Somatic DNA Mutations Research Articles

Mitochondrial DNA somatic mutation burden and heteroplasmy are associated with chronological age, smoking, and HIV infection.

The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low‐frequency mutations. We use primer ID‐based next‐generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D‐loop, in 164 women and girls aged 2–72 years, of whom 35% were smokers and 56% were HIV‐positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.

Proteogenomic landscape of squamous cell lung cancer

How genomic and transcriptomic alterations affect the functional proteome in lung cancer is not fully understood. Here, we integrate DNA copy number, somatic mutations, RNA-sequencing, and expression proteomics in a cohort of 108 squamous cell lung cancer (SCC) patients. We identify three proteomic subtypes, two of which (Inflamed, Redox) comprise 87% of tumors. The Inflamed subtype is enriched with neutrophils, B-cells, and monocytes and expresses more PD-1. Redox tumours are enriched for oxidation-reduction and glutathione pathways and harbor more NFE2L2/KEAP1 alterations and copy gain in the 3q2 locus. Proteomic subtypes are not associated with patient survival. However, B-cell-rich tertiary lymph node structures, more common in Inflamed, are associated with better survival. We identify metabolic vulnerabilities (TP63, PSAT1, and TFRC) in Redox. Our work provides a powerful resource for lung SCC biology and suggests therapeutic opportunities based on redox metabolism and immune cell infiltrates.

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available inpatient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Detection of a novel panel of somatic mutations in plasma cell-free DNA and its diagnostic value in hepatocellular carcinoma.

Background/aims: Circulating cell-free DNA (cfDNA) contains tumor-specific alterations and could potentially serve as “liquid biopsy”. The study was to identify a novel panel of hepatocellular carcinoma (HCC)-specific mutations in plasma cfDNA and to assess its value in the diagnosis of HCC.Materials and methods: 33 HCC tissue, 37 blood, and 37 swab specimens were collected from HCC patients and control individuals. Genomic DNA was subjected to next-generation sequencing. The selected mutations in the plasma cfDNA in the HCC versus control groups were compared, and the diagnostic performance of cfDNA mutations was evaluated.Results: A majority of selected mutations in the HCC tissue DNA, ranging from 52% to 84%, was detected in the matched plasma cfDNA. For the selected mutations, receiver operating characteristic (ROC) analysis revealed an area under the ROC curve (AUC) of 0.92, sensitivity of 65%, and specificity of 100% for the diagnosis of HCC regardless of alpha-fetoprotein (AFP) status. Detection of the selected mutations in cfDNA in combination with AFP exhibited better diagnosis performance, with AUC of 0.96, sensitivity of 73%, and specificity of 100% for AFP-negative patients, whereas the AUC was 0.86 with sensitivity of 53% and specificity of 100% for AFP-positive patients. Furthermore, the rates of the selected mutations were significantly greater in recurrent HCC than in non-recurrent HCC (P<0.05).Conclusions: This study has identified a novel panel of somatic mutations, and detection of the mutations in plasma cfDNA shows good diagnostic performance. Therefore, this approach holds promise as a novel tool for diagnosing HCC.

