Gene Copy Number Variation Research Articles

Association between the Prognosis of Stomach Adenocarcinoma (STAD) Patients and the Hotspots of Gene Copy Number Variation

Stomach Adenocarcinoma, with complex mechanisms about progression, has the biggest proportion in Stomach Cancer, which is one of the most common causes of death in the world. It is widely acknowledged that gene mutations have relationship with Stomach Adenocarcinoma. Copy number variation (CNV), producing identical sequence in a large amount, is associated with Stomach Adenocarcinoma. There are some hotspots of gene mutations in chromosome, CNV is no exception. Their positions may potentially have association with prognosis of patients, which is poorly understood. In this research, windows with fixed length were set; the number of start and end points of CNV in the windows were calculated. The rates of Poisson Distribution in different windows were calculated and the hotspots were found. Then by using proportional hazards model, The author learned the significance of the impact of CNV in certain areas on prognosis with data from TCGA. After analyzing 180 windows, the author found 14 significant windows. These discoveries may reveal new methods of targeted therapy, promoting the precise treatment of Stomach Adenocarcinoma patients

Microbial Diversity Biased Estimation Caused by Intragenomic Heterogeneity and Interspecific Conservation of 16S rRNA Genes.

The 16S rRNA gene has been extensively used as a molecular marker to explore evolutionary relationships and profile microbial composition throughout various environments. Despite its convenience and prevalence, limitations are inevitable. Variable copy numbers, intragenomic heterogeneity, and low taxonomic resolution have caused biases in estimating microbial diversity. Here, analysis of 24,248 complete prokaryotic genomes indicated that the 16S rRNA gene copy number ranged from 1 to 37 in bacteria and 1 to 5 in archaea, and intragenomic heterogeneity was observed in 60% of prokaryotic genomes, most of which were below 1%. The overestimation of microbial diversity caused by intragenomic variation and the underestimation introduced by interspecific conservation were calculated when using full-length or partial 16S rRNA genes. Results showed that, at the 100% threshold, microbial diversity could be overestimated by as much as 156.5% when using the full-length gene. The V4 to V5 region-based analyses introduced the lowest overestimation rate (4.4%) but exhibited slightly lower species resolution than other variable regions under the 97% threshold. For different variable regions, appropriate thresholds rather than the canonical value 97% were proposed for minimizing the risk of splitting a single genome into multiple clusters and lumping together different species into the same cluster. This study has not only updated the 16S rRNA gene copy number and intragenomic variation information for the currently available prokaryotic genomes, but also elucidated the biases in estimating prokaryotic diversity with quantitative data, providing references for choosing amplified regions and clustering thresholds in microbial community surveys. IMPORTANCE Microbial diversity is typically analyzed using marker gene-based methods, of which 16S rRNA gene sequencing is the most widely used approach. However, obtaining an accurate estimation of microbial diversity remains a challenge, due to the intragenomic variation and low taxonomic resolution of 16S rRNA genes. Comprehensive examination of the bias in estimating such prokaryotic diversity using 16S rRNA genes within ever-increasing prokaryotic genomes highlights the importance of the choice of sequencing regions and clustering thresholds based on the specific research objectives.

Network diffusion-based approach for survival prediction and identification of biomarkers using multi-omics data of papillary renal cell carcinoma

Identification of cancer subtypes based on molecular knowledge is crucial for improving the patient diagnosis, prognosis, and treatment. In this work, we integrated copy number variations (CNVs) and transcriptomic data of Kidney Papillary Renal Cell Carcinoma (KIRP) using a network diffusion strategy to stratify cancers into clinically and biologically relevant subtypes. We constructed GeneNet, a KIRP specific gene expression network from RNA-seq data. The copy number variation data was projected onto GeneNet and propagated on the network for clustering. We identified robust subtypes that are biologically informative and significantly associated with patient survival, tumor stage and clinical subtypes of KIRP. We performed a Singular Value Decomposition (SVD) analysis of KIRP subtypes, which revealed the genes/silent players related to poor survival. A differential gene expression analysis between subtypes showed that genes related to immune, extracellular matrix organization, and genomic instability are upregulated in the poor survival group. Overall, the network-based approach revealed the molecular subtypes of KIRP and captured the relationship between gene expression and CNVs. This framework can be further expanded to integrate other omics data.

Copy number variation of gasdermin D gene is associated with atrial fibrillation-related thromboembolic stroke.

