Frequent Copy Number Alterations Research Articles

Neoadjuvant talazoparib (TALA) in patients (pts) with germline BRCA1/2 (gBRCA1/2) mutation-positive, early HER2-negative breast cancer (BC): Exploration of tumor BRCA mutational status and zygosity and overall mutational landscape in a phase 2 study.

554 Background: TALA is a poly(ADP-ribose) polymerase inhibitor approved as monotherapy for adult pts with g BRCA1/2-mutated HER2-negative locally advanced/metastatic BC. We report biomarker analyses from a phase 2, nonrandomized, single-arm, open-label study (NEOTALA; NCT03499353) evaluating the efficacy and safety of TALA in the neoadjuvant setting for pts with early g BRCA1/2-mutated HER2− BC. Efficacy and safety results are presented separately. Methods: The biomarker analysis population was all pts treated with TALA for whom biomarker results are available. To support molecular eligibility, blood was tested using BRCAnalysis CDx (Myriad Genetics). Baseline tumor tissue was retrospectively tested using FoundationOne CDx, with BRCA1/2 zygosity assessed using somatic-germline-zygosity (SGZ; Sun et al. JCO PO, 2018). Germline mutational status of 14 non- BRCA DNA damage response (DDR) genes was retrospectively assessed in baseline saliva samples using Ambry CustomNext-Cancer. Mutations were defined as known/likely pathogenic/deleterious variants, including copy number alterations (CNAs). Association between mutational status of MYC or RAD21 and primary endpoint pathological complete response (pCR) as per Independent Central Review was investigated with logistic regression. Results: Of 52 evaluable tumor samples from 61 treated pts, 39 (75%) and 13 (25%) pts exhibited BRCA1 and BRCA2 mutations, respectively; 1 (2%) pt exhibited mutations in both genes, and 1 (2%) pt had mutations in neither. BRCA loss of heterozygosity (LOH) was seen in 42/43 (98%) evaluable BRCA-mutant tumors. Of 45 pts evaluable centrally for both germline and tumor, 44/45 (98%) pts exhibited the same BRCA mutation in tumor as originally detected in germline, with the remaining pt exhibiting a g BRCA1 mutation, but lacking a tumor BRCA mutation. None of 49 saliva-evaluable pts exhibited non- BRCA germline DDR mutations. TP53 (51 [98%] pts) was the most frequently mutated gene in tumors. MYC and RAD21 (each 14 [27%] pts) were the most frequent CNAs. No evidence of association between mutational status of MYC or RAD21 and pCR was found (odds ratio=0.39, 95% CI 0.12-2.30). Based on a cutoff of ≥16%, genomic LOH was elevated in 24/27 (89%) tumors evaluable for both gLOH and pCR, precluding assessment of the potential association of gLOH high/low status with pCR. Conclusions: Tumor BRCA mutations were evident in nearly all pts in the biomarker analysis population, with BRCA LOH evident in all but 1 BRCA-mutated tumor. No pts had non- BRCA germline DDR gene mutations; tumor TP53 mutations were near-universal. MYC and RAD21 each exhibited CNAs in 27% of tumors, with no association with pCR. These results support the central role of BRCA mutations in tumor pathobiology in this indication. Clinical trial information: NCT03499353.

Genomic heterogeneity and copy number variant burden are associated with poor recurrence-free survival and 11q loss in human papillomavirus-positive squamous cell carcinoma of the oropharynx.

Human papillomavirus-positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States. Favorable treatment outcomes have led to increased interest in treatment de-escalation to reduce treatment morbidity as well as the development of prognostic markers to identify appropriately low-risk patients. Intratumoral genomic heterogeneity and copy number alteration burden have been demonstrated to be predictive of poor outcomes in many other cancers; therefore, we sought to determine whether intratumor heterogeneity and genomic instability are associated with poor outcomes in HPV+ OPSCC. Tumor heterogeneity estimates were made based on targeted exome sequencing of 45 patients with HPV+ OPSCC tumors. Analysis of an additional cohort of HPV+ OPSCC tumors lacking matched normal sequencing allowed copy number analysis of 99 patient tumors. High intratumorally genomic heterogeneity and high numbers of copy number alterations were strongly associated with worse recurrence-free survival. Tumors with higher heterogeneity and frequent copy number alterations were associated with loss of distal 11q, which encodes key genes related to double-strand break repair, including ATM and MRE11A. Both intratumor genomic heterogeneity and high-burden copy number alterations are strongly associated with poor recurrence-free survival in patients with HPV+ OPSCC. The drivers of genomic instability and heterogeneity in these tumors remains to be elucidated. However, 11q loss and defective DNA double-strand break repair have been associated with genomic instability in other solid tumors. Copy number alteration burden and intratumoral heterogeneity represent promising avenues for risk stratification of patients with HPV+OPSCC.