PF542 MITOCHONDRIAL GENOMIC ANALYSIS IN PATIENTS WITH MYELOID MALIGNANCIES

Background: Mitochondrial DNA (mtDNA) somatic mutation frequently occur in neoplasms. Because of heteroplasmy of mDNA, it is difficult to determine whether genetic variations in mtDNA occur as germline variants or truly somatic variant. Genetic abnormalities in nuclear DNA in myelodysplastic syndrome (MDS) and other myeloid malignancies are massively investigated. However, the molecular abnormalities in mtDNA in MDS and other myeloid malignancies have not been poorly revealed. In this study, we analyzed full sequence of mtDNA using next generation sequencing (NGS) technique in patients with MDS or other myeloid malignancies to investigate possible variants in mtDNA in these patients. Aims: The aim of this study is to find mtDNA variants and to select possible somatic mtDNA variants and investigate possible association with disease characteristics. Methods: We collected total DNA from bone marrow or peripheral blood samples with malignant cells in 35 patients with various myeloid malignancies [15 MDS, 7 acute myeloid leukemia (AML) without recurrent genetic abnormalities, 6 primrary myelofibrosis (PMF), 4 myelomomocytic leukemia (CMML), and 2 atypical chronic myeloid leukemia (aCML)]. In addition, 49 patients with non-hematologic diseases are included as controls. Whole mitochondrial genomic sequencing was amplified, and NGS was performed by HiSeq 2000 (illumine). In addition, targeted sequencing using genomic DNA for 138 genes including well-known genes in MDS was also performed. Results: We selected candidate mtDNA single nucleotide variants (mt-SNVs) according to the heteroplasmy levels of variants and filtering from reference database and control patients’ results. We discarded small insertion or deletions from our analysis. When same selection criteria were applied in control samples, no candidate mt-SNVs were found except 1 known mutation in MELAS patients. We finally selected 53 candidate mt-SNVs in 26 patients (74.3%). The 40/53 (75.5%) of mt-SNVs were found in protein coding genes, 10/53 (18.9%) were found in rRNA genes, and 3/53 (5.7%) were found in tRNA genes. The mt-SNVs were most frequently found in CYTB and CO3, and ND3, ND4, ND4, RNR1, and RNR2 in descending order. mt-SNVs in CO3 were most frequently found in MPN patients, and variants in ND3 and ND4 were most frequently found in AML patients. Most of patients with ND3 variants had ASXL1 mutations. Summary/Conclusion: Our results suggested that somatic mtDNA may present different distributions among disease subtypes of myeloid malignancies, which may give new insight on the pathogenesis of myeloid malignancies.

PS1229 CHARACTERIZING THE ETIOLOGY OF PEDIATRIC ACUTE MYELOID LEUKEMIA BY STUDYING MUTATION ACCUMULATION IN NORMAL HEMATOPOIESIS

Background:The incidence of leukemia peaks early in life and decreases before rising again later in life. Considering that recurrent somatic DNA mutations are thought to be the driving force behind the genesis of leukemia, this phenomenon represents an apparent paradox as young cells have less somatic (oncogenic) mutations than adult cells. Although therapy and outcome have significantly improved over the last decades, pediatric acute myeloid leukemia (pAML) remains a cancer with a high relapse rate and poor prognosis. Approximately 60% of pAML has somatic chromosomal abnormalities like translocations and inversions, while the rest is cytogenetically normal (CN). The most common chromosomal translocation 8;21 (∼11% of pAML) fuses the AML1 gene on chromosome 21 to the ETO gene on chromosome 8, leading to the driver event of this pAML. In CN pAML, often single‐gene mutations can be found in driver genes like FLT3, WT1 and NPM1. The various chromosomal abnormalities and mutations in combination with the different ages of onset underscore the large genetic heterogeneity of pAML. However, all these pAML subtypes receive the same treatment regimen.Aims:To develop more targeted therapies aimed at improving survival, increased understanding of the etiology and genetic landscape of pAML is needed. We hypothesize that the underlying etiology of these different pAML subtypes differ in cellular origin and time of onset during development.Methods:To test this hypothesis, we characterized the somatic mutations in the genomes of pAML and matched normal hematopoietic stem and progenitor cells (HSPCs) of pediatric patients with either a CN‐ or an AML1‐ETO‐driven subtype. With this knowledge, we can identify and study the mutational processes underlying the genesis of pAML as well as track down the cellular origin of the disease using a phylogenetic approach. To identify somatic mutations in the normal HSPCs, we clonally expanded primary single cells from the bone marrow biopsies to obtain sufficient DNA for whole genome sequencing (WGS) analysis.Results:Previously, we have performed similar analyses in bone marrow‐ and umbilical cord blood‐derived HSPCs of healthy donors and created a healthy reference line for lifelong mutation accumulation. We found that the mutation rate is higher in the pAMLs compared to the healthy HSPCs of the same patient as well as our reference line. In addition, we performed WGS on 10 HSPC clones of one pAML patient to track down its clonal origin. For this, we genotyped the observed leukemic blast somatic mutations in healthy HSPCs of the same patient and determined the genetic relatedness between these cells and the blasts. Using this approach, we constructed a developmental lineage tree and tracked down the branch that gave rise to the pAML.Summary/Conclusion:This research provides novel insights into the etiology of pAML and differences therein between subtypes, which may contribute to the development of more targeted treatment.