Atrial fibrillation (AF) is one of the major causes of ischaemic stroke. In addition to clinical risk evaluated by the CHA2DS2-VASC score, the impact of genetic factors on the risk of AF-related thromboembolic stroke has been largely unknown. We found several copy number variations (CNVs) in novel genes that were associated with thromboembolic stroke risk in our AF patients by genome-wide approach. Among them, the gasdermin D (GSDMD) gene was related to inflammation. We aimed to test whether GSDMD deletion was associated with AF-related stroke. A total of 400 patients with documented non-familial AF were selected, of which 100 patients were diagnosed with ischaemic stroke. The baseline characteristics of age, sex, valvular heart disease, coronary artery disease, heart failure, and CHA2DS2-VASc scores were not statistically different between cases and controls. We found that individuals who carried GSDMD homozygous deletion genotype had a higher risk for ischaemic stroke (odds ratio 2.195; 95% confidence interval, 1.24-3.90; P = 0.007), even adjusted by CHA2DS2-VASc scores. We also validated the association of GSDMD with AF stroke in a large Caucasian population (UK Biobank). We found a link between the homozygous deletion of the GSDMD gene and an increased risk of stroke in patients with AF. This may implicate the use of therapy targeting GSDMD in the prevention of ischaemic stroke for AF patients.

Whole-genome resequencing in the sea louse Caligus rogercresseyi uncovers gene duplications and copy number variants associated with pesticide resistance

The sea louse Caligus rogercresseyi is a marine ectoparasite that constitutes one of the major threats to the salmon farming industry, where the primary control strategy is the use of delousing drugs through immersion treatments. The emergence of pharmacological resistance in this copepodid species has previously been described using transcriptome data. However, the molecular mechanisms underlying chromosome rearrangements have not yet been explored. This study aimed to identify structural genomic variations and gene expression in C. rogercresseyi associated with pesticide sensitivity. In this study, genome resequencing was conducted using Oxford Nanopore Technology on lice strains with contrasting sensitivity to azamethiphos to detect genome duplications. Transcriptome profiling of putative gene duplications was performed by Illumina sequencing. Copy Number Variants (CNVs) were identified through comparative coverage, and collinear/tandem gene duplications over all the chromosomal regions by sequence homology. Duplications or CNVs in functional genes were primarily identified in transposable elements and genes related to the drug response, with differential expression values calculated by RNA-seq analyses of the same strains. Notably, differentially duplicated genes were found in coding regions related to cuticle proteins, suggesting that a putative resistance mechanism may be associated with cuticular structure formation and the proteins involved. Collectively, the results revealed that the intensive use of pesticides on sea lice populations increases the frequency of gene duplication, expanding the molecular elements involved in drug response. This study is the first to report an association between genome rearrangements and pharmacological resistance in sea lice populations.

Copy number variation in tRNA isodecoder genes impairs mammalian development and balanced translation

The number of tRNA isodecoders has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-Seq, RNA-seq, ribo-profiling and proteomics we observed distinct molecular consequences of single tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoder genes buffer the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least six tRNA-Phe alleles is required for embryonic viability and tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA genes is required to buffer translation and viability in mammals.

Salivary amylase gene (AMY1) copy number variation has only a minor correlation with body composition in Chinese adults.

According to the WHO, about 39% of the global adult population were overweight or obese in 2016. Obesity has high heritability, with more than 1000 variants so far identified. There have been reports indicating that salivary amylase gene (AMY1) copy number was one of these variants, yet its association with obesity remains controversial. Our research aimed to provide more evidence on the relationship of AMY1 copy number variation (CNV) with body mass index (BMI) and body composition. We recruited 133 Chinese adults (65 males, 68 females, 18-25years old) with normal fasting blood glucose and blood pressure levels. 19 males were selected for a 10-week intervention to change body composition. After anthropometric measurements, BMI was calculated, and body composition was measured using dual energy X-ray absorptiometry (DEXA). For the 19 selected participants, we collected their height, weight, and body composition data one more time after intervention. All participants were required to leave their saliva samples and their AMY1 copy number was determined by real-time fluorescence quantitative PCR. We failed to find any significant difference in BMI and body composition between different copy number groups. Only a weak correlation was found between body muscle mass and body fat mass. After adjusted for height and weight, AMY1 CNV explained 4.83% of the variance and one single increase in AMY1 CNV can increase 0.214kg of the body muscle mass, while one single increase in AMY1 CNV can decrease 0.217kg of the body fat mass and explained 4.69% of the variance. As a genetic factor, the AMY1 gene copy number variation has only a minor correlation with BMI and body composition, and its effect can easily be hidden by other factors such as individual diet and exercise habit.

HOXA9 gene promotor methylation and copy number variation of SOX2 and HV2 genes in cell free DNA: A potential diagnostic panel for non-small cell lung cancer

BackgroundMost cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer.MethodsWe investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls.ResultsMethylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity.ConclusionsIn this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes.

CD86 Is Associated with Immune Infiltration and Immunotherapy Signatures in AML and Promotes Its Progression.

