognostic model based on the risk score system. Validation indicated that our model can independently predict the prognosis of LGG patients.

Prostate epithelial genes define therapy-relevant prostate cancer molecular subtype

Background and objectivesTranscriptomic landscape of prostate cancer (PCa) shows multidimensional variability, potentially arising from the cell-of-origin, reflected in serum markers, and most importantly related to drug sensitivities. For example, Aggressive Variant Prostate Cancer (AVPC) presents low PSA per tumor burden, and characterized by de novo resistance to androgen receptor signaling inhibitors (ARIs). Understanding PCa transcriptomic complexity can provide biological insight and therapeutic guidance. However, unsupervised clustering analysis is hindered by potential confounding factors such as stromal contamination and stress-related material degradation.Materials and methodsTo focus on prostate epithelial cell-relevant heterogeneity, we defined 1,629 genes expressed by prostate epithelial cells by analyzing publicly available bulk and single- cell RNA sequencing data. Consensus clustering and CIBERSORT deconvolution were used for class discovery and proportion estimate analysis. The Cancer Genome Atlas Prostate Adenocarcinoma dataset served as a training set. The resulting clusters were analyzed in association with clinical, pathologic, and genomic characteristics and impact on survival. Serum markers PSA and PAP was analyzed to predict response to docetaxel chemotherapy in metastatic setting.ResultsWe identified two luminal subtypes and two aggressive variant subtypes of PCa: luminal A (Adipogenic/AR-active/PSA-high) (30.0%); luminal S (Secretory/PAP-high) (26.0%); AVPC-I (Immune-infiltrative) (14.7%), AVPC-M (Myc-active) (4.2%), and mixed (25.0%). AVPC-I and AVPC-M subtypes predicted to be resistant to ARI and have low PSA per tumor burden. Luminal A and AVPC-M predicted to be resistant to docetaxel and have high PSA/PAP Ratio. Metastatic PCa patients with high PSA/PAP ratio (>20) had significantly shorter progression-free survival than those with low ratio (≤20) following docetaxel chemotherapy.ConclusionWe propose four prostate adenocarcinoma subtypes with distinct transcriptomic, genomic, and pathologic characteristics. PSA/PAP ratio in advanced cancer may aid in determining which patients would benefit from maximized androgen receptor inhibition or early use of antimicrotubule agents.

Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

Distinctive genomic characteristics in POLE/POLD1-mutant cancers can potentially predict beneficial clinical outcomes in patients who receive immune checkpoint inhibitor.

BackgroundMutations in POLE /POLD1 proofreading domain can cause deficiencies in DNA repair, conferring ultramutated cancer phenotypes. Preliminary clinical studies have revealed an association between POLE/POLD1 mutations and beneficial clinical outcomes to immune checkpoint inhibitor (ICI) therapy This study aims to investigate the genomic characteristics of POLE/POLD-mutant tumors and the prognostic value of POLE/POLD mutation for ICI treatment.MethodsGenomic data of 21,074 patients with 23 cancer types were retrieved from Burning Rock variant database (BR VarDB). The prevalence and spectra of POLE and POLD1 mutations were assessed and compared with that in The Cancer Genome Atlas (TCGA) samples. The correlations of POLE/POLD1 mutation with tumor mutational burden (TMB) and microsatellite instability (MSI) were investigated. The prognostic value of POLE/POLD1 mutations was also explored in 2,487 ICI-treated patients from published studies.ResultsBR VarDB samples displayed a similar mutational prevalence of POLE (3.2% vs. 3.2%) and POLD1 (1.4% vs. 1.6%, P=0.248) versusTCGA samples, but a slightly lower frequency of POLE and POLD1 co-mutations (0.21% vs. 0.43%, P<0.001). POLE/POLD1-mutant tumors harbored increased TCT→TAT and TCG→TTG transversions, and genomic signatures associated with DNA mismatch repair (MMR) deficiency and ultra-hypermuation. Furthermore, tumors with POLE/POLD1 proofreading mutation showed a significantly higher TMB than tumors with non-proofreading mutations (P<0.01), although both possessed a higher TMB than POLE/POLD1 wild-type (WT) tumors (P<0.0001 and P<0.0001, respectively). MSI was commonly observed in tumors harboring dominant clone of POLE/POLD1 mutation (10.2%), but occurred rarely in POLE/POLD1 WT tumors (0.5%) and tumors with accumulating sub-cloned POLE/POLD1 mutation (0%). Survival analysis revealed that POLE/POLD1 mutation was not independently correlated with longer survival after adjusting for TMB and other factors (HR =0.86, P=0.372). However, patients harboring POLE/POLD1 mutation demonstrated a higher response rate than patients with POLE/ POLD1 WT tumors (35.2% vs. 19.6%, P=0.0165).ConclusionsWe delineated distinctive genomic characteristics in POLE/POLD1-mutant tumors, suggesting the potential predictive role of POLE/POLD1 mutations, especially those in the proofreading domain, for beneficial outcomes of immunotherapy. Our results also suggest that MSI caused by a loss-of-function mutation in the MMR pathway tends to result from POLE/POLD1 proofreading deficiency in POLE/POLD1-mutant tumors with MSI.

