Landscape Of Driver Mutations Research Articles

Landscape of driver mutations and their clinical effects on Down syndrome–related myeloid neoplasms

AbstractTransient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer.

Colorectal cancer (CRC) is a prevalent malignancy with diverse molecular characteristics. The NGS-based approach enhances our comprehension of genomic landscape of CRC and may guide future advancements in precision oncology for CRC patients. In this research, we conducted an analysis using Next-Generation Sequencing (NGS) on samples collected from 111 individuals who had been diagnosed with CRC. We identified somatic and germline mutations and structural variants across the tumor genomes through comprehensive genomic profiling. Furthermore, we investigated the landscape of driver mutations and their potential clinical implications. Our findings underscore the intricate heterogeneity of genetic alterations within CRC. Notably, BRAF, ARID2, KMT2C, and GNAQ were associated with CRC prognosis. Patients harboring BRAF, ARID2, or KMT2C mutations exhibited shorter progression-free survival (PFS), whereas those with BRAF, ARID2, or GNAQ mutations experienced worse overall survival (OS). We unveiled 80 co-occurring and three mutually exclusive significant gene pairs, enriched primarily in pathways such as TP53, HIPPO, RTK/RAS, NOTCH, WNT, TGF-Beta, MYC, and PI3K. Notably, co-mutations of BRAF/ALK, BRAF/NOTCH2, BRAF/CREBBP, and BRAF/FAT1 correlated with worse PFS. Furthermore, germline AR mutations were identified in 37 (33.33%) CRC patients, and carriers of these variants displayed diminished PFS and OS. Decreased AR protein expression was observed in cases with AR germline mutations. A four-gene mutation signature was established, incorporating the aforementioned prognostic genes, which emerged as an independent prognostic determinant in CRC via univariate and multivariate Cox regression analyses. Noteworthy BRAF and ARID2 protein expression decreases detected in patients with their respective mutations. The integration of our analyses furnishes crucial insights into CRC's molecular characteristics, drug responsiveness, and the construction of a four-gene mutation signature for predicting CRC prognosis.

Driver mutations of intrahepatic cholangiocarcinoma shape clinically relevant genomic clusters with distinct molecular features and therapeutic vulnerabilities.

Purpose: To establish a clinically applicable genomic clustering system, we investigated the interactive landscape of driver mutations in intrahepatic cholangiocarcinoma (ICC).Methods: The genomic data of 1481 ICCs from diverse populations was analyzed to investigate the pair-wise co-occurrences or mutual exclusivities among recurrent driver mutations. Clinicopathological features and outcomes were compared among different clusters. Gene expression and DNA methylation profiling datasets were analyzed to investigate the molecular distinctions among mutational clusters. ICC cell lines with different gene mutation backgrounds were used to evaluate the cluster specific biological behaviors and drug sensitivities.Results: Statistically significant mutation-pairs were identified across 21 combinations of genes. Seven most recurrent driver mutations (TP53, KRAS, SMAD4, IDH1/2, FGFR2-fus and BAP1) showed pair-wise co-occurrences or mutual exclusivities and could aggregate into three genetic clusters: Cluster1: represented by tripartite interaction of KRAS, TP53 and SMAD4 mutations, exhibited large bile duct histological phenotype with high CA19-9 level and dismal prognosis; Cluster2: co-association of IDH/BAP1 or FGFR2-fus/BAP1 mutation, was characterized by small bile duct phenotype, low CA19-9 level and optimal prognosis; Cluster3: mutation-free ICC cases with intermediate clinicopathological features. These clusters showed distinct molecular traits, biological behaviors and responses to therapeutic drugs. Finally, we identified S100P and KRT17 as “cluster-specific”, “lineage-dictating” and “prognosis-related” biomarkers, which in combination with CA19-9 could well stratify Cluster3 ICCs into two biologically and clinically distinct subtypes.Conclusions: This clinically applicable clustering system can be instructive to ICC prognostic stratification, molecular classification, and therapeutic optimization.