Cluster of differentiation 86 (CD86), also known as B7-2, is a molecule expressed on antigen-presenting cells that provides the costimulatory signals required for T cell activation and survival. CD86 binds to two ligands on the surface of T cells: the antigen CD28 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). By binding to CD28, CD86-together with CD80-promotes the participation of T cells in the antigen presentation process. However, the interrelationships among CD86, immunotherapy, and immune infiltration in acute myeloid leukemia (AML) are unclear. The immunological effects of CD86 in various cancers (including on chemokines, immunostimulators, MHC, and receptors) were evaluated through a pan-cancer analysis using TCGA and GEO databases. The relationship between CD86 expression and mononucleotide variation, gene copy number variation, methylation, immune checkpoint blockers (ICBs), and T-cell inflammation score in AML was subsequently examined. ESTIMATE and limma packages were used to identify genes at the intersection of CD86 with StromalScore and ImmuneScore. Subsequently, GO/KEGG and PPI network analyses were performed. The immune risk score (IRS) model was constructed, and the validation set was used for verification. The predictive value was compared with the TIDE score. CD86 was overexpressed in many cancers, and its overexpression was associated with a poor prognosis. CD86 expression was positively correlated with the expression of CTLA4, PDCD1LG2, IDO1, HAVCR2, and other genes and negatively correlated with CD86 methylation. The expression of CD86 in AML cell lines was detected by QRT-PCR and Western blot, and the results showed that CD86 was overexpressed in AML cell lines. Immune infiltration assays showed that CD86 expression was positively correlated with CD8 T cell, Dendritic cell, macrophage, NK cell, and Th1_cell and also with immune examination site, immune regulation, immunotherapy response, and TIICs. ssGSEA showed that CD86 was enriched in immune-related pathways, and CD86 expression was correlated with mutations in the genes RB1, ERBB2, and FANCC, which are associated with responses to radiotherapy and chemotherapy. The IRS score performed better than the TIDE website score. CD86 appears to participate in immune invasion in AML and is an important player in the tumor microenvironment in this malignancy. At the same time, the IRS score developed by us has a good effect and may provide some support for the diagnosis of AML. Thus, CD86 may serve as a potential target for AML immunotherapy.

Whole-exome mutational landscape and molecular marker study in mucinous and clear cell ovarian cancer cell lines 3AO and ES2

BackgroundOvarian cancer is one of the most lethal cancers in women because it is often diagnosed at an advanced stage. The molecular markers investigated thus far have been unsatisfactory.MethodsWe performed whole-exome sequencing on the human ovarian cancer cell lines 3AO and ES2 and the normal ovarian epithelial cell line IOSE-80. Molecular markers of ovarian cancer were screened from shared mutation genes and copy number variation genes in the 6q21-qter region.ResultsWe found that missense mutations were the most common mutations in the gene (93%). The MUC12, FLG and MUC16 genes were highly mutated in 3AO and ES2 cells. Copy number amplification occurred mainly in 4p16.1 and 11q14.3, and copy number deletions occurred in 4q34.3 and 18p11.21. A total of 23 hub genes were screened, of which 16 were closely related to the survival of ovarian cancer patients. The three genes CCDC170, THBS2 and COL14A1 are most significantly correlated with the survival and prognosis of ovarian cancer. In particular, the overall survival of ovarian cancer patients with high CCDC170 gene expression was significantly prolonged (P < 0.001). The expression of CCDC170 in normal tissues was significantly higher than that in ovarian cancer tissues (P < 0.05), and its expression was significantly decreased in advanced ovarian cancer. Western blotting and immunofluorescence assays also showed that the expression of CCDC170 in ovarian cancer cells was significantly lower than that in normal cells (P < 0.001, P < 0.01).ConclusionsCCDC170 is expected to become a new diagnostic molecular target and prognostic indicator for ovarian cancer patients, which can provide new ideas for the design of antitumor drugs.

Abstract 5696: A prognostic machine learning model for early breast cancer which combines clinical and genetic data in patients treated with neo/adjuvant chemotherapy