Chr20q Amplification Defines a Distinct Molecular Subtype of Microsatellite Stable Colorectal Cancer.

Colorectal cancer is the third leading cause of cancer-related death in the United States. About 15% of colorectal cancers are associated with microsatellite instability (MSI) due to loss of function in the DNA mismatch repair pathway. This subgroup of patients has better survival rates and is more sensitive to immunotherapy. However, it remains unclear whether microsatellite stable (MSS) patients with colorectal cancer can be further stratified into subgroups with differential clinical characteristics. In this study, we analyzed The Cancer Genome Atlas data and found that Chr20q amplification is the most frequent copy number alteration that occurs specifically in colon (46%) and rectum (61%) cancer and is mutually exclusive with MSI. Importantly, MSS patients with Chr20q amplification (MSS-A) were associated with better recurrence-free survival compared with MSS patients without Chr20q amplification (MSS-N; P = 0.03). MSS-A tumors were associated with high level of chromosome instability and low immune infiltrations. In addition, MSS-A and MSS-N tumors were associated with somatic mutations in different driver genes, with high frequencies of mutated TP53 in MSS-A and mutated KRAS and BRAF in MSS-N. Our results suggest that MSS-A and MSS-N represent two subtypes of MSS colorectal cancer, and such stratification may be used to improve therapeutic treatment in an individualized manner. SIGNIFICANCE: This study shows that chromosome 20q amplification occurs predominately in microsatellite-stable colorectal cancer and defines a distinct subtype with good prognosis, high chromosomal instability, distinct mutation profiles, and low immune infiltrations.

Mutations and Copy Number Alterations in IDH Wild-Type Glioblastomas Are Shaped by Different Oncogenic Mechanisms.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and “Others” differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

Frequency and Correlation of Common Genes Copy Number Alterations in Childhood Acute Lymphoblastic Leukemia with Prognosis.

Objective:It was shown by genomic profiling that despite no detectable chromosomal abnormalities a proportion of children with pre-B acute lymphoblastic leukemia harbors copy number alterations (CNA) of genes playing role in B-cell development and function. The aim of the study was to determine the frequency of CNA in pediatric acute lymphoblastic leukemia and correlate these findings with clinical outcome. Methods:DNA extracted from peripheral blood or bone marrow at diagnosis/relapse of fifty newly diagnosed children with precursor B-cell acute lymphoblastic leukemia was analyzed for CNA with multiplex ligation-dependent probe amplification. Results:The analysis revealed 76 CNA in 24 patients most frequently found in PAR1 (17%), CDKN2A/B (15.7%) and PAX5 (14.4%) genes. There were significant CNA co-occurrences between PAX5, CDKN2A/B, BTG1, ETV6, PAR1 or XP22 genes, (p<0.020) and the high-risk group. There was a significant correlation between EBF1, RB1, and IKZF1 alterations and bone marrow relapse. Patients with CNA in screened genes are more likely to succumb to their disease except for those with PAR1 or XP22 genes (p<0.050). Conclusion:The multiplex ligation-dependent probe amplification could be considered as an independent diagnostic tool allowing prompt identification of patients at high risk of treatment failure and, subsequently, a more adequate treatment approach.

Distinct Genomic Alterations in Prostate Tumors Derived from African American Men.

We aim to understand, from acquired genetic alterations in tumors, why African American (AA) men are more likely to develop aggressive prostate cancer. By analyzing somatic mutations in 39 genes using deeper next-generation sequencing with an average depth of 2,522 reads for tumor DNA and genome-wide DNA copy-number alterations (CNA) in prostate cancer in a total of 171 AA/black men and comparing with those in 860 European American (EA)/white men, we here present several novel findings. First, >35% of AA men harbor damaging mutations in APC, ATM, BRCA2, KDM6A, KMT2C, KMT2D, MED12, ZFHX3, and ZMYM3, each with >1% of mutated copies. Second, among genes with >10% of mutated copies in tumor cells, ZMYM3 is the most frequently mutated gene in AA prostate cancer. In a patient's tumor with >96% frameshift mutations of ZMYM3, we find allelic imbalances in 10 chromosomes, including losses of five and gains of another four chromosomes, suggesting its role in maintaining genomic integrity. Third, when compared to prostate cancer in EA/white men, a higher frequency of CNAs of MYC, THADA, NEIL3, LRP1B, BUB1B, MAP3K7, BNIP3L and RB1, and a lower frequency of deletions of RYBP, TP53, and TMPRSS2-ERG are observed in AA/black men. Finally, for the above genes with higher frequency of CNAs in AA than in EA, deletion of MAP3K7, BNIP3L, NEIL3 or RB1, or gain of MYC significantly associates with both higher Gleason grade and advanced pathologic stage in AA/black men. Deletion of THADA associates with advanced pathologic stage only. IMPLICATIONS: A higher frequency of damaging mutation in ZMYM3 causing genomic instability along with higher frequency of altered genomic regions including deletions of MAP3K7, BNIP3L, RB1, and NEIL3, and gain of MYC appear to be distinct somatically acquired genetic alterations that may contribute to more aggressive prostate cancer in AA/black men.