A Novel Method for Identifying the Potential Cancer Driver Genes Based on Molecular Data Integration.

The identification of the cancer driver genes is essential for personalized therapy. The mutation frequency of most driver genes is in the middle (2-20%) or even lower range, which makes it difficult to find the driver genes with low-frequency mutations. Other forms of genomic aberrations, such as copy number variations (CNVs) and epigenetic changes, may also reflect cancer progression. In this work, a method for identifying the potential cancer driver genes (iPDG) based on molecular data integration is proposed. DNA copy number variation, somatic mutation, and gene expression data of matched cancer samples are integrated. In combination with the method of iKEEG, the "key genes" of cancer are identified, and the change in their expression levels is used for auxiliary evaluation of whether the mutated genes are potential drivers. For a mutated gene, the concept of mutational effect is defined, which takes into account the effects of copy number variation, mutation gene itself, and its neighbor genes. The method mainly includes two steps: the first step is data preprocessing. First, DNA copy number variation and somatic mutation data are integrated. Then, the integrated data are mapped to a given interaction network, and the diffusion kernel is used to form the mutation effect matrix. The second step is to obtain the key genes by using the iKGGE method, and construct the connection matrix by means of the gene expression data of the key genes and mutation impact matrix of the mutated genes. Experiments on TCGA breast cancer and Glioblastoma multiforme datasets demonstrate that iPDG is effective not only to identify the known cancer driver genes but also to discover the rare potential driver genes. When measured by functional enrichment analysis, we find that these genes are clearly associated with these two types of cancers.

Impact of prior chemotherapy or radiation therapy on tumor mutation burden in NSCLC.

2627 Background: Higher non-synonymous tumor mutation burden (TMB) in non-small cell lung cancer (NSCLC) is associated with a higher likelihood of response to checkpoint inhibitors. Tissue samples subject to TMB analysis may be obtained after exposure to cytotoxic chemotherapy or radiation therapy – both of which introduce somatic mutations in DNA and can influence the number of identified mutations. The role of TMB as a potential predictive marker for immunotherapy is evolving, and the impact of prior therapy on TMB could influence interpretation. Methods: Eligible cases were from patients with confirmed NSCLC, available clinical annotation and tumor molecular profiling including TMB analysis at a CLIA-certified genomics laboratory (Caris Life Sciences, Phoenix, AZ) using the Illumina NextSeq platform. TMB was calculated using only missense mutations that had not been previously reported as germline alterations. Treatment history was obtained for each patient under an IRB approved protocol to determine whether patients had had received chemotherapy or radiation therapy in the year prior to collection of the tissue subject to TMB analysis. Data analysis was performed using the chi-square test of deviance to evaluate whether TMB was statistically significantly different between groups, correcting for smoking status. Results: Out of 1,118 patients identified, 459 cases met all eligibility criteria and were evaluated. 76 patients (17%) received either chemotherapy or radiation prior to tissue collection. Samples acquired prior to any therapy had a median TMB of 10 mut/Mb vs. 11 mut/Mb in samples acquired after any therapy. After adjusting for smoking, there was no significant difference in TMB between these cohorts (p = 0.41). Secondary pair wise analysis showed no statistically significant difference in TMB from chemotherapy-naïve and chemotherapy-treated samples (p = 0.28). The same was true for radiation (p = 0.75). Collection of clinical data is ongoing and further analysis, including additional cases will be presented. Conclusions: Though cytotoxic chemotherapy and radiation therapy can introduce somatic mutations, prior exposure to either was not associated with a significant difference in TMB.