Abstract Background: Accurate modelling of the impact of patient-specific features and cancer treatments on survival allows the assignment of targeted therapy. There has not been any effort to build a multi-source model for the survival analysis of breast cancer. We show in this study a prognostic model, which integrates genetic (DNA), clinical and therapy inputs to predict survival for early breast cancer (stages 1-3) for all breast cancer subtypes. Methods: We used a data-driven Random Survival Forest approach, a statistical non-parametric ensemble learning method, that incorporates censor and time-to-event data. The learning is performed by creating numerous decision trees and selecting the model based on the correct responses in unseen data. We used The Cancer Genome Atlas Breast Cancer (TCGA) dataset and observed improvements in the accuracy when more sources of data were used, in line with the previous research. Integrating the impact of non-silent somatic tumor mutations (whole exome) and gene copy number variation (CNV) were analyzed on all mutations and per particular mutation. Results: Data from 1096 women with stage 1-3 early breast cancer were inputs to the model n=437 ER+ve HER2-ve, n=123 HER2+ve ER+ve, n=40 HER2+ ER-ve and n=126 TNBC. Pathological stage 1, n=183; stage 2, n=620; and stage 3, n=249. The following chemotherapy and hormonal treatments were used in the analysis: anthracycline, taxanes, platinum, alkylating and anti-metabolite agents, anti-oestrogen, aromatase inhibitors, ovarian suppression and HER2 antibody treatment. The model accuracy for predicting survival for early breast cancer using only clinical data was 0.78 for Area Under Curve (AUC) and c-index. The predictive accuracy improved stepwise by adding hormone, genetic and treatment data to AUC of 0.86 and c-index to 0.85. We observed the same trend if the proportion of test data increased from 0.25 to 0.75. Changes in median genes FGFR2 and CDKN2A copy number were strongly prognostic with p=0.0001 and p=0.002, and weaker signals for CBFB p=0.05, HRAS p=0.06, AKT p=0.07. Conclusion: Using public datasets, we developed a predictive survival model for an individual with early breast cancer up to 5 years from diagnosis using multi-source and patient-specific data. We show that using this approach for survival analysis yields good accuracy. Citation Format: Aidan (Amanzhol) Kubeyev, Andrea Giorni, Prabu Siva, Luiz Silva, Jordan Laurie, Matthew Foster, Matthew Griffiths, Uzma Asghar. A prognostic machine learning model for early breast cancer which combines clinical and genetic data in patients treated with neo/adjuvant chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5696.

Abstract 2494: Heterogeneous tumor-driven immune landscapes between lung adenocarcinomas in ever and never-smokers

Abstract To depict the genomic and immune landscapes of lung adenocarcinomas (ADCs) in ever and never-smokers and guide the design of immunotherapy strategies, we applied multiple deconvolution methods that allow the identification of immune cell infiltration and immune pathway enrichment using a whole exome and transcriptome sequencing-based approach in 145 patient-matched lung ADC and non-malignant lung parenchymal tissue pairs from ever and never smokers in our in-house cohort. Results were subsequently validated by an external lung ADC cohort, including 108 never-smokers and 61 ever-smokers. Firstly, we showed that ever-smokers had an increased abundance of neutrophils and macrophage infiltration, an elevated antigen processing and presentation capacity, and activated T-cell-mediated immunity. Secondly, ever-smokers revealed elevated macrophages in acinar ADCs, decreased mast cells in lepidic ADCs, and increased neutrophils in papillary ADCs, suggesting the tumor-driven immune characteristic discrepancies among various pathologic subtypes of ADC. Thirdly, we identified mutational disparities in tumor mutation burdens, subclonal single nucleotide variants, and the predicted and expressed neoantigens between ever and never-smokers. The mutant gene KRAS preferably occurred in ever-smokers, further contributing to the infiltration of macrophages and expression of cytokines. The copy number variations of immune-related genes (NFIB, TNFAIP3) were identified as conferring to the dysregulated immune environment in ever-smokers. Moreover, a higher genomic instability index was observed in ever-smokers (p = 0.008). Fourthly, the neoantigen was significantly increased in ever-smoker lung ADCs without nodal disease. While DNA immunoediting preferably occurred in never-smokers. Notably, in lung ADC patients with nodal disease, ever-smokers exhibited slightly more homogenous mutations (p = 0.094), consistent with the evolutionary bottleneck during cancer progression. Finally, the lower immunoediting and higher microenvironment scores significantly determined the more prolonged progression-free survival in never-smokers (p &amp;lt; 0.050). Our multiscale genomic and immune profiling analyses thus offer valuable knowledge of tumor-driven immune disparity arising from smoking and provide a robust solution to the rational design of immune therapies. Citation Format: Xiaozheng Kang, Xin Wang, Liyan Ji, Bingfa Yan, Xuan Gao, Xuefeng Xia, Xingsheng Hu. Heterogeneous tumor-driven immune landscapes between lung adenocarcinomas in ever and never-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2494.