Immunological classification of gliomas based on immunogenomic profiling

BackgroundGliomas are heterogeneous in the tumor immune microenvironment (TIM). However, a classification of gliomas based on immunogenomic profiling remains lacking.MethodsWe hierarchically clustered gliomas based on the enrichment levels of 28 immune cells in the TIM in five datasets and obtained three clusters: immunity-high, immunity-medium, and immunity-low.ResultsGlioblastomas were mainly distributed in immunity-high and immunity-medium, while lower-grade gliomas were distributed in all the three subtypes and predominated in immunity-low. Immunity-low displayed a better survival than other subtypes, indicating a negative correlation between immune infiltration and survival prognosis in gliomas. IDH mutations had a negative correlation with glioma immunity. Immunity-high had higher tumor stemness and epithelial-mesenchymal transition scores and included more high-grade tumors than immunity-low, suggesting that elevated immunity is associated with tumor progression in gliomas. Immunity-high had higher tumor mutation burden and more frequent somatic copy number alterations, suggesting a positive association between tumor immunity and genomic instability in gliomas.ConclusionsThe identification of immune-specific glioma subtypes has potential clinical implications for the immunotherapy of gliomas.

Immune-Related Gene-Based Novel Subtypes to Establish a Model Predicting the Risk of Prostate Cancer.

BackgroundThere is significant heterogeneity in prostate cancer (PCa), but immune status can reflect its prognosis. This study aimed to explore immune-related gene-based novel subtypes and to use them to create a model predicting the risk of PCa.MethodsWe downloaded the data of 487 PCa patients from The Cancer Genome Atlas (TCGA) database. We used immunologically relevant genes as input for consensus clustering and applied survival analysis and principal component analysis to determine the properties of the subtypes. We also explored differences of somatic variations, copy number variations, TMPRSS2-ERG fusion, and androgen receptor (AR) scores among the subtypes. Then, we examined the infiltration of different immune cells into the tumor microenvironment in each subtype. We next performed Gene Set Enrichment Analysis (GSEA) to illustrate the characteristics of the subtypes. Finally, based on the subtypes, we constructed a risk predictive model and verified it in TCGA, Gene Expression Omnibus (GEO), cBioPortal, and International Cancer Genome Consortium (ICGC) databases.ResultsFour PCa subtypes (C1, C2, C3, and C4) were identified on immune status. Patients with the C3 subtype had the worst prognosis, while the other three groups did not differ significantly from each other in terms of their prognosis. Principal component analysis clearly distinguished high-risk (C3) and low-risk (C1 + 2 + 4) patients. Compared with the case in the low-risk subtype, the Speckle-type POZ Protein (SPOP) had a higher mutation frequency and lower transcriptional level in the high-risk subtype. In C3, there was also a higher frequency of copy number alterations (CNA) of Clusterin (CLU) and lower CLU expression. In addition, C3 had a higher frequency of TMPRSS2-ERG fusion and higher AR scores. M2 macrophages also showed significantly higher infiltration in the high-risk subtype, while CD8+ T cells and dendritic cells had significantly higher infiltration in the low-risk subtype. GSEA revealed that MYC, androgen, and KRAS were relatively activated and p53 was relatively suppressed in high-risk subtype, compared with the levels in the low-risk subtype. Finally, we trained a six-gene signature risk predictive model, which performed well in TCGA, GEO, cBioPortal, and ICGC databases.ConclusionPCa can be divided into four subtypes based on immune-related genes, among which the C3 subtype is associated with a poor prognosis. Based on these subtypes, a risk predictive model was developed, which could indicate patient prognosis.

Multiomics integrative analysis for gene signatures and prognostic values of m6A regulators in pancreatic adenocarcinoma: a retrospective study in The Cancer Genome Atlas project.