Mutational Landscape of MDS Patients with HMA Failure Revealed By the Correlative Analysis from Inspire Trial

Mutational Landscape of MDS Patients with HMA Failure Revealed By the Correlative Analysis from Inspire Trial

The landscape of driver mutations in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma is a form of skin cancer originating from keratinocytes in the skin. It is the second most common type of cancer and is responsible for an estimated 8000 deaths per year in the United States. Compared to other cancer subtypes with similar incidences and death tolls, our understanding of the somatic mutations driving cutaneous squamous cell carcinoma is limited. The main challenge is that these tumors have high mutation burdens, primarily a consequence of UV-radiation-induced DNA damage from sunlight, making it difficult to distinguish driver mutations from passenger mutations. We overcame this challenge by performing a meta-analysis of publicly available sequencing data covering 105 tumors from 10 different studies. Moreover, we eliminated tumors with issues, such as low neoplastic cell content, and from the tumors that passed quality control, we utilized multiple strategies to reveal genes under selection. In total, we nominated 30 cancer genes. Among the more novel genes, mutations frequently affected EP300, PBRM1, USP28, and CHUK. Collectively, mutations in the NOTCH and p53 pathways were ubiquitous, and to a lesser extent, mutations affected genes in the Hippo pathway, genes in the Ras/MAPK/PI3K pathway, genes critical for cell-cycle checkpoint control, and genes encoding chromatin remodeling factors. Taken together, our study provides a catalog of driver genes in cutaneous squamous cell carcinoma, offering points of therapeutic intervention and insights into the biology of cutaneous squamous cell carcinoma.

Abstract 2233: Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies

Abstract Alterations in the signaling cascade of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways are common mechanisms of cancer initiation and progression. RAS proteins (KRAS/HRAS/NRAS), a crucial component of this cascade, are often found to be activated in cancer cells. Recent advancements in RAS targeting offer new treatment strategies for patients, however studies suggest that the clinical responses may be tissue-dependent. Thus, understanding the landscape of RAS alterations and the concomitant mutations observed in their effector pathways may be crucial in developing appropriate disease-specific therapeutic strategies for effectively addressing this biology. In this study, we utilized comprehensive genomic profiling data from 282,792 cancer patients across 77 tumor types, tested in the course of routine clinical care (FoundationOne) to assess the landscape of gene alterations within this signaling cascade. RAS alterations were identified in 27% of the cohort. Mutations in PI3K and PTEN were also frequent (&amp;gt;10%); alterations in FGFR1-4, EGFR, RAF (ARAF/BRAF/RAF1) and ERBB2 were each identified in over 5% of samples. A closer inspection of non-small cell lung carcinomas (NSCLC, n=55,893), colorectal carcinomas (CRC, n=35,104) and pancreatic cancers (PC, n=17,447), revealed distinct mutational spectrums. RAS alterations, particularly KRAS, exhibited varying prevalence: PC (85%), CRC (54%) and NSCLC (32%). In NSCLC, 35% of the KRAS alterations were identified to be G12C. In contrast, the predominant KRAS alterations in CRC and PC were G12D (29% CRC, 41% PC) and G12V (20% CRC, 30% PC). Further, only CRCs exhibited a high rate of KRAS G13D (16% of all KRAS alterations); KRAS G12R was found exclusively to PC (15%) and KRAS amplifications were most frequently identified in NSCLC (11%). Distinct patterns were also observed in genes upstream and downstream of the RAS proteins. Alterations in ALK, EGFR, STK11 and MET were significantly more prevalent in NSCLC (P &amp;lt; 10−5); whereas gene alterations in PI3K, RAF, MTOR and ERBB2 were more frequently identified in CRC (P &amp;lt; 10−5). In contrast, PC showed a predominant presence of RAS mutations, with limited mutations in other genes of the signaling cascade. Of note, PI3K alterations were identified at a much lower prevalence in PC (4%) compared to NSCLC (11%) and CRC (19%). Our findings suggest a tissue-dependent spectrum of driver mutations exist across the MAPK/PI3K/AKT signaling pathways. The observed mutational profiles may explain some of the efficacy differences observed by targeting RAS across these diseases and have implications on acquired resistance mechanisms in a tissue-specific manner. Additional insights may be gleaned by investigating immunotherapy related biomarkers with these alterations to suggest combination strategies for effectively targeting the RAS signaling cascade. Citation Format: Smruthy Sivakumar, Ethan S. Sokol, Garrett M. Frampton, Priti S. Hegde, David Fabrizio. Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2233.

Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia

AbstractRecent genetic studies using high-throughput sequencing have disclosed genetic alterations in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, their effects on clinical outcomes have not been fully investigated. To address this, we comprehensively examined genetic alterations and their prognostic impact in a large series of pediatric B-ALL cases. We performed targeted capture sequencing in a total of 1003 pediatric patients with B-ALL from 2 Japanese cohorts. Transcriptome sequencing (n = 116) and/or array-based gene expression analysis (n = 120) were also performed in 203 (84%) of 243 patients who were not categorized into any disease subgroup by panel sequencing or routine reverse transcription polymerase chain reaction analysis for major fusions in B-ALL. Our panel sequencing identified novel recurrent mutations in 2 genes (CCND3 and CIC), and both had positive correlations with ETV6-RUNX1 and hypodiploid ALL, respectively. In addition, positive correlations were also newly reported between TCF3-PBX1 ALL with PHF6 mutations. In multivariate Cox proportional hazards regression models for overall survival, TP53 mutation/deletion, hypodiploid, and MEF2D fusions were selected in both cohorts. For TP53 mutations, the negative effect on overall survival was confirmed in an independent external cohort (n = 466). TP53 mutation was frequently found in IGH-DUX4 (5 of 57 [9%]) ALL, with 4 cases having 17p LOH and negatively affecting overall survival therein, whereas TP53 mutation was not associated with poor outcomes among NCI (National Cancer Institute) standard risk (SR) patients. A conventional treatment approach might be enough, and further treatment intensification might not be necessary, for patients with TP53 mutations if they are categorized into NCI SR.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

Interplay between DNA damage repair and apoptosis shapes cancer evolution through aneuploidy and microsatellite instability

Driver mutations and chromosomal aneuploidy are major determinants of tumorigenesis that exhibit complex relationships. Here, we identify associations between driver mutations and chromosomal aberrations that define two tumor clusters, with distinct regimes of tumor evolution underpinned by unique sets of mutations in different components of DNA damage response. Gastrointestinal and endometrial tumors comprise a separate cluster for which chromosomal-arm aneuploidy and driver mutations are mutually exclusive. The landscape of driver mutations in these tumors is dominated by mutations in DNA repair genes that are further linked to microsatellite instability. The rest of the cancer types show a positive association between driver mutations and aneuploidy, and a characteristic set of mutations that involves primarily genes for components of the apoptotic machinery. The distinct sets of mutated genes derived here show substantial prognostic power and suggest specific vulnerabilities of different cancers that might have therapeutic potential.

Abstract 2725: The single-cell atlas of driver mutations in acute myeloid leukemia

Abstract Assessment of intratumor genetic heterogeneity by next-generation sequencing (NGS) is confounded by tumor purity and zygosity of mutations. Furthermore, bulk NGS cannot visualize the cell-level co-occurrence or exclusivity among multiple mutations, causing the inaccurate inference of subclonal architecture. Here, we performed single cell DNA sequencing (scDNA seq) in 82 bone marrow samples from 70 patients with acute myeloid leukemia (AML) using a novel microfluidics-based platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). All samples were concurrently sequenced by the bulk NGS using 295 gene exome capture sequencing. Selected cases were also analyzed by SNP array (Illuminia Omni 2.5 array) to obtain allele-specific copy number data. In total, 319,406 cells (median 3,755 cells/sample) were genotyped with median allele drop-out rate of 8.7% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 24x/cell. The scDNA seq detected 230 of 238 (97%) bulk NGS-confirmed mutations. RUNX1 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss-of-heterozygosity of RUNX1 in these cases. Additionally, in cases with homozygous FLT3-ITD mutations, SNP array detected allele-specific copy number gain of mutant loci, which likely resulted in homozygous calls. scDNA seq data unambiguously resolved the single-cell level co-occurrence of driver mutations in AML such as DNMT3A/FLT3-ITD/NPM1, SRSF2/IDH2, and ASXL1/RUNX1, confirming the cooperative function of these driver mutations. On the other hand, the data also revealed mutually exclusive relationships at cellular level between IDH1/IDH2, FLT3-ITD/TKD, NRAS/KRAS, NRAS/PTPN11, and SRSF2/EZH2 mutations, which supports the functional redundancy of these genes in leukemogenesis. Inference of phylogenetic trees using SCITE algorithm (Jahn et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the analyzed cases had a linear evolution pattern where the founder mutations linearly acquired sub-clonal mutations in a step-wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 11 patients revealed the remodeling of clonal architecture at relapse. Patients who had 2 or more major subclones at baseline had significantly worse overall survival than those with one subclone (2-year OS 13 vs. 70 months, p = 0.0493). For the first time our data provides a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single-cell level using high-throughput scDNA seq genotype information. Citation Format: Kiyomi Morita, Feng Wang, Katharina Jahn, Yuanqing Yan, Robert Durruthy-Durruthy, Anup Parikh, Jairo Matthews, Latasha Little, Curtis Gumbs, Jianhua Zhang, Xingzhi Song, Erika Thompson, Keyur Patel, Carlos Bueso-Ramos, Courtney DiNardo, Farhad Ravandi, Elias Jabbour, Michael Andreeff, Jorge Cortes, Marina Konopleva, Guillermo Garcia-Manero, Hagop Kantarjian, Dennis J. Eastburn, P Andrew Futreal, Niko Beerenwinkel, Koichi Takahashi. The single-cell atlas of driver mutations in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2725.

