Changes In Copy Number Variation Research Articles

Multi-omics approach for identifying CNV-associated lncRNA signatures with prognostic value in prostate cancer

BackgroundProstate cancer, the second most prevalent malignancy among men, poses a significant threat to affected patients’ well-being due to its poor prognosis. Novel biomarkers are required to enhance clinical outcomes and tailor personalized treatments. Herein, we describe our research to explore the prognostic value of long non-coding RNAs (lncRNAs) deregulated by copy number variations (CNVs) in prostate cancer. MethodsThe study employed an integrative multi-omics data analysis of the prostate cancer transcriptomic, CNV and methylation datasets to identify prognosis-related subtypes. Subtype-specific expression profiles of protein-coding genes (PCGs) and lncRNAs were determined. We analysed CNV patterns of lncRNAs across the genome to identify subtype-specific lncRNAs with CNV changes. LncRNAs exhibiting significant amplification or deletion and a positive correlation were designated CNV-deregulated lncRNAs. A prognostic risk score model was subsequently developed using these CNV-driven lncRNAs. ResultsSix molecular subtypes of prostate cancer were identified, demonstrating significant differences in prognosis (P = 0.034). The CNV profiles of subtype-specific lncRNAs were examined, revealing their correlation with CNV amplification or deletion. Six lncRNAs (CCAT2, LINC01593, LINC00276, GACAT2, LINC00457, LINC01343) were selected based on significant CNV amplifications or deletions using a rigorous univariate Cox proportional risk regression model. A robust risk score model was developed, stratifying patients into high-risk and low-risk categories. Notably, our prognostic model based on these six lncRNAs exhibited exceptional predictive capabilities for recurrence-free survival (RFS) in prostate cancer patients (P = 0.024). ConclusionsOur study successfully identified a prognostic risk score model comprising six CNV-driven lncRNAs that could potentially be prognostic biomarkers for prostate cancer. These lncRNA signatures are closely associated with RFS, providing promising prospects for improved patient prognostication and personalized therapeutic strategies for novel prostate cancer treatment.

Abstract P5-06-02: Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile

Abstract Background: Structural variation is a hallmark of breast cancer. Previous single-cell genomic analysis of 10 untreated early-stage primary TNBC tumors identified only one or two subclones per tumor, found that copy number variation (CNV) occurs early in the evolution of the tumor, and asserted that CNV changes do not contribute to genomic variation at later time points of a tumor’s growth. This PCNE model (punctuated copy number evolution) is analogous to the big-bang theory of colorectal cancer. However, cancer exists in a dynamic environment, and systemic chemotherapy provides evolutionary pressure, making stasis quite unlikely. An investigation of late-stage cancers could shed light on CNV evolution. Methods: We performed a longitudinal, prospective observational study of over 130 patients with metastatic breast cancer, with regular follow-up and detailed treatment histories. CTCs were enumerated by CellSearch, and samples with >20 CTCs in 7.5ml of whole blood were further processed for single CTC isolation via DEPArray, single-cell library construction, and whole-genome sequencing. CNV counts for each cell were obtained using previously published binning methods (Ginkgo). Three mathematical models were used to evaluate the evolution of CNVs: a punctuated model, a gradual model, and a gradual-on-punctuated hybrid model. Results: In total, 150 patient-years of data were collected. Among samples with sufficient CTC counts for study inclusion, a total of nine patients and 376 cells were isolated for sequencing. A mean sequencing depth of 0.8× was achieved from each single cell. The degree of CNV heterogeneity varies from patient to patient. CNV changes occurred over time in a gradual manner on a background of punctuated changes. The adjusted R2 value for the punctuated model was 0.46, the adj R2 value for the gradual model was 0.76, and the hybrid model achieved an adj R2 of 0.99. Patient samples with large CNV heterogeneity across single cells tend to be from patients with longer treatment histories and from the last blood draw (i.e., the blood collection closest to patient’s point of death). We also identified multiple whole-genome duplication (WGD) events from the same patient, in contrast to previous findings that WGD events are usually early clonal events in most instances. Finally, X loss events are among the most frequent chromosomal aberrations identified in CTCs (in up to 80% of CTCs in some patients), which supports previous literature on the loss of the Barr body in hastening metastatic spread. Discussion and Conclusion: In contrast to the previous study, our findings support a gradual-on-punctuated hybrid model, and this model can explain how copy number changes can be a source from which tumors gain resistance to systemic therapies. The model explains how CNV heterogeneities are seen in patients with longer treatment histories, suggesting that CNV continues to be a source of genetic variation at the metastatic stage of the disease course. Citation Format: Weelic Chong, Rui Luo, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Ronald Myers, Mariel Becker, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-02.