Abstract 5861: Tumor cell-intrinsic factors promoting fibrotic tumor microenvironment in gastric cancer

Abstract Background: Gastric cancer (GC) is one of the leading causes of death in the world. Evidence has shown over the past decade that cancer-associated​ fibroblasts (CAFs) play essential roles in various types of cancers, including GC, such as tumorigenesis, therapy resistance, and immunosuppression, which contribute to the progress of tumors. Although GC tumors are known to be enriched with stroma, the molecular mechanisms of how GC tumors foster the fibrotic milieu is still largely unknown. Here we analyzed a large-scale genomic and transcriptomic dataset of GC to identify GC tumor cell-intrinsic factors for developing fibroblast-rich microenvironments​. Methods: We analyzed GC patient samples with gene expression data and copy number variation profiles from the Asian Cancer Research Group (ACRG) (N = 271). The microenvironment cell populations-counter (MCP-counter) method was used for transcriptome deconvolution to estimate the population of fibroblasts and immune cells in GC tumors. Genes with q value &amp;lt; 0.05 and fold change &amp;gt; 1.5 were defined as differentially expressed genes (DEGs) and identified using the Subio Platform. The copy number amplified genes found in more than 3% of the samples were selected as amplified genes. Pathway and process enrichment analysis and protein-protein interaction analysis were performed using Metascape. Results: We analyzed MCP-counter outputs and observed a fibroblast-enriched population with few infiltrations of anti-tumor immune cells, such as CD8 T cells and cytotoxic T cells in GC patients, suggesting the immunosuppressive role of CAFs in the GC tumor. By analyzing 933 DEGs upregulated in fibroblast-high patients, we found 96 genes with frequent copy number amplification that were not listed on the curated stroma and extracellular matrix-related genes and thus potentially tumor cell-intrinsic genes correlated with the abundance of the stroma. Pathway and protein-interaction analyses revealed that G protein-coupled receptor (GPCR) signaling pathway was enriched in these genes, including HTR2A, AGTR1, FZD1, CALCRL, and NBEA with strong correlations between fibroblast score (Spearman's r &amp;gt; 0.55), implying that these signaling in GC tumor cells contribute to promoting fibroblast-enriched microenvironment. Conclusions: Bioinformatic analyses of a publicly available genomic and transcriptomic dataset revealed a fibroblast-rich and immune-excluded population in GC patients. Several GPCR signaling in tumor cells were strongly correlated with fibroblast abundance in tumors, and these signaling pathways have potential roles in promoting the stroma-rich GC microenvironment. Citation Format: Takashi Semba, Yiling Tong, Feng Wei, Atsuko Yonemura, Tadahito Yasuda, Tomoyuki Uchihara, Huaito Wang, Hideo Baba, Takatsugu Ishimoto. Tumor cell-intrinsic factors promoting fibrotic tumor microenvironment in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5861.

Abstract 883: Glutaminases expression and viral infection as potential prognostic factors in cervical, head and neck and liver cancers

Abstract Human Papilloma virus (HPV)-associated Cervical squamous cell carcinoma (CESC-HPV(+)), HPV-associated Head and Neck squamous cell carcinoma (HNSCC-HPV(+)), and Hepatitis Virus B/C (HBV/HCV)-associated Liver hepatocellular carcinoma (LIHC-HBV(+)/LIHC-HCV(+)) present metabolic changes during viral transformation to adjust energy demands of transformed cells. Glutamine is used as an important energy and carbon source by cancer cells through its degradation (glutaminolysis). This latter is regulated by Glutaminases 1 and 2 (GLS1/GLS2) that convert glutamine in glutamate. GLS1/2 expression is altered both in HNSCC and LIHC but it is undetermined in CESC. We aimed to indagate if the expression of GLS1/GLS2 genes and/or status of infection by HPV, HBV, and HCV, are associated to the overall survival (OS) in CESC, HNSCC and LIHC. The Cancer Genome Atlas (TCGA) was mined and both gene expression and copy number variation (CNV) of GLS1/GLS2 was analyzed across the CESC, HNSCC and LIHC cohorts. Changes in gene expression were determined by analyzing the Log2FC and by applying statistical analysis (Welch’s T-test or One-Way ANOVA). To learn if the genomic profile was associated with phenotype, gene status was correlated with demographic and clinical data such as age group, ethnicity, race, gender, BMI, viral subtype, histologic grade, clinical stage, and tobacco/alcohol consumption. We applyed chi-squared, one-way ANOVA, and Welch’s T-tests for those assays. Kaplan Meier (K-M) plots were obtained to figure out the association of genomic and phenotypic data with OS. Our results showed that GLS1/GLS2 had no CNVs across the analyzed cohorts. However, GLS1 was upregulated in cancerous tissues both in HNSCC and LIHC (p&amp;lt;0.05, Log2FC&amp;gt;2) whereas GLS2 was downregulated in cancerous tissues in LIHC (p&amp;lt;0.05, Log2FC=-2). Likewise, GLS1 was upregulated both in HNSCC-HPV(-) and LIHC-HBV/HCV(+) compared with HNSCC-HPV(+) and LIHC-HBV/HCV(-), respectively (p&amp;lt;0.05). Also, GLS2 was over-expressed in CESC-HPV(+) and HNSCC-HPV(+) patients, compared with CESC-HPV(-) and HNSCC-HPV(-) patients, respectively (p&amp;lt;0.05). In the other hand, GLS2 was under-expressed in LIHC-HBV/HCV(+) compared with LIHC-HBV/HCV(-) patients (p&amp;lt;0.05, Log2FC = -2.5). Finally, K-M plots showed that GLS2 expression is associated with OS in CESC patients whereas the HPV subtypes and HBV/HCV infection were associated with OS in HNSCC and LIHC patients, respectively (p&amp;lt;0.05). We concluded that the expression of glutaminases is abnormal in patients with CESC, HNSCC, and LIHC and it depends on the status of viral infection. Since the GLS2 expression and viral infection affects the OS in the analyzed tumors, our research provides with potential prognostic markers of CESC, HNSCC and LIHC that should be validated in future studies. Citation Format: Livisu Pajares Rojas, Claudia Machicado Rivero. Glutaminases expression and viral infection as potential prognostic factors in cervical, head and neck and liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 883.