An immune response-associated genomic model to predict early resistance to PD1/PD-L1 blockade among real-world TMB-high Chinese cancer patients.

e14167 Background: TMB-high was used as a favorable biomarker to predict the response to anti-PD-1/PD-L1 therapy, but a substantial portion of the TMB-high patients suffered from early resistance with limited clinical benefits but potential severe adverse effects. Methods: Whole-exome sequencing were performed in more than 200 advanced cancer patients, who received anti-PD-1 therapy alone or in combination with chemotherapy and targeted therapy across 20 provinces of China, with quarterly follow-ups. The genomic profiles of the patients with progressive disease (PD) and complete remission (CR) outcomes were individually analyzed and the potential resistance and hypersensitive biomarkers were identified. These biomarkers were formulated into an immune response-associated genomic (IRAG) model based on XGBoost algorithm to predict the sensitivity to PD-1 blockage and early resistance among TMB-high patients. Results: Non-synonymous somatic mutations (CTNNB1, B2M, PD-L1 and HLA class I genes), somatic gene amplifications (MDM2/4, CDK4, CCND1/FGF3/FGF4/FGF19, FLT3 and MYC), chromosome 9p21 deletion, and loss of heterozygosity in chromosome 6 HLA-I region were identified as potential resistance biomarkers in the PD group. TMB-high, microsatellite instability (MSI), TMB-low with indel-proportion high, POLE loss-of-function mutations, Epstein-Barr virus infection and DNA mismatch repair-deficient germline and somatic mutations were identified as potential hypersensitivity biomarkers in the CR group. By combining all genomic resistance and hypersensitivity biomarkers, we developed a quantitative IRAG model and separated the patients into three groups: TMB-low, TMB-high/IRAG-low and TMB-high/IRAG-high. The PFS in TMB-high/IRAG-low group was characterized with early clinical resistance, similar to the TMB-low group (median: 3.8 vs. 4.2 months, p = 0.22). The reanalysis of the patient set with known clinical outcomes showed that the TMB-high/IRAG-high group had a significantly longer PFS than the other two groups. (median: 10 vs. 3.8, 4.2 months, p &lt; 0.001). The TMB-high/IRAG-high group clearly showed higher chromosomal stability with fewer somatic copy number alterations. Conclusions: We developed a model based on one single genomic assay (whole-exome) to successfully identify those TMB-high patients who suffered from early resistance to PD-1 inhibitors. Larger cohort and independent analyses are necessary to verify the validity of these results.

Cardiovascular Manifestations of Mitochondrial Disease.

Genetic mitochondrial cardiomyopathies are uncommon causes of heart failure that may not be seen by most physicians. However, the prevalence of mitochondrial DNA mutations and somatic mutations affecting mitochondrial function are more common than previously thought. In this review, the pathogenesis of genetic mitochondrial disorders causing cardiovascular disease is reviewed. Treatment options are presently limited to mostly symptomatic support, but preclinical research is starting to reveal novel approaches that may lead to better and more targeted therapies in the future. With better understanding and clinician education, we hope to improve clinician recognition and diagnosis of these rare disorders in order to improve ongoing care of patients with these diseases and advance research towards discovering new therapeutic strategies to help treat these diseases.

Mucosal and Cutaneous Human Papillomavirus Infections and Cancer Biology.