N6-methyladenosine(m6A) is the most abundant post-transcriptional RNA modification in eukaryotes. However, little is known about its role in pancreatic adenocarcinoma (PAAD). The aim of our study was to identify gene signatures and prognostic values of m6A regulators in PAAD. Patients from 3 different datasets with complete genomic and transcriptomic sequencing data were enrolled. Survival analysis for different gene alterations was performed using log-rank tests and Cox regression model. The association between alteration of m6A regulators and clinicopathological characteristics was examined using chi-square test. Results showed a high frequency of copy number alterations (CNAs) of m6A regulatory genes in PAAD patients, but somatic mutations were rarely happened. CNAs and mutations of m6A regulatory genes was associated with patient’s gender, pathologic stage and resected tumor size. Patients with “gain of function” for m6A “reader” genes combined with copy number loss of “writers” or “erasers” had worse overall survival (OS) compared with other patterns. Moreover, copy number gain of m6A “reader” gene insulin growth factor 2 binding protein 2 (IGF2BP2) was an independent risk factor for OS (HR = 2.392, 95%CI: 1.392-4.112, p<0.001) and disease-free survival (DFS) (HR = 2.400, 95%CI: 1.236-4.659, p=0.010). Gene Set Enrichment Analysis (GSEA) indicated that IGF2BP2 was correlated with multiple biological processes associated with cancer, of which the most significant processes were relevant to cancer cell cycle, cell immortalization and tumor immunity. To sum up, a significant relationship was found between m6A genomic alterations and worse clinical outcomes. These innovative findings are expected to guide further research on the mechanism of m6A in PAAD.

Abstract 1991: Chromosomal instability of circulating tumor DNA predicts response to neoadjuvant chemotherapy in gastric cancer

Abstract Background: Neoadjuvant chemotherapy (NAC) was increasingly used for early-stage gastric cancer (GC) treatment to reduce the recurrence and mortality. However, clinical practices demonstrated that not all the patients underwent NAC prior to surgery could benefit from it. Current available methods for assessing the response to NAC, such as Tumor Regression Grading (TRG), CT, or serum tumor markers, can only evaluated after NAC or surgery. Novel markers for predicting the effect of neoadjuvant chemotherapy on prognosis before NAC is in urgent need. Gastric Cancer is featured by frequent somatic copy number alterations (SCNA) and high chromosomal instability. In addition, chromosomal instability has been demonstrated to effectively predict the therapeutic response to radiotherapy, chemotherapy, and targeted therapy. In this work, we investigated the chromosomal instability in GC patients undergoing NAC as a prognosis predictor. Methods: From Jan 2016 to Jun 2017, a cohort of 25 patients with Stage II/III gastric adenocarcinoma after surgical treatment at Department of Gastrointestinal Oncology, Peking University Cancer Hospital were enrolled in our study. ctDNA was collected before (pre-NAC) and after NAC (post-NAC). We analyzed the mutation profiles of 50 plasma samples collected from 25 patients using a 179 cancer-related gene panel (GenetronHealth; Beijing, China). Copy number instability (CNI) scores of ctDNA were calculated to reflect chromosome instability in ctDNA. Results: Based on the clinical evaluation of a combination of TRG, CT and serum tumor markers, 60% (15/25) patients were responsive to NAC. The pre-NAC CNI scores of ctDNA were significantly higher than that of post-NAC (median 127 vs. 113, Paired t test P=0.0403), which correlated well with the response rates and prognosis in the GC patients treated with NAC. 73.7% (14/19) patients with higher pre-NAC CNI score (CNI score &amp;gt;90) were responsive to NAC, while the response rate of the lower pre-NAC CNI score (CNI score &amp;lt;90) was only 16.7%(1/6). And the pre-NAC CNI scores of response group were much higher than that of the non-Response. (median 129 vs 86.5). Furthermore, comparing with the patients with lower pre-NAC CNI (CNI score &amp;gt;90), the higher ones had obviously better prognosis after NAC treatment (P=0.037, HR=9.358). Conclusions: To our knowledge, this is the first time demonstrating that the chromosomal instability quantification measured by CNI score of pre-NAC ctDNA may become a novel promising markers of response and prognosis to NAC in gastric cancer patients before NAC treatment. Additional larger cohort study aimed at understanding relationship between response to therapy and ctDNA instability are further required. Citation Format: Yongning Jia, Honglin Zhu, Fei Pang, Fei Shan, Shuangxi Li, Danhua Wang, Lin Li, Tonghui Ma, Ziyu Li. Chromosomal instability of circulating tumor DNA predicts response to neoadjuvant chemotherapy in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1991.

Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level.

Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer.

Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.

In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response.