Differential genetic mutations of ectoderm, mesoderm, and endoderm-derived tumors in TCGA database

BackgroundIn terms of biological behavior, gene regulation, or signaling pathways, there is a certain similarity between tumorigenesis and embryonic development of humans. Three germ layer structure exhibits the distinct ability to form specific tissues and organs.MethodsThe present study set out to investigate the genetic mutation characteristics of germ layer differentiation-related genes using the tumor cases of the cancer genome atlas (TCGA) database.ResultsThese tumor samples were divided into three groups, including the ectoderm, mesoderm, and endoderm. Children cases less than 9 years old accounted for a larger proportion for the cases in the ectoderm and mesoderm groups; whereas the middle-aged and elderly individuals (from 50 to 89 years old) were more susceptible to tumors of endoderm. There was a better prognosis for the cases of mesoderm, especially the male with the race of White, compared with the other groups. A missense mutation was frequently detected for the cases of ectoderm and endoderm, while deletion mutation was common for that of mesoderm. We could not identify the ectoderm, mesoderm, or endoderm-specific mutated genes or variants with high mutation frequency. However, there was a relatively higher mutation incidence of endoderm markers (GATA6, FOXA2, GATA4, AFP) in the endoderm group, compared with the groups of ectoderm and mesoderm. Additionally, four members (SMO, GLI1, GLI2, GLI3) within the Hedgehog signaling pathway genes showed a relatively higher mutation rate in the endoderm group than the other two groups.ConclusionsTCGA tumors of ectoderm, mesoderm, and endoderm groups exhibit the distinct subject distribution, survival status, and genomic alteration characteristics. The synergistic mutation effect of specific genes closely related to embryonic development may contribute to the tumorigenesis of tissues or organs derived from the specific germ layers. This study provides a novel reference for exploring the functional connection between embryogenesis and tumorigenesis.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Distinctive gene and protein characteristics of extremely piezophilic Colwellia

BackgroundThe deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia, including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea.ResultsSignificant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia. Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD+ homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems.ConclusionsWe identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.

Body weight and high-fat diet are associated with epigenetic aging in female members of the BXD murine family.

DNA methylation (DNAm) is shaped by genetic and environmental factors and modulated by aging. Here, we examine interrelations between epigenetic aging, body weight (BW), and life span in 12 isogenic strains from the BXD family of mice that exhibit over twofold variation in longevity. Genome‐wide DNAm was assayed in 70 liver specimens from predominantly female cases, 6–25 months old, that were maintained on normal chow or high‐fat diet (HFD). We defined subsets of CpG regions associated with age, BW at young adulthood, and strain‐by‐diet‐dependent life span. These age‐associated differentially methylated CpG regions (age‐DMRs) featured distinct genomic characteristics, with DNAm gains over time occurring in sites such as promoters and exons that have high CpG density and low average methylation. CpG regions associated with BW were enriched in introns, tended to have lower methylation in mice with higher BW, and were inversely correlated with gene expression (i.e., higher mRNA levels in mice with higher BW). CpG regions associated with life span were linked to genes involved in life span modulation, including the telomerase reverse transcriptase gene, Tert, which had both lower methylation and higher expression in long‐lived strains. An epigenetic clock defined from age‐DMRs revealed accelerated aging in mice belonging to strains with shorter life spans. Both higher BW and the HFD were associated with accelerated epigenetic aging. Our results highlight the age‐accelerating effect of heavier BW. Furthermore, we demonstrate that the measure of epigenetic aging derived from age‐DMRs can predict genotype and diet‐induced differences in life span among female BXD members.

Precise Populations’ Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 103 to 1.8 × 104 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems.

CDC20 is a novel potential therapeutic target in NF1-mutant melanoma.

e22075 Background: NF1-mutant melanoma (NF1-MT) is a distinct molecular subtype with worse overall prognosis than NF1-wild melanoma (NF1-WT). Despite its prevalence in 20% of patients and associated clinical disadvantage, there is no specific targeted therapy available to treat NF1-MT as its biological impact remains understudied. We tested the hypothesis that NF1-MT possess distinct genomic and transcriptomic characteristics from NF1-WT, which can potentially inform therapeutic innovation. Methods: Tumor DNA from metastatic melanoma (MM) patients treated with checkpoint inhibition at NYU was genotyped using exome sequencing. Microenvironment-related transcripts (n = 770) were then compared in NF1-MT versus NF1-WT using unsupervised clustering, differential gene expression, and gene set enrichment analyses. Verification of genomic and transcriptional findings was performed using qPCR and immunohistochemistry (IHC). TCGA melanoma (SKCM) data were analyzed as an independent cohort. Results: Pathogenic NF1 mutations were detected in 31/121 (27%) cases and were significantly associated with higher tumor mutational burden (P = 0.02). NF1-MT showed recurrent homozygous deletion in the membrane regulatory gene SYT1 and amplifications in oncogenes including CCND1 and MDM2, verified by qPCR. NF1-MT showed upregulation of proliferation with significantly increased expression of Ki-67 and cell division cycle protein 20 CDC20 (P = 0.03) validated by IHC (P = 0.03). Cell cycle was the top enriched pathway in NF1-MT (P = 0.003, FDR = 0.04). Analysis of TCGA melanoma data (n = 440) corroborated upregulation of MKI67 and CDC20 in NF1-MT melanoma (P = 0.01, P = 0.03). Conclusions: NF1-MT is associated with a hyper-proliferative tumor phenotype that cannot be only attributed to MAP kinase activation. Our data suggest that NF1-MT may be vulnerable to pharmacological inhibitors of cell cycle progression and proliferation, such as those targeting CDC20 or CDK4/6.