Papillomaviridae is a family of small non-enveloped icosahedral viruses with double-stranded circular DNA. More than 200 different human papillomaviruses (HPVs) have been listed so far. Based on epidemiological data, a subgroup of alphapapillomaviruses (alpha HPVs) was referred to as high-risk (HR) HPV types. HR HPVs are the etiological agents of anogenital cancer and a subset of head and neck cancers. The cutaneous HPV types, mainly from beta and gamma genera, are widely present on the surface of the skin in the general population. However, there is growing evidence of an etiological role of betapapillomaviruses (beta HPVs) in non-melanoma skin cancer (NMSC), together with ultraviolet (UV) radiation. Studies performed on mucosal HR HPV types, such as 16 and 18, showed that both oncoproteins E6 and E7 play a key role in cervical cancer by altering pathways involved in the host immune response to establish a persistent infection and by promoting cellular transformation. Continuous expression of E6 and E7 of mucosal HR HPV types is essential to initiate and to maintain the cellular transformation process, whereas expression of E6 and E7 of cutaneous HPV types is not required for the maintenance of the skin cancer phenotype. Beta HPV types appear to play a role in the initiation of skin carcinogenesis, by exacerbating the accumulation of UV radiation-induced DNA breaks and somatic mutations (the hit-and-run mechanism), and they would therefore act as facilitators rather than direct actors in NMSC. In this review, the natural history of HPV infection and the transforming properties of various HPV genera will be described, with a particular focus on describing the state of knowledge about the role of cutaneous HPV types in NMSC.

Cell-free DNA as a biomarker in diffuse large B-cell lymphoma: A systematic review.

Cell-free DNA (cfDNA), which is DNA released from cells into the circulation, is one of the most promising non-invasive biomarkers in cancer. This approach could be of interest for the management of Diffuse Large B-Cell Lymphoma (DLBCL) patients, which is the most common non-Hodgkin lymphoma. Then, the aim of this systematic review was to define the utility of cfDNA in this disease. Selected articles were classified in four groups, depending on the aspects of cfDNA studied, i.e. concentration, methylation, IgH gene rearrangements, and somatic mutations. While concentration and methylation of cfDNA need to be further analyzed, IgH gene rearrangements and somatic mutations seem to be the most promising biomarkers to date. Their detection has been shown to allow disease monitoring and early prediction of relapse. Although more efforts and standardization of techniques are needed, studying cfDNA in liquid biopsy may help improve the outcome of DLBCL patients.

Recent Advances in Liquid Biopsy in Precision Oncology Research.

Liquid biopsy is a minimally invasive test for cancer genetic status based on circulating tumor DNA (ctDNA), circulating tumor cells, or other tumor-derived materials in blood plasma. Although the minimal invasiveness and time resolution are attractive features of liquid biopsy, the limited amount of ctDNA in plasma poses problems. Recent developments in digital PCR and next-generation sequencing (NGS)-based technology have improved the accuracy of liquid biopsy. In particular, molecular barcoding technology in NGS-based methods, i.e., tagging of molecular barcodes to cell-free DNA before amplification, reduces technical errors by validating the consensus of sequences originating from a single molecule, leading to marked improvement of the accuracy and detection limit. However, substitutions caused by DNA damage and somatic mutations originating from normal cells are still obstacles to the sensitive detection of mutations on ctDNA. Since there have been only a few clinical applications, a deeper understanding of ctDNA biology and more advanced analytical technology are needed for the practical application of liquid biopsy.

Somatic mutations, DNA methylation, and expression of DNA repair genes in Arabidopsis thaliana treated with 5-azacytidine

An inhibitor of DNA methylation 5-azacytidine (5A) is a chemical analog of the nucleoside cytidine. This study investigated the influence of 5A-induced DNA hypomethylation on the accumulation of somatic DNA mutations (nucleotide substitutions, indels) in the Actin2 3′ untranslated region, nuclear internal transcribed spacer ITS1-5.8rRNA-ITS2, and the ribulose-1,5-bisphosphate carboxylase/oxygenase gene of Arabidopsis thaliana and analyzed concurrent changes in the expression of DNA methyltransferase and DNA repair genes. The 5A treatment (20 mg per 100 g of soil) decreased DNA methylation, and the detected 5A-induced demethylation was associated with the up-regulation of the DNA methyltransferase genes: chromomethylase AtCMT3, methyltransferase AtMETI, and domains rearranged methyltransferases AtDRM1 and AtDRM2. Cultivation of plants in the presence of 5A led to a considerable increase in the number of single nucleotide substitutions in the analyzed DNA regions of 5A-treated A. thaliana. The 5A treatment significantly increased the transcriptions of 7 DNA repair genes (endonuclease AtARP, DNA demethylases AtDME and AtDML2, DNA glycosylase AtMBD4, DNA damage-binding protein AtDDB1, and photolyases AtUVR2 and AtUVR3) out of the 17 analyzed genes from the base excision repair, nucleotide excision repair, and photoreactivation pathways. However, 5A decreased the transcription of DNA 3′-phosphatase AtZDP, DNA repair protein AtRad23a, mismatch repair proteins AtMsh2 and AtMsh3. It is possible that the changes in the transcription of the DNA repair genes contributed to the detected increase in the number of single nucleotide substitutions that accumulated in the 5A-treated A. thaliana. Taken together, the data indicate that there is an interaction between the processes of DNA methylation and mutation accumulation.

DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression.

BackgroundDNA mismatch repair (MMR) defects are a major factor in colorectal tumorigenesis in Lynch syndrome (LS) and 15% of sporadic cases. Some adenomas from carriers of inherited MMR gene mutations have intact MMR protein expression implying other mechanisms accelerating tumorigenesis. We determined roles of DNA methylation changes and somatic mutations in cancer-associated genes as tumorigenic events in LS-associated colorectal adenomas with intact MMR.MethodsWe investigated 122 archival colorectal specimens of normal mucosae, adenomas and carcinomas from 57 LS patients. MMR-deficient (MMR-D, n = 49) and MMR-proficient (MMR-P, n = 18) adenomas were of particular interest and were interrogated by methylation-specific multiplex ligation-dependent probe amplification and Ion Torrent sequencing.FindingsPromoter methylation of CpG island methylator phenotype (CIMP)-associated marker genes and selected colorectal cancer (CRC)-associated tumor suppressor genes (TSGs) increased and LINE-1 methylation decreased from normal mucosa to MMR-P adenomas to MMR-D adenomas. Methylation differences were statistically significant when either adenoma group was compared with normal mucosa, but not between MMR-P and MMR-D adenomas. Significantly increased methylation was found in multiple CIMP marker genes (IGF2, NEUROG1, CRABP1, and CDKN2A) and TSGs (SFRP1 and SFRP2) in MMR-P adenomas already. Furthermore, certain CRC-associated somatic mutations, such as KRAS, were prevalent in MMR-P adenomas.InterpretationWe conclude that DNA methylation changes and somatic mutations of cancer-associated genes might serve as an alternative pathway accelerating LS-associated tumorigenesis in the presence of proficient MMR.FundJane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid Juselius Foundation, and HiLIFE.

Hürthle Cell Carcinomas Exhibit Widespread Loss of Heterozygosity

Abstract Hürthle cell carcinomas (HCC) harbor recurrent somatic DNA mutations and mtDNA mutations.

PS02.195: CIRCULATING TUMOUR DNA HAS UTILITY IN THE CLINICAL MANAGEMENT OF ESOPHAGEAL CANCER

Abstract Background Cell-free DNA, a consequence of normal cell death and apoptosis, is found in the blood of all individuals. In cancer patients, a proportion of circulating cell free DNA will be derived from tumour cells, and is termed circulating tumour DNA (ctDNA).We have developed a blood-based ‘liquid biopsy’ to detect ctDNA in plasma of esophageal cancer patients. Methods Tumour, buffy-coat (germline) and plasma samples were obtained from 28 esophageal cancer patients at the time of diagnosis. Serial blood samples were collected from 8 patients. Somatic DNA mutations in 9 genes (TP53, ARID1A, SMAD4, CDKN2A, SMARCA4, NRG1, APC, PIK3CA and KRAS) were evaluated in tumour biopsies and plasma using targeted sequencing. Tumour specific mutations were confirmed by droplet digital PCR, which was used to track patient-specific ctDNA in plasma from serially collected blood samples. Results Somatic mutations in at least one of the targeted genes were detected in 19/28 (68%) tumour biopsies. The same mutations were detected in ctDNA from plasma in 9/19 (48%) patients. Additional mutations that were not detected in the tumour biopsies were detected in plasma DNA from 2 patients, highlighting the issues of intra-tumoural heterogeneity and sampling bias of tumour biopsies. ctDNA was more frequently detected in patients with advanced disease where it represented a greater proportion of the total cell-free plasma DNA than in patients with localised disease. In patients with serial samples, levels of ctDNA correlated with response to treatment, and rises in ctDNA could be detected prior to clinical evidence of relapse. Conclusion Detection of ctDNA using targeted sequencing and digital PCR is feasible in the majority of esophageal cancer patients, particularly those with advanced disease. Dynamic changes in ctDNA levels reflect response to treatment and disease relapse as determined by conventional clinical imaging. We conclude that ctDNA levels can provide supplemental evidence regarding disease staging, as well as prognostic and predictive information that can inform therapeutic pathway decisions in esophageal cancer patients. Disclosure All authors have declared no conflicts of interest.