Abstract 2589: TROP2 gene copy number variations are a hallmark of glioblastoma and MEN-2A cancers

Abstract TROP2 mutations are not required to trigger activation of tumor growth and metastasis. However, expression of Trop-2 is affected by DNA methylation in choriocarcinomas, i.e. trophoblast-derived malignancies, and in lung adenocarcinomas, suggesting that loss of TROP2 expression may provide a selective advantage in specific tumor types. Intriguingly, amplification of the TROP1/EPCAM and TROP2/TACSTD2 genes was found to be driven by alterations of DNA methylation, with parallel LOH in sister cell clones. Consistent, mutations of TP53 were shown to induce amplification of TROP genes through loss of DNA methylation. Hence, we investigated whether LOH/DNA methylation of the TROP2 gene and gene amplification are selectively associated with specific tumor types. Our findings indicate that TROP2 amplification occurs in a subgroup of aggressive glioblastomas (15.7%, n=3/19), suggesting impact on malignant progression. On the other hand, TROP2 LOH was shown to occur in the majority of low-grade gliomas (80%, n=4/5). Investigation on the TROP2 gene in MEN-2A revealed that LOH of TROP2 was somatically acquired in 25% of phaechromocytomas and medullary thyroid carcinomas (n=6/24), but did not appear as a heritable trait in the germline. TROP2 gene copy number variations were rarely detected in other cancer types, suggesting tumor subtype-specific impact. Citation Format: Saverio Alberti, Marco Trerotola, Antonino Moschella, Emanuela Guerra. TROP2 gene copy number variations are a hallmark of glioblastoma and MEN-2A cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2589.

Abstract 2312: Single-cell low pass whole genome sequencing for copy-number profiling in circulating tumor cells from prostate, non-small cell lung and breast cancers

Abstract Background: Circulating tumor cells (CTCs) have been considered as an alternative to tissue biopsy for providing both germline-specific and tumor-derived genetic variants. Copy number variations (CNVs) play an important role in molding the genomes of cancers and have been shown the clinical value for prognostic signature and therapeutic target. The current study aimed to evaluate CNVs at single-cell resolution and explore the potential clinical utility of tracking this key mechanism of cancer progression. Methods: Blood samples were collected in Streck tubes from 40 stage II-IV cancer patients (5 prostate cancer, 15 non-small lung cancer (NSCLC), 20 breast cancer (BC)) before treatment. CTCs was identified as cells containing an intact nucleus, without CD45 expression, and with cytokeratin expression. Genomic analysis of chromosomal CNVs by low pass whole genome sequencing (Lp-WGS) and number of large-scale transitions (LSTs) calculation was performed in the CTCs. Results: A total of 9 CTCs from 4 prostate cancer, 70 CTCs from 7 NSCLC, 22 CTCs from 9 breast cancer patients, representing a range of sizes, shapes, and protein expression levels of cytokeratin were identified. A total of 84 individual CTCs were successfully sequenced from 20 patients (1-6 CTCs/patient). The selected highlights for gene CNVs include: 1) CXCR4 and ATM were detected as loss on single cell CTC (scCTC) level in BC; 2) MED12, an important regulator for maintaining accurate cytokinesis and survival, was detected as loss on scCTC level in NSCLC. A wide range of LST scores were observed among the 20 patient samples. Cell morphology analysis, phenotypic heterogeneity biomarker analysis and homologous recombination deficiency (HRD) biomarker analysis using a previously developed HRD classifier1 trained on genomic markers is still ongoing. Conclusion: Our data demonstrated that Lp-WGS of DNA from CTCs can detect CNVs loss and assess disease heterogeneity at the single-cell level. Since genome-wide CNV profiles can be used to characterize genomic instability, it is anticipated that the integrative analysis of morphology and genomics in CTCs can potentially complement tissue and/or ctDNA analysis to determine HRD, enabling in-depth investigation of tumor heterogeneity and individualized clinical assessment. Citation Format: Danyi Wang, Giuseppe Locatelli, Ioannis Gounaris, Juergen Scheuenpflug, Zheng Feng. Single-cell low pass whole genome sequencing for copy-number profiling in circulating tumor cells from prostate, non-small cell lung and breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2312.

