BACKGROUND: The CDKN2A gene is frequently affected by somatic copy number variations (SCNVs, including deletions and amplifications [SCNdel and SCNamp]) in the cancer genome. Using surgical gastric margin tissue samples (SMs) as the diploid reference in SCNV analysis via CDKN2A/P16-specific real-time PCR (P16-Light), we previously reported that the CDKN2A SCNdel was associated with a high risk of metastasis of gastric carcinoma (GC). However, the status of CDKN2A SCNVs in SMs and their clinical significance have not been reported.
METHODS: Peripheral white blood cell (WBC) and frozen GC and SM tissue samples were collected from patients (n = 80). Droplet digital PCR (ddPCR) was used to determine the copy number (CN) of the CDKN2A gene in tissue samples using paired WBCs as the diploid reference.
RESULTS: A novel P16-ddPCR system was initially established with a minimal proportion (or limit, 10%) of the detection of CDKN2A CN alterations. While CDKN2A SCNamp events were detected in both SMs and GCs, fewer CDKN2A SCNdel events were detected in SMs than in GCs (15.0% vs. 41.3%, P = 4.77E-04). Notably, significantly more SCNamp and fewer SCNdel of the CDKN2A gene were detected in SMs from GC patients without metastasis than in those from patients with lymph node metastasis by P16-ddPCR (P = 0.023). The status of CDKN2A SCNVs in SM samples was significantly associated with overall survival (P = 0.032). No cancer deaths were observed among the 11 patients with CDKN2A SCNamp.
CONCLUSIONS: CDKN2A SCNVs in SMs identified by P16-ddPCR are prevalent and significantly associated with GC metastasis and overall survival.

Somatic copy number alterations (SCNAs) are a predominant type of oncogenomic alterations that affect a large proportion of the genome in the majority of cancer samples. Current technologies allow high-throughput measurement of such copy number aberrations, generating results consisting of frequently large sets of SCNA segments. However, the automated annotation and integration of such data are particularly challenging because the measured signals reflect biased, relative copy number ratios. In this study, we introduce labelSeg, an algorithm designed for rapid and accurate annotation of CNA segments, with the aim of enhancing the interpretation of tumor SCNA profiles. Leveraging density-based clustering and exploiting the length-amplitude relationships of SCNA, our algorithm proficiently identifies distinct relative copy number states from individual segment profiles. Its compatibility with most CNA measurement platforms makes it suitable for large-scale integrative data analysis. We confirmed its performance on both simulated and sample-derived data from The Cancer Genome Atlas reference dataset, and we demonstrated its utility in integrating heterogeneous segment profiles from different data sources and measurement platforms. Our comparative and integrative analysis revealed common SCNA patterns in cancer and protein-coding genes with a strong correlation between SCNA and messenger RNA expression, promoting the investigation into the role of SCNA in cancer development.

Colorectal cancer (CRC) represents the second deadliest malignancy worldwide. Around 75% of CRC patients exhibit high levels of chromosome instability that result in the accumulation of somatic copy number alterations. These alterations are associated with the amplification of oncogenes and deletion of tumor-ppressor genes and contribute to the tumoral phenotype in different malignancies. Even though this relationship is well known, much remains to be investigated regarding the effect of said alterations in long non-coding RNAs (lncRNAs) and, in turn, the impact these alterations have on the tumor phenotype. The present study aimed to evaluate the role of differentially expressed lncRNAs coded in regions with copy number alterations in colorectal cancer patient samples. We downloaded RNA-seq files of the Colorectal Adenocarcinoma Project from the The Cancer Genome Atlas (TCGA) repository (285 sequenced tumor tissues and 41 non-tumor tissues), evaluated differential expression, and mapped them over genome sequencing data with regions presenting copy number alterations. We obtained 78 differentially expressed (LFC > 1|< -1, padj < 0.05) lncRNAs, 410 miRNAs, and 5028 mRNAs and constructed a competing endogenous RNA (ceRNA) network, predicting significant lncRNA-miRNA-mRNA interactions. Said network consisted of 30 lncRNAs, 19 miRNAs, and 77 mRNAs. To understand the role that our ceRNA network played, we performed KEGG and GO analysis and found several oncogenic and anti-oncogenic processes enriched by the molecular players in our network. Finally, to evaluate the clinical relevance of the lncRNA expression, we performed survival analysis and found that C5orf64, HOTAIR, and RRN3P3 correlated with overall patient survival. Our results showed that lncRNAs coded in regions affected by SCNAs form a complex gene regulatory network in CCR.

Stomach Adenocarcinoma (STAD) is a leading cause of death worldwide. Somatic Copy Number Alterations (SCNAs), which result in Homologous recombination (HR) deficiency in double-strand break repair, are associated with the progression of STAD. However, the landscape of frequent breakpoints of SCNAs (hotspots) and their functional impacts remain poorly understood. In this study, we aimed to explore the frequency and impact of these hotspots in 332 STAD patients and 1,043 cancer cells using data from the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE). We studied the rates of DSB (Double-Strand Breaks) loci in STAD patients by employing the Non-Homogeneous Poisson Distribution (λ), based on which we identified 145 DSB-hotspots with genes affected. We further verified DNA cytosine deamination as a critical process underlying the burden of DSB in STAD. Finally, we illustrated the clinical impact of the significant biological processes. Our findings highlighted the relationship between DNA cytosine deamination and SCNA in cancer was associated with recurrent Somatic Copy Number Alterations in STAD.