S833 THE SINGLE‐CELL ATLAS OF DRIVER MUTATIONS IN AML

Background:Assessment of genetic intratumor heterogeneity using next generation sequencing (NGS) can underestimate the complexity of subclonal architecture, since it is confounded by tumor purity, zygosity, and cell‐level co‐occurrence and exclusivity among multiple mutations.Aims:To thoroughly dissect the subclonal architecture, we performed single cell DNA sequencing (scDNA‐seq) in 98 bone marrow samples from 80 patients with acute myeloid leukemia (AML).Methods:We used a novel microfluidics‐based scDNA‐seq platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). As a reference, all samples were concurrently sequenced by the bulk NGS using 295‐gene exome capture sequencing. Allele‐specific copy number data was obtained from SNP array data (Illuminia Omni 2.5 array). Droplet digital PCR (Bio‐Rad QX200 Droplet Digital PCR system) was used to estimate the sensitivity of our scDNA‐seq platform.Results:Median 6,786 cells/sample were sequenced with median allele drop‐out rate of 7.2% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 26x/cell. The scDNA‐seq detected all of the bulk NGS‐confirmed mutations. RUNX1 and FLT3 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss‐of‐heterozygosity of the mutant loci, which likely resulted in homozygous calls. The scDNA‐seq also uniquely detected driver mutations that were not detected by the bulk NGS but were confirmed by droplet digital PCR, suggesting that scDNA‐seq is more sensitive than the conventional bulk NGS.scDNA‐seq data unambiguously visualized the single‐cell level co‐occurrence of driver mutations (i.e. DNMT3A/FLT3‐ITD/NPM1 and SRSF2/IDH2), confirming the cooperative function of these mutations. scDNA‐seq data also revealed the cellular‐level mutual exclusivity between functionally redundant mutations such as IDH1/IDH2, FLT3‐ITD/TKD and NRAS/KRAS (Figure 1).Inference of phylogenetic trees using SCITE algorithm (Jahn, et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the cases showed a linear evolution pattern where the founder mutations linearly acquired sub‐clonal mutations in a step‐wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 15 patients revealed the remodeling of clonal architecture in AML. For example, scDNA‐seq data for a previously‐untreated therapy‐related AML case revealed that a FLT3p.D835Y clone, which was originally presented as a small sub‐clone, survived the induction therapy consisting of azacitidine and sorafenib, and significantly expanded at relapse, while all other FLT3 and KRAS mutations were eradicated. This clonal remodeling is consistent with differential sensitivity of various FLT3 mutations to sorafenib (Smith et al. Leukemia 2015), of which FLT3 p.D835Y mutation has been shown to be more resistant to sorafenib compared to p.D835E and ITD mutations, demonstrating the differential behavior of sub‐clones and clonal selection under molecularly targeted therapy (Figure 2).Summary/Conclusion:We performed a high‐throughput scDNA‐seq and described a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single‐cell resolution.image

Abstract 2354: Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes

Abstract A comprehensive understanding of the mutations that drive tumorigenesis and disease progression is essential to understanding tumor biology and designing precision therapies. The landscape of driver mutations in the protein-coding genome has been well-characterized by large exome-sequencing studies. Surprisingly, many tumors do not have mutations in any known protein-coding driver. Non-coding driver mutations are hypothesized to explain many of these cases, but aside from a few regions, like the TERT promoter, our understanding of drivers in the complex regulatory genome remains limited. To fill this gap, we analyzed 150,000 cis-regulatory modules with clustered transcription factor binding sites in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using a new statistical method, ActiveDriverWGS, we identified dozens of frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR&amp;lt;0.05) with many structural rearrangements and focal copy number alterations in additional samples. The FMREs were enriched in super-enhancers, long-range chromatin interactions and H3K27ac marks derived from primary tumors, suggesting a gene regulatory role of these mutations through three-dimensional genome organization. The interaction network of chromatin loops and FMREs revealed putative target genes located dozens to hundreds of kilobases away from the mutated regulatory elements. We found known and putative oncogenes and tumor suppressors whose expression significantly correlated with mutations in FMREs, suggesting novel oncogenic mechanisms. Most of the FMREs were also confirmed by additional driver discovery methods, lending confidence to our statistical approach. We also validated ActiveDriverWGS on protein-coding sequence and accurately recovered known driver genes. The non-coding regulatory genome is characterized by diverse mutational processes, regional hypermutations and technically challenging areas with suboptimal sequencing coverage. Thus our findings, most of which are reported for the first time, should be carefully vetted and experimentally validated in future studies. Our integrative analysis of somatic mutations, cis-regulatory regions and long-range chromatin interaction networks is a novel framework for cancer discovery and reveals the currently largest set of potential non-coding drivers in a pan-cancer cohort. Citation Format: Juri Reimand. Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes [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 2354.