A MYB Transcription Factor Atlas Provides Insights into the Evolution of Environmental Adaptations in Plants.

The MYB transcription factor superfamily includes key regulators of plant development and responses to environmental changes. The diversity of lifestyles and morphological characteristics exhibited by plants are potentially associated with the genomic dynamics of the MYB superfamily. With the release of the plant genomes, a comprehensive phylogenomic analysis of the MYB superfamily across Viridiplantae is allowed. The present study performed phylogenetic, phylogenomic, syntenic, horizontal gene transfer, and neo/sub-functionalization analysis of the MYB superfamily to explore the evolutionary contributions of MYB members to species diversification, trait formation, and environmental adaptation in 437 different plant species. We identified major changes in copy number variation and genomic context within subclades across lineages. Multiple MYB subclades showed highly conserved copy number patterns and synteny across flowering plants, whereas others were more dynamic and showed lineage-specific patterns. As examples of lineage-specific morphological divergence, we hypothesize that the gain of a MYB orthogroup associated with flower development and environmental responses and an orthogroup associated with auxin and wax biosynthesis in angiosperms were correlated with the emergence of flowering plants, unbiased neo-/sub-functionalization of gene duplicates contributed to environmental adaptation, and species-specific neo-/sub-functionalization contributed to phenotype divergence between species. Transposable element insertion in promoter regions may have facilitated the sub-/neo-functionalization of MYB genes and likely played a tissue-specific role contributing to sub-/neo-functionalization in plant root tissues. This study provides new insights into the evolutionary divergence of the MYB superfamily across major flowering and non-flowering lineages and emphasizes the need for lineage-/tissue-specific characterization to further understand trait variability and environmental adaptation.

Gene signatures, immune infiltration, and drug sensitivity based on a comprehensive analysis of m6a RNA methylation regulators in cervical cancer

BackgroundCervical cancer is the fourth most common cancer in women. N6-dimethyladenosine (m6A) mRNA methylation is closely associated with cervical cancer.MethodsUsing TCGA database, we studied the expression and mutation of m6A-related genes in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and obtained genetic characteristics based on an m6A risk model and prognostic value of m6A. We studied the effects of the m6A risk score on immune features and genomic changes of patients with CESC, evaluated the sensitivity of patients with CESC to different small-molecule drugs based on the m6A risk score, and established a clinical prediction model.ResultsTen m6A-related genes were differentially expressed between CESC and normal tissues. High-risk patients had a low overall survival (OS) and significantly low immune scores but showed no significantly altered stromal scores. The tumor mutation burden (TMB) and tumor neoantigen levels significantly differed between the high- and low-risk groups. In the high-risk group, copy number variation (CNV) changes mainly led to gene amplification, while in the low-risk group, CNV changes primarily manifested as gene copy number deletions. ZC3H13 expression was low in CESC tissues. ZC3H13 knockdown promoted CESC cell proliferation, migration, and invasion, reducing the RNA methylation levels. Rapamycin suppressed the CESC cell proliferation, migration, and invasion abilities, increasing the m6A levels.Conclusionm6A mRNA methylation is closely related to the occurrence, development, immune invasion, drug sensitivity, and prognosis of cervical cancer. The prognostic m6A feature model of m6A signature genes can accurately predict the OS of patients with CESC. Drugs targeting factors regulating m6A mRNA methylation might offer a good prospect for treating cervical cancer.