Pan-Cancer Analysis Reveals Differential Susceptibility of Bidirectional Gene Promoters to DNA Methylation, Somatic Mutations, and Copy Number Alterations.

Bidirectional gene promoters affect the transcription of two genes, leading to the hypothesis that they should exhibit protection against genetic or epigenetic changes in cancer. Therefore, they provide an excellent opportunity to learn about promoter susceptibility to somatic alteration in tumors. We tested this hypothesis using data from genome-scale DNA methylation (14 cancer types), simple somatic mutation (10 cancer types), and copy number variation profiling (14 cancer types). For DNA methylation, the difference in rank differential methylation between tumor and tumor-adjacent normal matched samples based on promoter type was tested by the Wilcoxon rank sum test. Logistic regression was used to compare differences in simple somatic mutations. For copy number alteration, a mixed effects logistic regression model was used. The change in methylation between non-diseased tissues and their tumor counterparts was significantly greater in single compared to bidirectional promoters across all 14 cancer types examined. Similarly, the extent of copy number alteration was greater in single gene compared to bidirectional promoters for all 14 cancer types. Furthermore, among 10 cancer types with available simple somatic mutation data, bidirectional promoters were slightly more susceptible. These results suggest that selective pressures related with specific functional impacts during carcinogenesis drive the susceptibility of promoter regions to somatic alteration.

Abstract B42: Identification of CPT1A as a novel driver of proliferation in luminal breast cancer

Abstract Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor-positive, luminal breast tumors. Slower-growing luminal breast tumors are often successfully treated using endocrine-based therapies, resulting in a relatively good prognosis for these patients. However, more highly proliferative luminal tumors, even with the recent approval of CDK4/6 inhibitors, are often resistant, or become resistant, to current therapies, leading to a worse outcome for these women. Therefore, it is necessary to identify genetic events responsible for tumorigenesis and to identify potential druggable genetic alterations and/or pathways in order to improve clinical outcome. To address this question, we recently developed and used an innovative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA and METABRIC studies. By using an 11-gene mRNA-based gene expression signature of proliferation as a conceptual framework, we identified DNA copy number alterations and somatic mutations associated with luminal breast cancer proliferation. In order to identify the subset of amplified genes that are essential for cell viability, we analyzed a dataset of 27 breast cancer cell lines with available mRNA expression and genome-wide shRNA proliferation data. By integrating DNA copy number and shRNA analyses, we identified amplified carnitine palmitoyltransferase 1A (CPT1A) as a novel driver of proliferation in luminal breast cancer. CPT1A was found to be amplified in 40.2% of highly proliferative (top quartile) luminal tumors compared to 24.7% of all other samples and CPT1A DNA copy number status correlated with mRNA expression levels. Importantly, CPT1A was shown to be essential in luminal breast cancer cell lines with a high proliferation signature score. By analyzing RPPA data from luminal breast tumors, we confirmed CPT1A amplified tumors have increased expression of protein markers of proliferation. Since CPT1A is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during fatty acid β oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. Interestingly, our analyses of RNA-seq data (n=1,031) demonstrated that highly proliferative luminal tumors have significantly higher CPT1A mRNA expression than either basal-like, which are largely synonymous with TNBC, or less proliferative luminal tumors, suggesting that these tumors may have a greater dependency on CPT1A activity. CPT1A has two major variants: cytoplasmic Variant 1 is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during FAO while nuclear Variant 2 is purported to mediate histone deacetylase activity. Analyses of RNAseq data (n=819) demonstrated that Variant 1 represents &amp;gt;99.2% of CPT1A mRNA transcripts in a given tumor, suggesting the FAO-associated role of CPT1A is the more critical function of this protein in breast cancer. Consistent with this observation, we determined that breast cancer cell lines with high CPT1A protein expression are significantly more sensitive to etomoxir, a CPT1A-specific inhibitor known to repress FAO, when compared to cell lines with low CPT1A protein levels. Finally, we determined that under glucose-depleted conditions, cell lines with high CPT1A expression showed no difference in etomoxir response whereas cells with low CPT1A become significantly more sensitive. Collectively, these data suggest that CPT1A amplification and overexpression contribute to proliferation. Given that a number of approved or experimental drugs target CPT1A or FAO, our data suggest that CPT1A and/or FAO may represent a novel therapeutic target for luminal breast cancer. Citation Format: Kristina Mastrioanni, Kimberly Parker, Michael L. Gatza. Identification of CPT1A as a novel driver of proliferation in luminal breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B42.