Copy number variations of cytochrome P450 genes in Kinh Vietnamese.

The cytochrome P450 (CYP450) family is well known as a major group of drug metabolizing enzymes. The polymorphism of CYP450 genes is the main factor having an impact on the interindividual difference in drug response, including drug efficacy and drug safety. The single nucleotide polymorphism (SNPs) of Vietnamese Kinh has been widely studied, but information about the copy number variations (CNVs) of other CYP450 genes is still unknown. To identify the CNV variability of CYP450 in 154 healthy unrelated Kinh Vietnamese, except eCYP2D6, which was previously reported. Multiplex Ligation-Dependent Probe Amplification (MLPA) was applied for determination of copy number of 10 CYP450 genes. Later, PCR or quantitative PCR (qPCR) was used to confirm the detected CNVs in randomly chosen subjects. Of the 154 subjects, along with CYP2D6, 4 other CYP450 genes showed CNVs including duplications (CYP1B1), deletions (CYP2A6 and CYP2C9), and both duplications and deletions (CYP2E1). Among these, CYP2A6 exhibited the greatest frequency of CNVs compared with other CYP450, in which CYP2A6Del accounted for 11%. Meanwhile, allele CYP2E1Del showed the lowest frequency with only 0.3%. The present study provides new insight into CYP450 CNVs in the Kinh Vietnamese cohort. Our data have contributed to genetic profiling of CYP450 CNVs in Vietnam, which would be helpful for facilitating implementation of pharmacogenetics in drug dosing adjustment in Vietnam.

PD46-05 GENETIC ABERRATIONS IDENTIFIED IN FOCAL BARBOTAGES– a COMPLEMENTARY DIAGNOSTIC TOOL IN UPPER TRACT UROTHELIAL CARCINOMA (UTUC)

You have accessJournal of UrologyCME1 Apr 2023PD46-05 GENETIC ABERRATIONS IDENTIFIED IN FOCAL BARBOTAGES– a COMPLEMENTARY DIAGNOSTIC TOOL IN UPPER TRACT UROTHELIAL CARCINOMA (UTUC) Tomas Axelsson, Filip Sydén, Jesper Eisfeldt, Ylva Eriksson, Gustav Göthner Lundberg, Alexandra Grahn, Emma Tham, and Marianne Brehmer Tomas AxelssonTomas Axelsson More articles by this author , Filip SydénFilip Sydén More articles by this author , Jesper EisfeldtJesper Eisfeldt More articles by this author , Ylva ErikssonYlva Eriksson More articles by this author , Gustav Göthner LundbergGustav Göthner Lundberg More articles by this author , Alexandra GrahnAlexandra Grahn More articles by this author , Emma ThamEmma Tham More articles by this author , and Marianne BrehmerMarianne Brehmer More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: UTUC is divided into low- and high risk UTUC. Despite radical surgery, 10-20% in the low-risk and 50% of the patients in the high-risk group respectively, recur and die from UTUC. Although tumour stage and grade are known prognostic factors, the mechanisms of recurrence in each risk group respectively are not entirely known. In a previous study of genetic analysis of tumour tissue from nephroureterectomy (NU) we showed that gene mutations in some cases had a stronger prognostic value than histopathology. We have also previously shown that cytology taken as focal barbotage, at ureterorenoscopy (URS), renders good diagnostic material. The aim of this study was to investigate if gene analysis of focal barbotages obtained at URS is possible. In addition, we tried to identify gene mutations and copy number variations (CNV) that might signify a more aggressive tumour. METHODS: This prospective study included 41 patients with UTUC confirmed at URS from 2018 to 2021. Patients were followed until October 2022. At URS, barbotage specimens from the ureter and renal pelvis were collected and sent for cytology analysis and for gene sequencing. When possible, tumour biopsies were taken. Final tumour grades (WHO-classifications 1999 and 2004) were determined from cytology and/or biopsy, or from radical NU samples when available. A total of 41 barbotage samples were sent for genetic analysis. Next generation sequencing using a 388-gene panel was performed. Manual filtering of the sequencing data from each sample was performed to identify possible pathogenic mutations. CNV was analysed using WisecondorX. RESULTS: 41 patients, 26 men and 15 women were included. Mean age at inclusion was 75 years. In 14 patients the tumour was low grade (8 G1, 5 G2 and one unclear) and in 27 patients the tumour was high grade (14 G2 and 13 G3). During the follow-up period, 5 patients died from UTUC and 7 patients had tumour progression. Out of 41 focal barbotages analysed we identified known and suspected pathogenic mutations in 39 and CNVs in 32. Known pathogenic mutations in the fibroblast growth receptor 3 (FGFR3) gene were common in grade 1 and 2 tumours but not present in any G3 tumour. No patients with a FGFR3 mutation died during follow-up. Seven patients had a pathogenic mutation in tumour protein 53 (TP53), 6 of these had high grade tumours, 2 of whom died from UTUC during follow-up. One patient with a TP53 mutation had a low grade, G1-tumor, that progressed with metastasis during follow up. CONCLUSIONS: Gene mutations and CNVs can be identified in focal barbotage samples obtained at URS and may be complementary to cytologic analysis. The technique may be helpful in identifying low risk tumours that might be more at risk for disease progression or death than previously thought. Source of Funding: Nothing to declare © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1171 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomas Axelsson More articles by this author Filip Sydén More articles by this author Jesper Eisfeldt More articles by this author Ylva Eriksson More articles by this author Gustav Göthner Lundberg More articles by this author Alexandra Grahn More articles by this author Emma Tham More articles by this author Marianne Brehmer More articles by this author Expand All Advertisement PDF downloadLoading ...