Copy Number Variation of Circulating Tumor DNA (ctDNA) Detected Using NIPT in Neoadjuvant Chemotherapy-Treated Ovarian Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) can be used to characterize and monitor cancers. Recently, non-invasive prenatal testing (NIPT) as a new next-generation sequencing (NGS)-based approach has been applied for detecting ctDNA. This study aimed to investigate the copy number variations (CNVs) utilizing the non-invasive prenatal testing in plasma ctDNA from ovarian cancer (OC) patients who were treated with neoadjuvant chemotherapy (NAC). The plasma samples of six patients, including stages II–IV, were collected during the pre- and post-NAC treatment that were divided into NAC-sensitive and NAC-resistant groups during the follow-up time. CNV analysis was performed using the NIPT via two methods “an open-source algorithm WISECONDORX and NextGENe software.” Results of these methods were compared in pre- and post-NAC of OC patients. Finally, bioinformatics tools were used for data mining from The Cancer Genome Atlas (TCGA) to investigate CNVs in OC patients. WISECONDORX analysis indicated fewer CNV changes on chromosomes before treatment in the NAC-sensitive rather than NAC-resistant patients. NextGENe data indicated that CNVs are not only observed in the coding genes but also in non-coding genes. CNVs in six genes were identified, including HSF1, TMEM249, MROH1, GSTT2B, ABR, and NOMO2, only in NAC-resistant patients. The comparison of these six genes in NAC-resistant patients with The Cancer Genome Atlas data illustrated that the total alteration frequency is amplification, and the highest incidence of the CNVs (≥35% based on TCGA data) is found in MROH1, TMEM249, and HSF1 genes on the chromosome (Chr) 8. Based on TCGA data, survival analysis showed a significant reduction in the overall survival among chemotherapy-resistant patients as well as a high expression level of these three genes compared to that of sensitive samples (all, p < 0.0001). The continued Chr8 study using WISECONDORX revealed CNV modifications in NAC-resistant patients prior to NAC therapy, but no CNV changes were observed in NAC-sensitive individuals. Our findings showed that low coverage whole-genome sequencing analysis used for NIPT could identify CNVs in ctDNA of OC patients before and after chemotherapy. These CNVs are different in NAC-sensitive and -resistant patients highlighting the potential application of this approach in cancer patient management.

A Comprehensive Analysis of Pyroptosis-Related lncRNAs Signature Associated With Prognosis and Tumor Immune Microenvironment of Pancreatic Adenocarcinoma.

Background: Globally, pancreatic adenocarcinoma (PAAD) is a common and highly devastating gastrointestinal malignancy that seriously threatens human health. Pyroptosis refers to an emerging form of programmed cell death that has been discovered in recent years, and studies have demonstrated that long non-coding RNA (lncRNA) may act as a moderator in the pyroptosis process of cancer cells. However, relevant explorations about lncRNAs and pyroptosis are still insufficient in PAAD. Therefore, our research is designed to make a comprehensive analysis of the potential values of pyroptosis-related lncRNAs in PAAD. Methods: By integrating the RNA-sequencing, somatic mutation, and copy number variation (CNV) datasets, as well as the clinicopathological features, we established and validated a risk signature based on pyroptosis-related lncRNAs, and comprehensively analyzed its clinical significance and the potential connection with the tumor immune microenvironment (TIME). Consequences: The genetic variation landscape displayed that the somatic mutations were rare while CNV changes were general and mainly concentrated on copy number amplification of these 52 pyroptosis-related genes. Subsequently, a risk signature consisting of 10 lncRNAs (TRAF3IP2-AS1, LINC00519, LINC01133, LINC02251, AC005332.6, AL590787.1, AC090114.2, TRPC7-AS1, MIR223HG, and MIR3142HG) was constructed and patients were divided into different subgroups according to the median risk score; patients with high-risk scores presented worse outcomes compared to those with low-risk scores in the training, testing, and entire cohorts. Furthermore, patients at low-risk scores possessed a higher infiltration abundance of immune cells compared with high-risk patients, which was consistent with the expression levels of lncRNAs between the high/low-risk groups. Drug sensitivity analysis showed that low-risk scores were related to anti-cancer agents like AICAR and Axitinib, whereas high-risk scores were connected with certain drugs such as AUY922. These results demonstrated that our risk signature could be used for prognosis prediction; additionally, it was also related to the TIME that might act as a potential indicator to instruct immunotherapeutic strategies. Conclusion: This work explored the significance of the risk model constructed by pyroptosis-related lncRNAs in prognosis prediction and its internal link with the immune microenvironment of PAAD. The results are expected to assist in the diagnosis, prognostic assessment, and management of patients with PAAD.