Increased expression of YTHDF1 and HNRNPA2B1 as potent biomarkers for melanoma: a systematic analysis

BackgroundThe incidence and mortality of melanoma is increasing around the world. To deeply explain the mechanism insight into it, we conducted a systematic analysis to examine the levels of regulatory genes of the common RNA epigenetic modification-N6-methyladenosine (m6A) in patients with melanoma compared by the healthy.MethodsWe analyzed the expression of m6A Eraser, Writer, and Reader genes based on publicly available datasets on Oncomine and validated the results with a gene expression omnibus dataset. Hub genes were identified with Cytohubba and the frequency of copy number alterations was analyzed with the cBioPortal tool.ResultsThe results revealed the up-regulation of YTHDF1 and HNRNPA2B1 in melanoma. Combining the two genes improved the efficacy in diagnosing melanoma by about 10% compared to each gene alone. Hub genes identified with four analysis methods were compared and the overlapping genes were selected. These genes were enriched in several gene ontology terms. Genes related to p53-signaling consisted of CDK2, CDK1, RRM2, CCNB1, and CHEK1. All five genes were positively correlated with either YTHDF1 or HNRNPA2B1, suggesting that both genes may affect m6A modification by the five genes, further up-regulating their expression and facilitate their roles in inhibiting p53 to suppress tumorigenesis. We also observed major mutations in YTHDF1 and HNRNPA2B1 that led to their amplification in melanoma. Significant differences were observed in the clinical characteristics of patients with altered and unaltered m6A regulatory genes such as tumor stage and treatment response.ConclusionsWe, for the first time, identified a combination of m6A regulatory genes to diagnose melanoma. We also analyzed m6A-related genes more comprehensively based on systematic complete data. We found that YTHDF1 and HNRNPA2B1 were altered in melanoma and might influence the development of the disease through signaling pathways such as p53.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential

BackgroundMetastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome.MethodsPNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121).ResultsThe CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene “metastasis” signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression.ConclusionsMeasuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

Patterns of chromosome 18 loss of heterozygosity in multifocal ileal neuroendocrine tumors.

Ileal neuroendocrine tumors (NETs) represent the most common neoplasm of the small intestine. Although up to 50% of patients with ileal NETs are diagnosed with multifocal disease, the mechanisms by which multifocal ileal NETs arise are not yet understood. In this study, we analyzed genome‐wide sequencing data to examine patterns of copy number variation in 40 synchronous primary ileal NETs derived from three patients. Chromosome (chr) 18 loss of heterozygosity (LOH) was the most frequent copy number alteration identified; however, not all primary tumors from the same patient had evidence of this LOH. Our data revealed three distinct patterns of chr18 allelic loss, indicating that primary tumors from the same patient can present different LOH patterns including retention of either parental allele. In conclusion, our results are consistent with the model that multifocal ileal NETs originate independently. In addition, they suggest that there is no specific germline allele on chr18 that is the target of somatic LOH.

Next-generation sequencing identifies recurrent copy number variations in invasive breast carcinomas from Ghana.

African and African American (AA) women have a higher incidence of triple negative breast cancers (TNBC) with high histological grade and aggressive clinical behavior, but the reasons are not fully understood. We recently found that the oncogenic protein EZH2 is overexpressed in Ghanaian breast cancer, with 16% of the tumors expressing cytoplasmic EZH2. Understanding the molecular underpinnings of these aggressive tumors lead to the identification of potential targetable oncogenic drivers. We characterized the copy number variations of 11 Ghanaian breast tumors by targeted multiplexed PCR-based DNA next generation sequencing (NGS) of over 130 cancer-relevant genes. While the DNA quality was not optimal for mutation analysis, 90% of the tumors had frequent recurrent copy number alterations (CNAs) of 17 genes: SDHC, RECQL4, TFE3, BCL11A, BCL2L1, PDGFRA, DEK, SMUG1, AKT3, SMARCA4, VHL, KLF6, CCNE1, G6PD, FGF3, ABL1, and CCND1, with the top oncogenic functions being mitotic G1–G1/S phase regulation, gene transcription, apoptosis, and PI3K/AKT pathway. The most common recurrent high level CNAs were gains of RECQL4 and SDHC, in 50% and 60% of cases, respectively. Network analyses revealed a significant predicted interaction among 12 of the 17 (70.6%) genes with high level CNAs (p=5.7E-07), which was highly correlated with EZH2 expression (r=0.4–0.75). By immunohistochemistry, RECQL4 and SDHC proteins were upregulated in 53 of 86 (61.6%) and 48 of 86 (56%) of Ghanaian invasive carcinomas tissue samples. In conclusion, our data show that invasive carcinomas from Ghana exhibit recurrent CNAs in 17 genes with functions in oncogenic pathways including PI3K/AKT and G1–G1/S regulation, which may have implications for the biology and treatment of invasive carcinomas in African and AA women.