Abstract 5372: The mutational landscape of primary cutaneous melanoma

Abstract Large-scale melanoma genome profiling studies to date have mainly focused on metastatic tumors. To better understand the link between genomic changes and outcome, primary tumors must be studied. We have designed a comprehensive targeted genomic panel to study genetic alterations in melanoma, and applied this to a large collection of 536 primary melanomas, part of the Leeds Melanoma Cohort. Patients were invited to participate 3 months after diagnosis of primary melanoma. 1% received targeted therapies or immunotherapy and thus, our analysis is reflective of the landscape of primary melanoma in the treatment naïve setting. The sequencing panel allowed us to study genetic alterations in over 500 genes, including more than 150 genes frequently mutated in melanoma and an additional 250 genes linked to other solid tumor types. Both tumor and germline DNA were analyzed for most samples. The average somatic mutation rates we observed was 15 mutations/MB (0 to 236 mutations/MB), while the nonsynonymous mutational load ranged from 0 to 57 mutations/MB (average 4 mutations/MB). We found that a high mutational burden (highest 20%, &amp;gt;45 nonsynonymous mutations/Mb) positively correlated with better survival (Cox proportional hazards model with age, sex and AJCC stage as additional covariates). Among the top recurrently mutated genes were well established melanoma driver genes such as BRAF (44%), NRAS (28%), NF1 (10%), CDKN2A (15%), TP53 (11%) and ARID2 (8%). We also found hotspot mutations in less well documented melanoma genes, including RQCD1 p.P131L (4.5%). Altogether, our preliminary data shows a strong correlation between survival and high mutational load in primary melanomas. Our data also suggests a similar landscape of driver mutations in primary and metastatic melanoma, with some variants/genes possibly reflecting the differences in disease stage. We will further investigate the mutational landscape in our cohort of primary melanomas, including analyses of gene co-occurrence and mutual exclusivity. Additionally, we will use extensive clinical parameters and transcriptomics to examine the possibility of predicting survival and other clinically relevant parameters in our cohort based on patient mutational profiles. Citation Format: Sofia Y. Chen, Rashid Mamunur, Jon Laye, D. Timothy Bishop, Julia A. Newton-Bishop, David J. Adams. The mutational landscape of primary cutaneous melanoma [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 5372.

Abstract 1426: Targeted sequencing of colorectal cancer in search for prognostic biomarkers