Dynamic Tumor Mutational Profiling Using a Novel Non-Invasive Swab Technique for Serial Whole Exome Sequencing of Cervical Tumors During Chemoradiation Therapy

Clinically relevant genetic predictors of radiation response for cervical cancer are understudied due to the morbidity of repeat invasive biopsies required to obtain genetic material. We have developed a novel noninvasive cervical swab technique to collect tumor DNA with adequate throughput to perform whole exome sequencing (WES) at serial timepoints over the course of chemoradiation therapy (CRT). We hypothesize that characterization of dynamic changes in the mutational landscape of cervical tumors during the course of CRT will allow for identification of molecular signatures correlating with therapy response.A total of 69 patients with FIGO stage IB-IVA cervical cancer who underwent definitive CRT were prospectively enrolled on an IRB approved clinical trial. Cervical tumor DNA samples were collected by a noninvasive swab technique at baseline, and after 1, 3, and 5 weeks of CRT. Matched buccal DNA swabs were obtained to filter somatic mutations for each patient. An initial cohort included 24 patients (81 samples) and a second cohort included 45 patients (135 samples). WES was performed on the Illumina HiSeq 4000 series. Raw sequencing data were aligned to a human reference genome and MuTect was applied to detect somatic single nucleotide variants (SNV). Tumor purity (TP) was estimated from the distribution of variant allelic fractions of SNVs. In patients with pelvic MRIs at baseline and week 5 of CRT, 3D tumor volumes (TV) were generated (n = 6,8). Regression analysis was used to evaluate the goodness of fit between TP and TV.Estimated TP was highly correlated with TV in both cohorts (R2 = 0.969 and 0.858). Between the two cohorts, there was overlap in 4 of the top 5 genes (80%) containing the greatest number of exonic mutations across all timepoints (PIK3CA, MUC4, TPRXL, TTN). The number of altered genes present at baseline decreased over time in both cohorts. When normalized to TP, relative mutational burden decreased among standard and exceptional responders (< 20% TV remaining at week 5 of CRT) and increased among poor responders (> 20% TV remaining at week 5). The top ranked genes by magnitude of change at week 5 included MUC2, PARP10 and PIK3CA. Pathway analysis of these top ranked genes mapped to key radiation response pathways including regulation of the G1/S transition.We validated this method of performing serial WES over the course of CRT in 2 independent prospective patient cohorts, highlighting the robustness of our novel noninvasive technique. This allowed for the identification of a panel of potential genes involved in CRT resistance. Future directions include examination of dynamic changes in copy number variation, indels, and tumor mutational burden towards a comprehensive analysis of clinically relevant drivers of CRT resistance. This may allow future targeted treatment for patients with a radioresistant genetic profile, or concurrent targeted therapy for tumors with actionable mutations.

Whole-genome copy number and immunohistochemical analyses on surgically resected intracholecystic papillary neoplasms.

Intracholecystic papillary neoplasms are newly defined precancerous lesions. According to Classification of the World Health Organization, they have four histological morphologies, which are biliary, gastric, intestinal, and oncocytic. This study evaluated 17 patients with resected intracholecystic papillary neoplasms in terms of histological, immunohistochemical, and copy number variation (CNV). The histological subtypes included 5 cases of low-grade (5 gastric) and 12 cases of high-grade (6 gastric and 6 biliary) neoplasms. Most cases showed high expression of MUC1, MUC5AC, and CK7, moderate expression of MUC6 and Ki-67, and low expression of CK20, MUC2, and CDX2. The CNV profile identified gain of 7q in 12%, and loss of 1p (18%), 5q (29%), 9p (35%), 12p (17%), 17p (24%), and 19p (18%). No CNVs were observed in low-grade neoplasms, whereas high-grade ones had increasing abnormalities. β-catenin was often expressed in the nucleus of neoplasms with gastric morphology, suggesting the involvement of the Wnt/β-catenin pathway. However, it was not expressed among those with biliary morphology, which instead exhibited high p53 expression. Neoplasms with biliary morphology showed more CNV changes (9p, 17p, 19p losses). Distinct immunological and CNV patterns were seen in both morphologies, suggesting differences in their pathogenesis. More CNVs accumulated with tumor progression.

Screening for Copy Number Variations of the 15q13.3 Hotspot in CHRNA7 Gene and Expression in Patients with Migraines.