Abstract PD8-06: The genomic landscape of metastatic special histologic types of breast cancer

Abstract Introduction: Breast cancer (BC) is heterogeneous and comprises diverse entities with distinctive phenotypes, biology and clinical presentations. There are 21 special histologic types, accounting for approximately 20% of invasive BCs. Large-scale sequencing studies have focused preferentially on common histologic forms, and data on the genomic landscape of special histologic types, particularly in the metastatic setting, are scarce. Here, we sought to define the repertoire of somatic genetic alterations of metastatic special histologic types of BC. Materials and methods: We conducted a reanalysis of MSK-IMPACT targeted sequencing data of 346 special histologic types of BC from Razavi et al (Cancer Cell 2018), including invasive lobular (ILC; primary, n=129; metastasis, n=150), mucinous (primary n=11, metastasis n=5), micropapillary (primary n=8, metastasis n=13) and metaplastic carcinomas (primary, n=6; metastasis, n=11). Somatic single nucleotide variants (SNVs) and copy number alterations (CNAs) were inferred using a validated bioinformatics pipeline, and mutational signatures were assessed with SigMA. Genetic alterations of metastatic special histologic type BCs were compared to those of primary tumors and metastatic invasive ductal carcinomas of no special type (IDC-NSTs) matched by age, menopausal status and estrogen receptor (ER)/HER2 status at a 1:2 ratio for ILCs and at a 1:3 ratio for the remaining BCs. Results: The most frequently mutated genes in metastatic ILCs were CDH1 (71%), PIK3CA (49%), ESR1 (20%) and KMT2C (19%). ESR1 mutations were more frequent in metastatic than in primary ILCs (20% vs 3%; P&amp;lt;0.01). Metastatic ILCs, as compared to matched metastatic IDC-NSTs, had a higher frequency of mutations in CDH1 (71% vs 2%; P&amp;lt;0.001), KMT2C (19% vs 10%; P&amp;lt;0.05), TBX3 (16% vs 5%; P&amp;lt;0.001), ERBB2 (12% vs 3%; P&amp;lt;0.01) and FOXA1 (10% vs 2%; P&amp;lt;0.05), and a lower frequency of mutations in TP53 (16% vs 36%; P&amp;lt;0.001) and GATA3 (6% vs 16%; P&amp;lt;0.01). GATA3, NCOR1 and ESR1 (each, 40%) were the most frequently mutated genes in metastatic mucinous BCs. ESR1 and NCOR were numerically more frequent in metastatic than in primary mucinous BCs (40% vs 0%; P&amp;gt;0.05). Metastatic mucinous BCs had a numerically higher frequency of mutations in ESR1 (40% vs 6%; P&amp;gt;0.05) and GATA3 (40% vs 0%; P&amp;gt;0.05), and lower frequency of TP53 mutations (20% vs 53%; P&amp;gt;0.05) than matched metastatic IDC-NSTs. PIK3CA and TP53 were the most recurrently mutated genes in metastatic micropapillary BCs and were numerically more frequent in metastatic than in primary tumors (PIK3CA, 61% vs 25%; TP53, 46% vs 25%; P&amp;gt;0.05). PIK3CA was more frequently mutated in metastatic micropapillary BCs than IDC-NSTs (61% vs 23%; P&amp;lt;0.05). Metastatic metaplastic BCs most frequently harbored mutations in TP53 (72%) and PIK3CA (45%), with comparable frequencies in primary metaplastic BCs. ATM (27% vs 3%; P&amp;lt;0.05), MAP3K13 and YAP1 (27% vs 0%; P&amp;lt;0.05) were more frequently mutated in metastatic metaplastic BCs than in matched IDC-NSTs. No differences in the frequency of CNAs were seen between metastatic BCs of special histologic type and matched primary tumors or metastatic IDC-NSTs. Akin to metastatic IDC-NSTs, aging or APOBEC were the predominant mutational SNV signatures in metastatic ILCs, micropapillary and metaplastic BCs. Conclusions: The repertoire of somatic genetic alterations of primary and metastatic special histologic type BCs markedly overlaps. Nonetheless, there is an enrichment of mutations in ESR1 in metastatic BCs of typically ER-positive special histologic type. Metastatic BCs of special histologic type show an enrichment of similar genetic alterations as primary tumors, such as a higher frequency of mutations in CDH1, FOXA1 and TBX3 in ILCs, a lower frequency of PIK3CA and TP53 mutations in mucinous BCs and a higher frequency of PIK3CA and TP53 mutations in micropapillary and metaplastic BCs. Citation Format: Fresia Pareja, Simon SK Lee, Francisco Beca, Lorenzo Ferrando, Pier Selenica, David N Brown, Hannah Y Wen, Hong Zhang, Edi Brogi, Pedram Razavi, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. The genomic landscape of metastatic special histologic types of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-06.