Abstract Introduction Extensive genetic studies of recent years using next generation sequencing have clarified a comprehensive registry of genetic alteration in colorectal cancer (CRC). However, the clinical impacts of these genetic alterations have not fully been investigated, except for a better response to immune checkpoint blockade and favorable prognosis in patients with microsatellite instability (MSI) cancer. In this study, we investigated the effects of common genetic alterations on survival in CRC patients using large-scale mutation profiling. Methods We performed targeted-capture sequencing to common driver genes (n=128) in CRC in a total of 544 patients with stage I (n = 36), II (n = 236) and III (n = 272) CRC. All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Genome-wide copy numbers, fusion genes and MSI were also interrogated using baits for 1,605 SNPs, 7 introns and 18 microsatellites, respectively. RT-PCR was performed for the detection of PTPRK-RSPO3 fusion gene. Results The median age at diagnosis was 65 (32-95) with a median observation period of 1250 days. Of the 156 patients with recurrence, 15, 61 and 80 patients had stage I, II and III diseases, respectively. APC, TP53, KRAS, and PIK3CA represented the most frequently mutated, while common focal copy number alterations involved PTEN, SMAD4, SMAD2, FGFR1 and IGF2. Novel fusion genes including PTPRK-RSPO3 were detected in only 7 (1.5%) samples. Fifty-five patients had more than 20 mutations, comprising a group of hypermutated CRC. Among these, 44 were thought to have MSI based on the microsatellite analysis, while the remaining 11 were explained by POLE mutations affecting the proof reading domain. CRCs with POLE mutations were diagnosed earlier in life, with the median age of 49 (35-66). The frequency of CRCs with MSI was significantly lower than previous reports from the western population. In contrast to mutation frequency, copy number alterations were infrequent, which most hypermutated tumors showed Genomic Instability Index (GII) below 0.2. FBXW7 mutation positively affected tumor free survival. BRAF mutation, SMAD4 mutation, or existence of fusion genes did not have any effect on prognosis. In accordance with previous reports, only 4 patients (9.1%) with MSI had recurrence, confirming a better prognosis of MSI-positive patients. Similarly, only one of the 11 patients (9.1%) with POLE mutation had recurrence. The low relapse rate was also observed among an additional cohort of 819 patients, which included 6 POLE mutated cases, none of whom had recurrence, suggesting that POLE mutation was associated with a favorable prognosis. Conclusions We revealed the landscape of driver mutations in patients with stage I, II and III CRC with their implication in clinical outcomes. In addition to FBXW7 mutations, the MSI status and somatic POLE mutations are significantly associated with a better prognosis. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Kenichi Chiba, Tetsuichi Yoshizato, Yuichi Shiraishi, Satoru Miyano, Satoshi Nagayama, Yoshiharu Sakai, Seishi Ogawa. Targeted sequencing of colorectal cancer in search for prognostic biomarkers [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 1426.

Whole genome sequencing analysis for cancer genomics and precision medicine.

Explosive advances in next‐generation sequencer (NGS) and computational analyses have enabled exploration of somatic protein‐altered mutations in most cancer types, with coding mutation data intensively accumulated. However, there is limited information on somatic mutations in non‐coding regions, including introns, regulatory elements and non‐coding RNA. Structural variants and pathogen in cancer genomes remain widely unexplored. Whole genome sequencing (WGS) approaches can be used to comprehensively explore all types of genomic alterations in cancer and help us to better understand the whole landscape of driver mutations and mutational signatures in cancer genomes and elucidate the functional or clinical implications of these unexplored genomic regions and mutational signatures. This review describes recently developed technical approaches for cancer WGS and the future direction of cancer WGS, and discusses its utility and limitations as an analysis platform and for mutation interpretation for cancer genomics and cancer precision medicine. Taking into account the diversity of cancer genomes and phenotypes, interpretation of abundant mutation information from WGS, especially non‐coding and structure variants, requires the analysis of large‐scale WGS data integrated with RNA‐Seq, epigenomics, immuno‐genomic and clinic‐pathological information.

Abstract 4385: Landscape of driver gene mutations in stage II and stage III colorectal cancer

Abstract Introduction Colorectal cancer (CRC) is the second leading cause of cancer death in Japan. The overall survival rate is approximately 85% and 70% for stage II and stage III CRCs, respectively, where over 45,000 deaths were ascribed to recurrence or metastasis. Survival benefit of adjuvant therapy is well defined only among stage III CRC patients, whereas that for stage II CRC patients remains modest, or according to recent reports, might be even deleterious among those with microsatellite instability (MSI). In an attempt to reveal the landscape of driver mutations in CRC and their association with clinical outcomes, we conducted targeted-capture sequencing of known driver mutations in stage II and III CRCs. Methods A total of 248 patients with stage II (n = 145) and stage III (n = 103) CRC, were enrolled. All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Targeted-capture sequencing was performed using RNA baits designed for detection of oncogenic variants in 128 known driver genes in CRC. Additional baits for 1,605 SNPs, 7 introns and 18 microsatellites were also included to assess genome-wide copy numbers, fusion genes and MSI, respectively. Results The median age at diagnosis was 64 (32-95) with a median observation period of 1,460 days. Of the 101 patients with recurrence, 59 and 42 patients had stage II and III diseases, respectively. Sixty percent of stage III patients received adjuvant chemotherapy, whereas only 10% of stage II patients received adjuvant chemotherapy. The average depth of the targeted-capture sequencing was 909×. In total, 2,243 somatic mutations (median 5 per patient, range 0-106) were detected in 243 patients (97.9%). VTI1A-TCF7L2 translocation was identified in one sample. Except for a modest overrepresentation of TP53 mutations, frequency distribution of mutations was similar to those reported in previous reports, where APC (83%), TP53 (79%), KRAS (45%), and PIK3CA (18%) represented the most frequent mutational targets. Although none of these mutations were significantly associated with tumor recurrence or overall survival, we found that FBXW7 mutations had a positive correlation with tumor free survival. Twenty-one patients had more than 20 mutations, suggesting the presence of defects in DNA repair and/or replication. In fact, analysis of 18 microsatellites suggested the presence of MSI in 12 of the 21 samples (4%). The remaining patients with hypermutated cancer were explained by somatic mutations in POLE gene. Conclusions We revealed the landscape of driver mutations in Japanese patients with stage II and III CRC with their implication in clinical outcomes. Hypermutated samples accounted for 8% of the whole cohort, which were explained by MSI or replication errors due to somatic POLE mutations. Our findings help to understand the relationships between driver mutations and recurrence for CRCs after curative resection. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Yuichi Shiraishi, Kenichi Chiba, Keisuke Kataoka, Hiroo Ueno, Hiroko Tanaka, Satoshi Nagayama, Satoru Miyano, Yoshiharu Sakai, Seishi Ogawa. Landscape of driver gene mutations in stage II and stage III colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4385. doi:10.1158/1538-7445.AM2017-4385