Copy number variations and protein expression of Cyclin E1 and Epithelial cell transforming sequence 2 genes predict the chemotherapeutic response in patients with serous ovarian carcinoma.

Serous ovarian carcinoma (SOC) is characterized by extreme genomic instability, chromosomal rearrangements and copy number variations (CNVs) leading to the development of early metastasis and chemo-resistance. The present study was designed to observe the role of CNVs of Cyclin E1 (CCNE1) and Epithelial cell transforming sequence- 2 (ECT2) genes and their encoded proteins in predicting the chemotherapeutic response in SOC patients. This observational analytical study was conducted at University of Health Sciences, Lahore, Pakistan from December 2019 till June 2022.The study included twenty-five SOC patients with resectable ovarian tumors and twenty-five control subjects. The patients were followed-up for six months for their response to chemotherapy. The CNVs in CCNE1 and ECT-2 genes were determined by real time PCR while serum levels of encoded proteins were determined in controls and cases, before and after six months of treatment, through ELISA. The response to chemotherapy was categorized as sensitive or resistant based on serum CA-125 levels and radiological scans. The copy number variations in CCNE1 and ECT2 genes showed association with the clinic-pathological characteristics and chemotherapy response. Statistically significant difference was found between the mean pre-chemotherapy protein levels of CCNE1 in cases than controls (p-value <0.001) and between the mean pre and post-chemotherapy protein levels of CCNE1 and ECT2 (p-value <0.001) in SOC patients. The copy number variations of CCNE1 and ECT2 genes and their protein expression are positively associated with chemotherapeutic response in SOC patients.

First report of glyphosate resistance in an Amaranthus palmeri population from Europe

AbstractThe invasive weed Amaranthus palmeri is spreading throughout Spain, with Catalonia being one of the most affected regions. For this species, acetolactate synthase‐inhibiting herbicide‐resistant populations have been reported, and now glyphosate resistance is also suspected. Glyphosate targets and inhibits the enzyme 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), but A. palmeri has evolved different resistant mechanisms leading to plant survival. One of the most effective is the EPSPS overexpression due to copy number variation (CNV). Gene copies accumulate within the EPSPS cassette that is an extrachromosomal circular DNA displaying unique structural polymorphisms. This study aims to determine the response to glyphosate of a suspected resistant population collected from a roadside and investigate the resistance mechanism involved. The herbicide bioassay confirmed that 40% of the plants survived glyphosate applied at 540 g a.i. ha−1. No known mutations endowing glyphosate resistance were found at EPSPS amongst confirmed resistant plants, while in most of them (70%) specific molecular markers revealed the presence of the EPSPS cassette. All these results indicate that this population is glyphosate resistant and it is very likely that the EPSPS gene CNV is the main resistance mechanism. This is the first case of glyphosate resistance in A. palmeri in Europe whose introduction is likely due to importation of contaminated seed with glyphosate‐resistant Palmer amaranth from the Americas. This introduction poses a significant danger to summer crops in our continent.