Chromosome copy number imbalances, otherwise known as aneuploidies, are a common but poorly understood feature of cancer. Here, we describe recent advances in both detecting and manipulating aneuploidies that have greatly advanced our ability to study their role in tumorigenesis. In particular, new clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have been developed that allow the creation of isogenic cell lines with specific chromosomal changes, thereby facilitating experiments in genetically controlled backgrounds to uncover the consequences of aneuploidy. These approaches provide increasing evidence that aneuploidy is a key driver of cancer development and enable the identification of multiple dosage-sensitive genes encoded on aneuploid chromosomes. Consequently, measuring aneuploidy may inform clinical prognosis, while treatment strategies that target aneuploidy could represent a novel method to counter malignant growth.

Retinoblastoma (RB) is a childhood cancer that forms in the developing retina of young children; this tumor cannot be biopsied due to the risk of provoking extraocular tumor spread, which dramatically alters the treatment and survival of the patient. Recently, aqueous humor (AH), the clear fluid in the anterior chamber of the eye, has been developed as an organ-specific liquid biopsy for investigation of in vivo tumor-derived information found in the cell-free DNA (cfDNA) of the biofluid. However, identifying somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) of the RB1 gene, typically requires either: (1) two distinct experimental protocols-low-pass whole genome sequencing for SCNAs and targeted sequencing for SNVs-or (2) expensive deep whole genome or exome sequencing. To save time and cost, we applied a one-step targeted sequencing method to identify both SCNAs and RB1 SNVs in children with RB. High concordance (median = 96.2%) was observed in comparing SCNA calls derived from targeted sequencing to the traditional low-pass whole genome sequencing method. We further applied this method to investigate the degree of concordance of genomic alterations between paired tumor and AH samples from 11 RB eyes. We found 11/11 AH samples (100%) had SCNAs, and 10 of them (90.1%) with recurrent RB-SCNAs, while only nine out of 11 tumor samples (81.8%) had positive RB-SCNA signatures in both low-pass and targeted methods. Eight out of the nine (88.9%) detected SNVs were shared between AH and tumor samples. Ultimately, 11/11 cases have somatic alterations identified, including nine RB1 SNVs and 10 recurrent RB-SCNAs with four focal RB1 deletions and one MYCN gain. The results presented show the feasibility of utilizing one sequencing approach to obtain SCNA and targeted SNV data to capture a broad genomic scope of RB disease, which may ultimately expedite clinical intervention and be less expensive than other methods.

Inflammation is undoubtedly a hallmark of cancer development. Its maintenance within tumors and the consequences on disease aggressiveness are insufficiently understood.
Data of 27 tumor entities (about 5000 samples) were downloaded from the TCGA and GEO databases. Multi-omic analyses were performed on these and in-house data to investigate molecular determinants of tumor aggressiveness. Using molecular loss-of-function data, the mechanistic underpinnings of inflammation-induced tumor aggressiveness were addressed. Patient specimens and in vivo disease models were subsequently used to validate findings.
There was significant association between somatic copy number alterations (sCNAs) and tumor aggressiveness. SOX2 amplification was the most important feature among novel and known aggressiveness-associated alterations. Mechanistically, SOX2 regulates a group of genes, in particular the AP1 transcription factor FOSL2, to sustain pro-inflammatory signaling pathways, such as IL6-JAK-STAT3, TNFA and IL17. FOSL2 was found overexpressed in tumor sections of specifically aggressive cancers. In consequence, prolonged inflammation induces immunosuppression and activates cytidine deamination and thus DNA damage as evidenced by related mutational signatures in aggressive tumors. The DNA damage affects tumor suppressor genes such as TP53, which is the most mutated gene in aggressive tumors compared to less aggressive ones (38% vs 14%), thereby releasing cell cycle control. These results were confirmed by analyzing tissues from various tumor types and in vivo studies.
Our data demonstrate the implication of SOX2 in promoting DNA damage and genome instability by sustaining inflammation via FOSL2/IL6, resulting in tumor aggressiveness.

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

In patients with retinoblastoma, gains of chromosome 6p have been associated with less differentiated tumors. In cell-free DNA from the aqueous humor (AH), 6p gain has been associated with an increased risk of enucleation. While the identification of somatic copy number alterations (SCNAs) via the AH has been well established, these alterations are not routinely identified in the blood due to low tumor fraction.
SCNAs were considered positive at 20% deflection from the baseline. Somatic RB1 pathogenic variants were identified with targeted sequencing using a panel including all RB1 exons.
A 24-month-old patient presented with unilateral retinoblastoma (Group D/AJCC Stage cT2B) and was treated with primary enucleation. In the peripheral blood, a heterozygous mutation (c.3920T>A) in the APC gene was reported. Genomic analysis of the tumor and AH revealed two novel somatic RB1 mutations (c.1589_1590del and c.2330dupC). Both also demonstrated highly recurrent RB-related SCNAs. Chromosome 6p gain was detected in the blood with an amplitude suggesting approximately 12% tumor fraction. At a follow-up of 24 months, there has been no evidence of metastatic disease.
To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting in some advanced eyes there may be a high enough tumor fraction to detect these alterations (>5% needed). It remains unclear whether 6p gain or increased tumor fraction in the blood is indicative of increased risk of metastatic disease or new primary cancer; studies to address this are ongoing.