Abstract P4-05-08: Genomic landscape of breast cancer occurring in elderly individuals

Abstract Introduction: There is evidence to suggest that the biology of breast cancer (BC) might vary according to age, as BCs in elderly patients might be associated with accumulation of DNA damage and BCs in younger individuals might be enriched for genetic alterations affecting cancer predisposition genes. Whether the genetic landscape of BC in elderly individuals differs is yet to be determined. Here, we sought to describe the spectrum of somatic genetic alterations and mutational signatures in BC according to age. Materials and Methods: We conducted a re-analysis of two cohorts of BC: i) MSK-IMPACT targeted sequencing cohort (primary, n=918; metastatic, n=1,000) and ii) whole-exome sequencing data of primary BCs from The Cancer Genome Atlas (TCGA) BC study (n=1087). BCs of younger (&amp;lt;65 years old) and elderly individuals (≥ 65 years old) were compared, matched by estrogen receptor (ER)/HER2 status at a 2:1 ratio (MSK-IMPACT) or a 1:1 ratio (TCGA). Somatic single nucleotide variants and copy number alterations (CNAs) were determined using a validated bioinformatics pipeline, and mutational signatures were defined using DeconstructSigs and SigMA. Two-tailed Fisher’s exact test was performed. Results: Primary BCs in elderly and younger patients, matched by ER/HER2 status, from the MSK-IMPACT (n=220 and n=440) and TCGA (n=290, each) cohorts, and metastatic BCs from elderly (n=98) and younger (n=196) patients from the MSK-IMPACT cohort, matched by ER/HER2 status, were compared. In both cohorts of primary BCs, PIK3CA, TP53 and CDH1 were the most frequently mutated genes. Compared to younger patients, primary BCs in elderly patients from the MSK-IMPACT cohort displayed a lower frequency of mutations in TP53 (23% vs 34%, P&amp;lt;0.01), AKT1 (2% vs 6%, P&amp;lt;0.05) and MAP2K4 (0.4% vs 4%, P&amp;lt;0.01). In the TCGA cohort, somatic mutations in AKT1 were found to be less frequent in elderly than in younger patients (1% vs 5%, P&amp;lt;0.05), whereas NF1 (2% vs 6%, P&amp;lt;0.05) mutations were more frequent in elderly than in younger patients with primary BCs. Our analyses of BC metastases subjected to MSK-IMPACT revealed that elderly patients harbored a higher frequency of mutations in NCOR1 (11% vs 4%, P&amp;lt;0.05) and RUNX1 (9% vs 2%, P&amp;lt;0.05) than younger patients. The frequency of bi-allelic inactivation of homologous recombination DNA repair deficiency (HRD) genes (i.e. BRCA1, BRCA2, PALB2, RAD51C and RAD51D) in primary BCs of elderly and younger individuals in the TCGA cohort was 2% and 5%, respectively. No differences in the frequency of CNAs were observed between the two groups. The most frequent mutational signatures found in primary BCs of elderly and younger patients were aging, APOBEC and HRD in the MSK-IMPACT cohort, and aging, APOBEC and HRD in primary BCs of the TCGA cohort, with APOBEC being enriched in younger patients compared to elderly patients. The most frequent mutational signatures in metastatic BCs in elderly and younger individuals of the MSK-IMPACT cohort were APOBEC followed by aging and HRD. Conclusion: Our findings show that BCs in elderly patients harbor a lower frequency of somatic mutations in TP53, AKT1 and MAP2K4 than younger individuals. In contrast, BCs in elderly patients more frequently harbor somatic mutations in the transcription factors RUNX1 and NCOR1. Bi-allelic alterations affecting HRD-related genes was numerically more frequent in younger than in older patients. As expected, BCs in elderly patients show a numerically higher incidence of the aging signature, whereas primary BCs affecting younger patients displayed a higher frequency of the APOBEC signature than primary BCs from elderly individuals. Citation Format: Pier Selenica, Fresia Pareja, Audree Tadros, Lorenzo Ferrando, David N Brown, Hong Zhang, Pedram Razavi, Diana Mandelker, Mark E Robson, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. Genomic landscape of breast cancer occurring in elderly individuals [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-08.

Abstract P6-10-21: Evaluation of genomic changes in ductal carcinoma in situ as potential biomarkers of recurrence risk