Mutation profiling of uterine lavage fluid detects early-stage endometrial cancers and discovers a prevalent landscape of driver mutations in women without cancer

Mutation profiling of uterine lavage fluid detects early-stage endometrial cancers and discovers a prevalent landscape of driver mutations in women without cancer

Genomic Analysis of Uterine Lavage Fluid Detects Early Endometrial Cancers and Reveals a Prevalent Landscape of Driver Mutations in Women without Histopathologic Evidence of Cancer: A Prospective Cross-Sectional Study.

BackgroundEndometrial cancer is the most common gynecologic malignancy, and its incidence and associated mortality are increasing. Despite the immediate need to detect these cancers at an earlier stage, there is no effective screening methodology or protocol for endometrial cancer. The comprehensive, genomics-based analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) revealed many of the molecular defects that define this cancer. Based on these cancer genome results, and in a prospective study, we hypothesized that the use of ultra-deep, targeted gene sequencing could detect somatic mutations in uterine lavage fluid obtained from women undergoing hysteroscopy as a means of molecular screening and diagnosis.Methods and FindingsUterine lavage and paired blood samples were collected and analyzed from 107 consecutive patients who were undergoing hysteroscopy and curettage for diagnostic evaluation from this single-institution study. The lavage fluid was separated into cellular and acellular fractions by centrifugation. Cellular and cell-free DNA (cfDNA) were isolated from each lavage. Two targeted next-generation sequencing (NGS) gene panels, one composed of 56 genes and the other of 12 genes, were used for ultra-deep sequencing. To rule out potential NGS-based errors, orthogonal mutation validation was performed using digital PCR and Sanger sequencing.Seven patients were diagnosed with endometrial cancer based on classic histopathologic analysis. Six of these patients had stage IA cancer, and one of these cancers was only detectable as a microscopic focus within a polyp. All seven patients were found to have significant cancer-associated gene mutations in both cell pellet and cfDNA fractions. In the four patients in whom adequate tumor sample was available, all tumor mutations above a specific allele fraction were present in the uterine lavage DNA samples. Mutations originally only detected in lavage fluid fractions were later confirmed to be present in tumor but at allele fractions significantly less than 1%. Of the remaining 95 patients diagnosed with benign or non-cancer pathology, 44 had no significant cancer mutations detected. Intriguingly, 51 patients without histopathologic evidence of cancer had relatively high allele fraction (1.0%–30.4%), cancer-associated mutations. Participants with detected driver and potential driver mutations were significantly older (mean age mutated = 57.96, 95% confidence interval [CI]: 3.30–∞, mean age no mutations = 50.35; p-value = 0.002; Benjamini-Hochberg [BH] adjusted p-value = 0.015) and more likely to be post-menopausal (p-value = 0.004; BH-adjusted p-value = 0.015) than those without these mutations. No associations were detected between mutation status and race/ethnicity, body mass index, diabetes, parity, and smoking status. Long-term follow-up was not presently available in this prospective study for those women without histopathologic evidence of cancer.ConclusionsUsing ultra-deep NGS, we identified somatic mutations in DNA extracted both from cell pellets and a never previously reported cfDNA fraction from the uterine lavage. Using our targeted sequencing approach, endometrial driver mutations were identified in all seven women who received a cancer diagnosis based on classic histopathology of tissue curettage obtained at the time of hysteroscopy. In addition, relatively high allele fraction driver mutations were identified in the lavage fluid of approximately half of the women without a cancer diagnosis. Increasing age and post-menopausal status were associated with the presence of these cancer-associated mutations, suggesting the prevalent existence of a premalignant landscape in women without clinical evidence of cancer. Given that a uterine lavage can be easily and quickly performed even outside of the operating room and in a physician’s office-based setting, our findings suggest the future possibility of this approach for screening women for the earliest stages of endometrial cancer. However, our findings suggest that further insight into development of cancer or its interruption are needed before translation to the clinic.