Background: a migraine is a neurological disease. Copy number variation (CNV) is a phenomenon in which parts of the genome are repeated. We investigated the effects of the CNV and gene expression at the location 15q13.3 in the Cholinergic Receptor Nicotinic Alpha 7 Subunit (CHRNA7) gene, which we believe to be effective in the migraine clinic. Methods: we evaluated changes in CHRNA7 gene expression levels and CNV of 15q13.3 in patients with migraine (n = 102, with aura, n = 43; without aura, n = 59) according to healthy controls (n = 120) by q-PCR. The data obtained were analyzed against the reference telomerase reverse transcriptase (TERT) gene with the double copy number by standard curve analysis. Copy numbers were graded as a normal copy (2), gain (2>), and loss (<2). Results: we analyzed using the 2−ΔΔCT calculation method. The CHRNA7 gene was significantly downregulated in patients (p < 0.05). The analysis of CNV in the CHRNA7 gene was statistically significant in the patient group, according to healthy controls (p < 0.05). A decreased copy number indicates a dosage loss. However, no significant difference was observed among gain, normal, and loss copy numbers and expression values in patients (p > 0.05). The change in CNV was not associated with the downregulation of the CHRNA7 gene. Conclusion: Downregulation of the CHRNA7 gene may contribute to the formation of migraine by inactivation of the alpha-7 nicotinic receptor (α7nAChR). The association of CNV gains and losses with migraines will lead to better understanding of the molecular mechanisms and pathogenesis, to better define the disease, to be used as a treatment target.

CNV-MEANN: A Neural Network and Mind Evolutionary Algorithm-Based Detection of Copy Number Variations From Next-Generation Sequencing Data.

Copy number variation (CNV), is defined as repetitions or deletions of genomic segments of 1 Kb to 5 Mb, and is a major trigger for human disease. The high-throughput and low-cost characteristics of next-generation sequencing technology provide the possibility of the detection of CNVs in the whole genome, and also greatly improve the clinical practicability of next-generation sequencing (NGS) testing. However, current methods for the detection of CNVs are easily affected by sequencing and mapping errors, and uneven distribution of reads. In this paper, we propose an improved approach, CNV-MEANN, for the detection of CNVs, involving changing the structure of the neural network used in the MFCNV method. This method has three differences relative to the MFCNV method: (1) it utilizes a new feature, mapping quality, to replace two features in MFCNV, (2) it considers the influence of the loss categories of CNV on disease prediction, and refines the output structure, and (3) it uses a mind evolutionary algorithm to optimize the backpropagation (neural network) neural network model, and calculates individual scores for each genome bin to predict CNVs. Using both simulated and real datasets, we tested the performance of CNV-MEANN and compared its performance with those of seven widely used CNV detection methods. Experimental results demonstrated that the CNV-MEANN approach outperformed other methods with respect to sensitivity, precision, and F1-score. The proposed method was able to detect many CNVs that other approaches could not, and it reduced the boundary bias. CNV-MEANN is expected to be an effective method for the analysis of changes in CNVs in the genome.

Clinical impact of copy number variation changes in bladder cancer samples.

The aim of the present study was to detect copy number variations (CNVs) related to tumour progression and metastasis of urothelial carcinoma through whole-genome scanning. A total of 30 bladder cancer samples staged from pTa to pT4 were included in the study. DNA was extracted from freshly frozen tissue via standard phenol-chloroform extraction and CNV analysis was performed on two alternative platforms (CytoChip Oligo aCGH, 4x44K and Infinium OncoArray-500K BeadChip; Illumina, Inc.). Data were analysed with BlueFuse Multi software and Karyostudio, respectively. The results highlight the role of genomic imbalances in regions containing genes with metastatic and proliferative potential for tumour invasion. A high level of genomic instability in uroepithelial tumours was observed and a total of 524 aberrations, including 175 losses and 349 gains, were identified. The most prevalent genetic imbalances affected the following regions: 1p, 1q, 2q, 4p, 4q, 5p, 5q, 6p, 6q, 7q, 8q, 9p, 9q, 10p, 10q, 11q, 13q and 17q. High-grade tumours more frequently harboured genomic imbalances (n=227) than low-grade tumours (n=103). A total of 36 CNVs in high-grade bladder tumours were detected in chromosomes 1-5, 8-11, 14, 17, 19 and 20. Furthermore, five loss of heterozygosity variants containing 176 genes were observed in high-grade bladder cancer and may be used as potential targets for precision therapy. Revealing specific chromosomal regions related to the metastatic potential of uroepithelial tumours may lay a foundation for implementing molecular CNV profiling of bladder tumours as part of a routine progression risk estimation strategy, thus expanding the personalized therapeutic approach.

Epigenome-Wide Analysis of Methylation Changes in the Sequence of Gallstone Disease, Dysplasia, and Gallbladder Cancer.

Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-income and middle-income countries, and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide. To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of formalin-fixed, paraffin-embedded gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Preprocessed, quality-controlled data from 82 samples (gallstones n=32, low-grade dysplasia n=13, high-grade dysplasia n=9, GBC n=28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs). The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of cytosine-guanine dinucleotide islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected cyclin-dependent kinase inhibitor 2A, MDM2 proto-oncogene, tumor protein P53, and cyclin D1 genes. Gains in the targetable Erb-B2 receptor tyrosine kinase 2 gene were detected in 14% of GBC samples. Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (high-grade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.

Improving high-resolution copy number variation analysis from next generation sequencing using unique molecular identifiers

BackgroundRecently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct.ResultsHere, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization.ConclusionWe provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

A Mechanism Leading to Changes in Copy Number Variations Affected by Transcriptional Level Might Be Involved in Evolution, Embryonic Development, Senescence, and Oncogenesis Mediated by Retrotransposons

In recent years, more and more evidence has emerged showing that changes in copy number variations (CNVs) correlated with the transcriptional level can be found during evolution, embryonic development, and oncogenesis. However, the underlying mechanisms remain largely unknown. The success of the induced pluripotent stem cell suggests that genome changes could bring about transformations in protein expression and cell status; conversely, genome alterations generated during embryonic development and senescence might also be the result of genome changes. With rapid developments in science and technology, evidence of changes in the genome affected by transcriptional level has gradually been revealed, and a rational and concrete explanation is needed. Given the preference of the HIV-1 genome to insert into transposons of genes with high transcriptional levels, we propose a mechanism based on retrotransposons facilitated by specific pre-mRNA splicing style and homologous recombination (HR) to explain changes in CNVs in the genome. This mechanism is similar to that of the group II intron that originated much earlier. Under this proposed mechanism, CNVs on genome are dynamically and spontaneously extended in a manner that is positively correlated with transcriptional level or contract as the cell divides during evolution, embryonic development, senescence, and oncogenesis, propelling alterations in them. Besides, this mechanism explains several critical puzzles in these processes. From evidence collected to date, it can be deduced that the message contained in genome is not just three-dimensional but will become four-dimensional, carrying more genetic information.

Comprehensive Analysis of Differentially Expressed lncRNAs in Gastric Cancer.

Gastric cancer (GC) is the fourth most common malignant tumor. The mechanism underlying GC occurrence and development remains unclear. Previous studies have indicated that long non-coding RNAs (lncRNAs) are significantly associated with gastric cancer, but a systematic understanding of the role of lncRNAs in gastric cancer is lacking. In recent years, with the development of next-generation sequencing technology, tens of thousands of lncRNAs have been discovered. However, a large number of unannotated lncRNAs remain unidentified in different tissues, including potential gastric cancer-related lncRNAs. In this study, RNA sequencing (RNA-seq) data from 16 samples of eight gastric cancer patients were obtained and analyzed. A total of 1,854 previously unannotated lncRNAs were identified by ab initio assembly, and 520 differentially expressed lncRNAs were validated in the TCGA expression dataset. Methylation and copy number variation (CNV) array data from the same sample were integrated in the analysis. Changes in DNA methylation levels and CNVs may be responsible for the differential expression of 91 lncRNAs. Differentially expressed lncRNAs were enriched in coexpressed clusters of genes related to functions such as cell signaling, cell cycle, immune response, metabolic processes, angiogenesis, and regulation of retinoic acid (RA) receptors. Finally, a differentially expressed lncRNA, AC004510.3, was identified as a potential biomarker for the prediction of the overall survival of gastric cancer patients.

Phylogenomic analysis of the APETALA2 transcription factor subfamily across angiosperms reveals both deep conservation and lineage-specific patterns.