Abstract Routine breast cancer screening has increased detection of pre-invasive lesions such as ductal carcinoma in situ (DCIS). Currently, DCIS cases are mostly treated with wide local excision combined with adjuvant radiotherapy and/or hormonal therapy, even though only 25% of DCIS treated with surgery alone recur with 50% of recurrences being invasive. At present, prediction of recurrence risk is based on histopathological features and patient characteristics but reliable identification of individual recurrence risk is still challenging. Certain genetic aberrations (i.e. copy number variation, somatic mutations) are associated with breast cancer progression. While most recurrences are assumed to be genetically related to the primary tumour (“clonal recurrence”), the prevalence of ipsilateral independent primary events (“non-clonal recurrence”) after treatment for DCIS is uncertain. If this is a substantial proportion of cases, their presence would compromise attempts to find tumour intrinsic biomarkers of recurrence. To enable the discovery of better recurrence biomarkers, this project initially aims to identify cases with primary DCIS and later clonal ipsilateral recurrence. Sections from formalin-fixed paraffin-embedded blocks from primary DCIS cases and their subsequent recurrences were obtained from Nottingham City Hospital (UK). Two pathologists reviewed representative sections to identify areas for microdissection. Tumour epithelial cells were needle-microdissected and DNA was isolated. Sequencing libraries were prepared using a) a custom capture panel of 110 breast cancer-related genes or b) NEBNext Ultra II for low-coverage whole-genome sequencing (LC-WGS) depending on DNA quality and quantity. Sequencing was then performed on Illumina NextSeq 500 system with an average coverage of a) 400x and b) 1.3x, respectively. Data was processed by an in-house bioinformatics pipeline to detect and filter for high confidence somatic variants. Copy number profiles were obtained using CopywriteR (panel off-target reads), PureCN (panel on-target reads) and Control-FREEC (LC-WGS). Of 33 patients initially ascertained on the basis of a recurrent breast event, 30 had an ipsilateral recurrent tumour after 4.7 years on average, and 24 primary/recurrence pairs had sufficient data available to determine clonality. Eighteen pairs (75%) were classified as clonal by the following criteria: occurring in similar locations, showing a similar or higher tumour grade, and sharing ≥ 3 copy number breakpoints or likely somatic variants in the genes included in the panel. Six pairs (25%) did not meet this criteria and were classified as non-clonal. Preliminary data analysis shows that invasive recurrences (n = 12) were detected on average 3 years later than DCIS recurrences (n = 6), despite constant surveillance. The most commonly observed somatic mutations occurred in the TP53 and PIK3CA genes. Genetic variants in TP53 and PIK3CA were equally common in primary tumours with clonal and non-clonal recurrence. Frequent copy number alterations involved losses on 16q as well as gains on 1q, 8q and 17q, whereat the 8q gain was less profound. The presence of 25% non-clonal recurrences shows that differentiating between clonal and non-clonal recurrences might be crucial for further recurrence risk biomarker discovery. Thus, this project will proceed with comparing genetic variation in primary DCIS with clonal recurrence to DCIS cases without later recurrence. These samples are currently being obtained and analysed. Citation Format: Dorothea Lesche, Hugo Saunders, Sakshi Mahale, Magnus Zenthoven, Siobhan Huges, Tanjina Kader, Niko Thio, Islam M Miligy, Jia-Min Pang, Andrew R Green, Emad A Rakha, Ian G Campbell, Stephen B Fox, Kylie Gorringe. Evaluation of genomic changes in ductal carcinoma in situ as potential biomarkers of recurrence risk [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-21.

Systematic Analyses Reveals the Functional Roles of Ferroptosis Across Cancers

Background: Ferroptosis is a newly discovered type of cell death that is closely related to cancer, however, the characteristics of ferroptosis and its regulators in cancers are still largely unknown. Methods: Based on the cancer omics data in The Cancer Genome Atlas, we comprehensively analyzed the alterations of ferroptosis regulator genes (FRGs) and computationally established the ferroptosis potential index (FPI) to reveal the functional roles of ferroptosis in cancer. Findings: We found that the mutation rates of FRGs were generally low, but most FRGs were differentially expressed in tumors among various cancer types. Frequent copy number alterations (CNA) and differential DNA methylation of FRGs contributed greatly to their aberrant expression. In most cancers, the FPI was higher in tumors than in the adjacent normal tissues and was associated with histological/molecular subtypes and clinical features. We observed that the FPI was negatively correlated with a number of metabolism pathways such as terpenoid backbone biosynthesis, but positively associated with metastasis-related pathways such as epithelial-mesenchymal transition (EMT), WNT beta-catenin signaling and angiogenesis. The FPI was also correlated immune microenvironment since it was positively correlated with pathways such as IL6 JAK-STAT signaling, inflammatory response, complement. Furthermore, it was observed that high FPI predicted significantly worse overall survival in glioblastoma multiforme, renal clear/papillary cell carcinoma, hepatocellular carcinoma, and lung adenocarcinoma, while FPI and FRGs were intensively correlated with cancer drug sensitivity. Interpretation: Our study presents a systematical analysis of ferroptosis and its regulatory genes across cancers, and highlights the potential of ferroptosis-based cancer therapy. Funding Statement: This work was supported by grants from National Key RD and Science and Technology Program of Guangdong (2019B020227002 to RH.X.). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Not required.