Landscape of Driver Mutations and Their Clinical Impacts in Pediatric Acute Lymphoblastic Leukemia

IntroductionB-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner.MethodsWe enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection.ResultsThe median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]).Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation.Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified.Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL.ConclusionsWe revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Archetypes of AML Defined Using Whole Exome Sequencing and Clinical Characteristics in a Diverse Group of Patients

Background: Acute myeloid leukemia (AML) comprises a heterogeneous collection of morphologically related diseases. The identification and study of somatic mutations in AML using next-generation sequencing has led to insights into the pathogenesis of AML. Constellations of specific mutations may correlate with clinical phenotype and reveal the most relevant cooperating molecular pathways. Understanding AML biological pathways will contribute to more personalized, directed therapies and improve outcomes. Most sequencing efforts have focused on select groups of AML patients or select gene panels. Characterization of mutations identified by whole exome sequencing for AML patients from a variety of clinical scenarios has not been the subject of one study.Methods: Whole exome sequencing (WES) was performed, in addition to complete clinical molecular diagnostics, on leukemia samples from a diverse group of 141 consecutive AML patients who underwent complete clinical evaluation at a single quaternary leukemia care institution (MD Anderson Cancer Center, MDACC). The cohort included adults of all ages, treatment histories (treated/untreated), and ontogenies (de novo/secondary/therapy-related). To facilitate the identification of driver mutations from WES data, an automatic pipeline was developed to filter and annotate raw variants, and then select non-polymorphism, protein-coding changing mutations. A relational mutation/clinical database was created to analyze gene mutations and gene categories in the context of well-annotated mutations and complete clinical indices including treatments and outcomes.Results: We characterized the landscape of driver mutations in an unselected group of AML patients, and classified mutations by 9 predefined functional categories of known aberrant pathways implicated in AML (Figure 1). The frequency and distribution of gene mutations and gene categories were determined and compared to TCGA AML whole exome sequencing mutation data, derived from a de novo, untreated AML cohort. Forty-six separate genes/fusions were mutated in the MDACC cohort, including 326 mutations. The MDACC cohort included 23 cases (16%) with a TP53 mutation. Overall, a mean of 2.3+/-1.4 mutations-of-interest were identified per patient. For the MDACC dataset, the likelihood of co-occurrence and mutual exclusivity was determined for each gene (mutated in greater than 2 cases, n=34) and each gene category (n=9) versus every other gene, gene category, as well as each individual annotated clinical characteristic (n=30). 156 statistically significant co-occurring pairs and 71 statistically significant mutually exclusive pairs were found. We analyzed hierarchically clustered, strength-of-association correlograms to identify likely mutational frameworks with similarly associated clinical/molecular characteristics. We identified patterns of predominant genetic alterations that defined 4 distinct AML archetypes: 1) Transcription factor [TRNSXN] (16% of cases), 2) Nucleolar/Methylation/Signaling [NMS] (40%), 3) Spliceosome [SPLICE] (21%), and 4) TP53-mutated [TP53] (16%). Ten cases had no identifiable mutation-of-interest, compared to 4 cases without mutation in TCGA data. The defining mutational characteristics of each AML archetype, and select associated characteristics are shown in Table 1.Conclusions: Analysis of whole exome sequencing data along with clinical information from all-comer AML patients identifies archetypal classes of the disease, each with particular tendencies for specific mutational, clinical, and outcome characteristics. Our approach demonstrates that genomic characterization of mutations in a variety of diverse clinical scenarios has the potential to identify representative mechanisms of AML development across the spectrum of disease presentations. [Display omitted] [Display omitted] DisclosuresDaver:Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Ariad: Research Funding; Kiromic: Research Funding; BMS: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. DiNardo:Agios: Other: advisory board, Research Funding; Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Abbvie: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.