Global Copy Number Alterations Research Articles

Abstract A25: Cell-free DNA for noninvasive molecular profiling and response monitoring in pediatric cancers

Abstract Background: Cell-free DNA (cfDNA) is a promising tool to noninvasively profile the cancer genome and track response to treatment. Its utility in adult cancers has been widely demonstrated; however, evidence in pediatric cancers is relatively limited. To address that, we performed targeted sequencing to screen for actionable mutations in plasma, and evaluated the utility of shallow whole-genome sequencing (sWGS) as (i) a tool to estimate cfDNA tumor fractions to guide data interpretation in plasma, and (ii) a mutation-agnostic approach to monitor treatment responses and at the same time screen for actionable somatic copy number alterations from cfDNA, in pediatric cancer patients with neuroblastoma (NB), osteosarcoma (OS), and Wilms’ tumor (WT). Methods: In the first part of the study, we analyzed cfDNA by a ~400 gene targeted sequencing assay, MSK-IMPACT, and found mutations in 62% (31/50) of patients. In parallel, we performed sWGS (~0.1x) in the same cfDNA samples to estimate the fraction of tumor-derived DNA (cfDNA tumor fraction), which is established based on global copy number alterations and distinct tumor-derived cfDNA size profiles as features, that we learned in an independent adult cancer dataset using logistic regression model. Then we further evaluated the utility of sWGS-estimated tumor fractions to track responses in 28 patients (18 OS, 7 WT, and 3 NB) for whom longitudinal plasma samples were available. Results: Plasma samples with an estimated high tumor fraction tend to show better agreement between tumor and plasma. Among the three cancer types, MSK-IMPACT targeted sequencing analysis of cfDNA from relapsed neuroblastoma revealed actionable mutations, including alterations in ALK, NRAS, and TSC1. Wilms’ tumor cfDNA revealed prognostic biomarkers, such as somatic mutations in TP53 and 1q gains, which are associated with worse prognosis and require more aggressive therapy. The sWGS analysis also revealed somatic focal copy number amplifications that are potentially actionable such as CDK4 and MDM2 from the OS patients. When analyzing longitudinal plasma samples, we found that sWGS-derived cfDNA tumor fractions closely tracked the dynamics of disease response to treatment and surgical resection. In two OS patients where no mutations were detected by targeted sequencing but cfDNA tumor fraction was high, we performed exome sequencing on the cfDNA and found BRCA signature, which is potentially actionable in OS. Conclusions: Our results show that plasma profiling reveals actionable alterations across different types of pediatric cancers. Gene-panel targeted sequencing can reveal important somatic mutations, and low-pass sWGS allows longitudinal monitoring of tumor responses and detects clinically relevant copy number alterations. It also helps to identify cfDNA samples that have sufficient tumor-derived DNA for more comprehensive molecular profiling such as exome sequencing to screen for mutational signature that has clinical implication. Citation Format: Prachi Kothari, Julie Yang, Caitlin Stewart, Erika Gedvilaite, Dennis Stephens, Michael F. Berger, Michael V. Ortiz, Neerav Shukla, Emily Slotkin, Shakeel Modak, Dana W. Y. Tsui. Cell-free DNA for noninvasive molecular profiling and response monitoring in pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A25.

Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.