SummaryThe APETALA2 (AP2) subfamily of transcription factors are key regulators of angiosperm root, shoot, flower and embryo development. The broad diversity of anatomical and morphological structures is potentially associated with the genomic dynamics of the AP2 subfamily. However, a comprehensive phylogenomic analysis of the AP2 subfamily across angiosperms is lacking. We combined phylogenetic and synteny analysis of distinct AP2 subclades in the completed genomes of 107 angiosperm species. We identified major changes in copy number variation and genomic context within subclades across lineages, and discuss how these changes may have contributed to the evolution of lineage‐specific traits. Multiple AP2 subclades show highly conserved patterns of copy number and synteny across angiosperms, while others are more dynamic and show distinct lineage‐specific patterns. As examples of lineage‐specific morphological divergence due to AP2 subclade dynamics, we hypothesize that loss of PLETHORA1/2 in monocots correlates with the absence of taproots, whereas independent lineage‐specific changes of PLETHORA4/BABY BOOM and WRINKLED1 genes in Brassicaceae and monocots point towards regulatory divergence of embryogenesis between these lineages. Additionally, copy number expansion of TOE1 and TOE3/AP2 in asterids is implicated with differential regulation of flower development. Moreover, we show that the genomic context of AP2s is in general highly specialized per angiosperm lineage. To our knowledge, this study is the first to shed light on the evolutionary divergence of the AP2 subfamily subclades across major angiosperm lineages and emphasizes the need for lineage‐specific characterization of developmental networks to understand trait variability further.

Genomic and transcriptomic comparison of post-radiation versus sporadic sarcomas

Post-radiation sarcomas are rare secondary cancers arising from radiation therapies. To date, few genetic specificities have been described for such malignancies and the oncogenesis of sarcomas with complex genetics (both sporadic and post-radiation) remains largely misunderstood. We performed genomic and transcriptomic analyses on 77 post-radiation sarcomas using DNA-array and RNA sequencing. Consequently, we were able to investigate changes in copy number variations, transcriptome profiling, fusion gene expression, and mutational landscapes. We compare these data to a reference cohort of 93 sporadic sarcomas. At genomic level, similar chromosomal complexity was observed both in post-radiation and sporadic sarcomas with complex genetics. We found more frequent CDKN2A and CDKN2B (coding for p14/p16 and p15 proteins, respectively; at 9p21.3) losses in post-radiation (71%) than in sporadic tumors (39%; P = 6.92e−3). Among all detected fusion genes and punctual variations, few specificities were observed between these groups and such alterations are not able to drive a strong and specific oncogenesis. Recurrent MYC amplifications (96%) and KDR variants (8%) were detected in post-radiation angiosarcomas, in agreement with the literature. Transcriptomic analysis of such angiosarcomas revealed two distinct groups harboring different genomic imbalances (in particular gains of 17q24.2-17qter) with different clinical courses according to patient's vital status. Differential gene expression analysis permitted to focus on the immune response as a potential actor to tumor aggressiveness. Histochemistry validated a lower inflammation and lower immune infiltrate at tumor periphery for highly aggressive angiosarcomas. Our results provide new genomic and transcriptomic information about post-radiation sarcomas. The techniques we used (RNA-seq and DNA-arrays) did not highlight major differences in sarcomas with complex genetics depending on the radiation context, revealing similar patterns of transcriptomic profiles and chromosomal copy number variations. Additional characterizations, particularly whole genome sequencing, could measure changes in DNA following radiation therapy in such malignancies and may precise their oncogenesis.

Integrated landscape of copy number variation and RNA expression associated with nodal metastasis in invasive ductal breast carcinoma.

BackgroundLymph node metastasis (NM) in breast cancer is a clinical predictor of patient outcomes, but how its genetic underpinnings contribute to aggressive phenotypes is unclear. Our objective was to create the first landscape analysis of CNV-associated NM in ductal breast cancer. To assess the role of copy number variations (CNVs) in NM, we compared CNVs and/or associated mRNA expression in primary tumors of patients with NM to those without metastasis.ResultsWe found CNV loss in chromosomes 1, 3, 9, 18, and 19 and gains in chromosomes 5, 8, 12, 14, 16-17, and 20 that were associated with NM and replicated in both databases. In primary tumors, per-gene CNVs associated with NM were ten times more frequent than mRNA expression; however, there were few CNV-driven changes in mRNA expression that differed by nodal status. Overlapping regions of CNV changes and mRNA expression were evident for the CTAGE5 gene. In 8q12, 11q13-14, 20q1, and 17q14-24 regions, there were gene-specific gains in CNV-driven mRNA expression associated with NM.MethodsData on CNV and mRNA expression from the TCGA and the METABRIC consortium of breast ductal carcinoma were utilized to identify CNV-based features associated with NM. Within each dataset, associations were compared across omic platforms to identify CNV-driven variations in gene expression. Only replications across both datasets were considered as determinants of NM.ConclusionsGains in CTAGE5, NDUFC2, EIF4EBP1, and PSCA genes and their expression may aid in early diagnosis of metastatic breast carcinoma and have potential as therapeutic targets.