Abstract 4542: Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay

Abstract Introduction: Liquid biopsy-based detection and monitoring of tumor-specific somatic mutations in cell-free DNA has great potential to improve patient care by detecting cancer early, assisting adjuvant therapy decision-making, monitoring residual disease, determining treatment effects, assessing relapse and the consequent need for follow-up intervention. We have developed a novel highly sensitive and specific approach for the detection and quantification of circulating tumor DNA (ctDNA) by tracking personalized cancer signatures in plasma. We recently described our ability to detect ctDNA presence longitudinally in patients with non-small cell lung cancer (NSCLC) and have validated this personalized approach for detection of tumor DNA in plasma, and made it available for research use only (RUO). Summary of Experimental Procedures: This study describes results of the Signatera RUO assay's analytical validation test titrations of cancer cell lines and their matched normal counterparts. Multiplex-PCR (mPCR) assay pools specific to the tumor mutational signatures were derived from whole-exome data analysis of the tumor cell line and its corresponding matched normal cell line. Tumor DNA spike-in mixtures were amplified using the mPCR assays and then barcoded and sequenced at ultra-high depth on the Illumina HiSeq instrument (average &amp;gt; 100,000 reads per target). This study also demonstrates the ability to detect ctDNA using Signatera RUO assay's personalized somatic mutations in patients with epithelial and non-epithelial ovarian cancer. Results/Data Summary: The Signatera RUO analytical validation results demonstrate a variant-level sensitivity of ~60% at spiked-in tumor DNA concentrations of 0.03% along with an assay specificity of 99.9% in wild-type DNA. By targeting 16 somatic mutations present in the tumor, an estimated LOD of 0.01% tumor DNA in a patient's plasma is achieved. The Signatera RUO assay also demonstrates the ability to detect ctDNA in plasma down to less than 0.1% VAF at baseline (prior to treatment) in epithelial and non-epithelial ovarian cancer. Conclusion: The Signatera RUO assay provides a unique method to noninvasively detect ctDNA by assaying for personalized cancer signatures in plasma by ultra-high-depth sequencing of custom-made multiplex PCR assays, with high sensitivity and high specificity. Our study demonstrates the potential of this technology to improve the current standard of care by enabling early recurrence detection and the monitoring of treatment effectiveness across several tumor types. Citation Format: Himanshu Sethi, Raheleh Salari, Samantha Navarro, Prashanthi Natarajan, Ramya Srinivasan, Scott Dashner, Tony Tin, Mustafa Balcioglu, Ryan Swenerton, Bernhard Zimmermann. Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4542.