Integrative analysis of the microRNA-mRNA response to radiochemotherapy in primary head and neck squamous cell carcinoma cells.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a very heterogeneous disease resulting in huge differences in the treatment response. New individualized therapy strategies including molecular targeting might help to improve treatment success. In order to identify potential targets, we developed a HNSCC radiochemotherapy cell culture model of primary HNSCC cells derived from two different patients (HN1957 and HN2092) and applied an integrative microRNA (miRNA) and mRNA analysis in order to gain information on the biological networks and processes of the cellular therapy response. We further identified potential target genes of four therapy-responsive miRNAs detected previously in the circulation of HNSCC patients by pathway enrichment analysis.ResultsThe two primary cell cultures differ in global copy number alterations and P53 mutational status, thus reflecting heterogeneity of HNSCC. However, they also share many copy number alterations and chromosomal rearrangements as well as deregulated therapy-responsive miRNAs and mRNAs. Accordingly, six common therapy-responsive pathways (direct P53 effectors, apoptotic execution phase, DNA damage/telomere stress induced senescence, cholesterol biosynthesis, unfolded protein response, dissolution of fibrin clot) were identified in both cell cultures based on deregulated mRNAs. However, inflammatory pathways represented an important part of the treatment response only in HN1957, pointing to differences in the treatment responses of the two primary cultures. Focused analysis of target genes of four therapy-responsive circulating miRNAs, identified in a previous study on HNSCC patients, revealed a major impact on the pathways direct P53 effectors, the E2F transcription factor network and pathways in cancer (mainly represented by the PTEN/AKT signaling pathway).ConclusionsThe integrative analysis combining miRNA expression, mRNA expression and the related cellular pathways revealed that the majority of radiochemotherapy-responsive pathways in primary HNSCC cells are related to cell cycle, proliferation, cell death and stress response (including inflammation). Despite the heterogeneity of HNSCC, the two primary cell cultures exhibited strong similarities in the treatment response. The findings of our study suggest potential therapeutic targets in the E2F transcription factor network and the PTEN/AKT signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1865-x) contains supplementary material, which is available to authorized users.

THE TREATMENT-RESISTANT MESENCHYMAL SIGNATURE IN GLIOBLASTOMA DERIVES FROM TUMOR CELLS INDEPENDENT OF STROMA

BACKGROUND: Glioma sphere-forming cells (GSCs) derived from surgical specimens are a fundamental resource to study glioblastoma (GBM) biology. Mesenchymal-expressing GSCs have been proposed as a source of treatment resistance and mesenchymal tumors correlate with poorer survival. Recently, we found that the anti-angiogensis drug bevacizumab appeared to provide no benefit to patients with mesenchymal tumors, in contradiction to expectations that a mesenchymal microenvironment may benefit from anti-angiogenesis therapy. We have developed a collection of GSCs that have undergone comprehensive genomic characterization, similar to that performed by the Cancer Genome Atlas (TCGA) for whole tumor specimens. We hypothesized that the genomic landscape of GSCs would recapitulate what was observed by TCGA. METHODS: 47 GSCs were obtained from primary culture of fresh tumor specimens obtained at surgery and cultured as 3-dimensional spheres in the absence of serum. All lines were subjected to RNAseq (75bp paired-end, 100X coverage), copy number analysis (Affymetrix Oncoscan 2.0), whole methylome (Illumina Infinium 450k bead array), and targeted resequencing of known cancer-associated genes. Whole exome sequencing was performed for 22 GSCs. Gene expression was determined by reads per kilobase per million (RPKM) using an RNA sequencing data analysis pipeline (PRADA) and somatic mutations identified by a commonly used detection method (MuTech). Consensus clustering based on none-negative matrix factorization (CNMF) was performed on expression data and correlation to TCGA clusters determined by single-sample gene set enrichment analysis (ssGSEA). RESULTS: While global copy number alterations such as gain of chromosome 7 at the EGFR locus or loss of chromosome 10 at the PTEN locus were shared between tumor and matched GSC, the rate of somatic events was significantly higher in GSCs compared to tumors (range 47-570, median 124 vs range 2-255, median 65). Optimization of CNMF identified a total of 5 gene-expression clusters. GSCs in only one of these clusters showed enrichment for a unique TCGA class, mesenchymal. GSCs in other clusters were divided among multiple TCGA classes. CONCLUSIONS: Mesenchymal glioblastomas are derived from mesenchymal GSCs, suggesting that the tumor component is the largest contributor to the aggressive biology of this subtype. GSCs from other tumor subtypes correlate to multiple TCGA classes, suggesting that tumor stroma may contribute to the expression phenotype in those cases. Therapeutics targeting the microenvironment, such as anti-angiogenesis drugs, may have a greater role in non-mesenchymal tumors where the stromal contribution is more prominent. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.