MS31.03 Targeting the Hippo Pathway

The prognosis for malignant pleural mesothelioma (MPM) remains poor and many anticipated advances in MPM treatment have been disappointing. One reason for the failure of conventional cytotoxic drugs is that they do not address the cancer stem cell population or the stem cell pathways that drive tumor resistance and resurgence following treatment. Cancer stem cells are defined by their properties of self-renewal, pluripotency, a high proliferative capacity and the ability to resist standard chemotherapy and radiation. Among the stem cell pathways, Hippo has proven to be critical to driving growth in MPM with mutations along this pathway implicated in the majority of MPM tumors. The Hippo pathway is a highly conserved regulator of organ size by regulating contact inhibition and of stem cell proliferation and maintenance.(1) The largest and most comprehensive genomic analysis to date of transcriptomes, whole exomes and targeted exomes from 216 MPM samples found Hippo pathway signaling to be the number one most significantly mutated pathway in mesothelioma with a Q-value of 1.70E-17, driven by mutations, copy number variations and fusions in NF2, LATS1, LATS2 and MST1.(2) One of the most frequently mutated genes in MPM is Neurofibromatosis type 2 (NF2) tumor suppressor, located at chromosome 22q12, and is detected in 40% to 50% of MPM tumors.(3) Large tumor suppressor homolog 2 (LAST2) gene, which is located at chromosome 13q12, is another frequently inactivated gene that is detected in 13% of MPM tumors. (3, 4) Inactivation of NF2 and LATS2 by deletion and/or mutation often contribute to dysregulation of Hippo pathway.(5) In addition to LATS2, its closest gene family member LATS1 another Hippo pathway gene, has also recently been identified to be dysregulated in MPM, though less commonly than LATS2. LATS1 is located on chromosome 6 and changes in copy number variation and fusions to Presenilin 1 (PSEN1) on chromosome 14 have been observed in MPM.(2, 3) Mammalian sterile-20 like kinase 1 (MST1) is an important upstream kinase in the Hippo pathway that has also been found to be dysregulated in MPM.(2) Given the high frequency of mutations and dysregulation in the Hippo pathway, it is a promising potential area of drug development. One specific target along the Hippo pathway is blocking Yes-associated protein (YAP) activity. In normal cells, Merlin, a protein encoded by NF2, and LATS2 contribute to the phosphorylation of the transcription factor YAP at S127,(6) resulting in YAP ubiquitination and activation of Hippo pathway to control cell proliferation. In MPM tumor cells, inactivation of NF2 and LATS2 prevent the phosphorylation of YAP at S127, which results in YAP relocation from the cytosol to nucleus where it interacts with TEA domain transcription factors (TEAD). In addition, constitutively activation of YAP has been identified in over 70% of primary MPM tumors, (3, 5, 7) and YAP activation leads to Hippo signaling attenuation and transcription of downstream target genes, such as connective tissue growth factor (CTGF) and Cyr61.(8) Low Merlin expression (NF2), results in YAP1 activation, and has been shown to be associated with worse clinical outcomes with shorter times to recurrence and shorter overall survival times in patients with MPM.(9) Blocking YAP activity, either via upstream inhibition of one of the several pathways that regulate YAP and Hippo or via direct YAP/TEAD inhibition is an area of active interest.(10) inhibitors are in preclinical development with promising results and may enter clinical trials in the near future.

Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining

BackgroundAlthough intratumoral heterogeneity is commonly observed in several cancers, few studies have shown its presence in EGFR-mutated lung cancer. We performed immunohistochemistry to analyze the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer and performed targeted sequencing in specific cases. We discuss the effects of intratumoral heterogeneity and acquired resistance to EGFR-TKI. MethodsTwenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody and the samples were scored by staining intensity (0–3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing was performed. Single nucleotide variations (SNVs) and copy number variations (CNVs) were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining. ResultsAmong 20 cases, 3 showed heterogeneous staining. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part compared to the negative part, and an increase in the copy number of CCNE1 was observed in the IHC-positive part compared to the negative part in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part compared to the negative part, and an increase in the copy number of MDM2 was observed in the IHC-positive part compared to the negative part. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes. ConclusionThese cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity.