Targeted Capture Sequencing Research Articles

Characteristics of gene mutations in Chinese single-center pulmonary adenocarcinoma patients and comparison with European and American pulmonary adenocarcinoma populations

Objective To explore the characteristics and significances of gene mutations in pulmonary adenocarcinoma, and to provide evidence for targeted medication. Methods High throughput sequencing based target-capture sequencing was performed in 104 patients with pulmonary adenocarcinoma to detect the mutational status of 56 cancer-related genes. All patients were diagnosed in the First People's Hospital of Kunshan from May 2017 to August 2018. The mutational characteristics of pulmonary adenocarcinoma was analyzed and compared with European and American pulmonary adenocarcinoma populations. The correlations between mutational characteristics and clinical features were analyzed, and the mutation sites for targeted medication were screened. Results Among 104 patients with pulmonary adenocarcinoma, totally 34 mutational genes were detected in 84 patients (81%, 84/104). Highly frequent mutations included epidermal growth factor receptor (EGFR) (49%, 51/104), TP53 (21%, 22/104), KRAS (13%, 14/104), and BRAF (6%, 6/104). Among all the 187 variants, 76% (142/187) were non-synonymous missense mutations, 13% (24/187) were small fragment deletions, 6% (12/187) were copy number variants, 3% (5/187) were small fragment insertions, and 2% (4/187) were nonsense site mutations. Among 104 patients with pulmonary adenocarcinoma, 34 targeted drug-associated mutations of 13 genes were detected in 68 patients (65%), and 19 (18%) patients harbored ≥ 2 targeted drug-associated mutations. EGFR mutations were more common in female patients than in male patients [62% (34/55)vs. 35% (17/49), χ2= 7.629, P= 0.006], while KRAS mutations were more frequent in male patients than in female patients [22% (11/49) vs. 5% (3/55), χ2= 6.424, P= 0.011]. The mutation frequencies of gene EGFR, TP53, KRAS, and CDKN2A in Chinese single-center (the First People's Hospital of Kunshan) and European and American adenocarcinoma populations were significantly different (all P < 0.05). Conclusions The molecular mutational characteristics of pulmonary adenocarcinoma are complex, and vary greatly among different populations. High throughput sequencing-based multiple-gene detection can reveal its mutational features comprehensively, and that has important roles in personal targeted medication guidance, drug-resistance monitoring and prognosis evaluation. Key words: Lung neoplasms; Adenocarcinoma; High-throughput nucleotide sequencing; Mutation; Molecular targeted therapy

Familial autosomal recessive bestrophinopathy: identification of a novel variant in BEST1 gene and the specific metabolomic profile

BackgroundAutosomal recessive bestrophinopathy (ARB) is a retinal degenerative disorder caused by BEST1 mutations with autosomal recessive inheritance. We aim to map a comprehensive genomic and metabolomic profile of a consanguineous Chinese family with ARB.MethodsOphthalmic examinations were performed on the affected patients with ARB. The proband was screened for potential causative mutations in a panel with 256 known retinal disease genes by using target capture sequencing. The related mutation was further validated and segregated in the family members by Sanger sequencing. In silico prediction tools were used for pathogenicity assessment. A UHPLC-MS/MS metabolomic analysis was performed to explore the disease-associated metabolic feature.ResultsThe affected patients from this family were characterized by low vision, the presence of subretinal fluid, macular edema, and hyperopia with coincidental angle closure. DNA sequencing identified a novel missense mutation in the BEST1 gene c.646G > A (p.Val216Ile) of the proband. Sanger sequencing further confirmed the mutation. The missense mutation was co-segregation across the pedigree and predicted to be deleterious by SIFT (0.017). The blood metabolic profiles were highly similar among all family members probably because of the same lifestyle, habitat and genomic background. However, ARB patients presented a significant deregulation of metabolites, such as citric acid, L-Threonic acid, and eicosapentaenoic acid.ConclusionsWe identified a novel disease-associated variant in the BEST1 gene as well as a disease-specific metabolic feature in familial ARB. Our findings helped improve the understanding of ARB mechanisms.

Findings from a Genotyping Study of Over 1000 People with Inherited Retinal Disorders in Ireland.

The Irish national registry for inherited retinal degenerations (Target 5000) is a clinical and scientific program to identify individuals in Ireland with inherited retinal disorders and to attempt to ascertain the genetic cause underlying the disease pathology. Potential participants first undergo a clinical assessment, which includes clinical history and analysis with multimodal retinal imaging, electrophysiology, and visual field testing. If suitable for recruitment, a sample is taken and used for genetic analysis. Genetic analysis is conducted by use of a retinal gene panel target capture sequencing approach. With over 1000 participants from 710 pedigrees now screened, there is a positive candidate variant detection rate of approximately 70% (495/710). Where an autosomal recessive inheritance pattern is observed, an additional 9% (64/710) of probands have tested positive for a single candidate variant. Many novel variants have also been detected as part of this endeavor. The target capture approach is an economic and effective means of screening patients with inherited retinal disorders. Despite the advances in sequencing technology and the ever-decreasing associated processing costs, target capture remains an attractive option as the data produced is easily processed, analyzed, and stored compared to more comprehensive methods. However, with decreasing costs of whole genome and whole exome sequencing, the focus will likely move towards these methods for more comprehensive data generation.

Target Capture Sequencing Unravels Rubus Evolution.

Rubus (Rosaceae) comprises more than 500 species with additional commercially cultivated raspberries and blackberries. The most recent (> 100 years old) global taxonomic treatment of the genus defined 12 subgenera; two subgenera were subsequently described and some species were rearranged. Intra- and interspecific ploidy levels and hybridization make phylogenetic estimation of Rubus challenging. Our objectives were to estimate the phylogeny of 94 taxonomically and geographically diverse species and three cultivars using chloroplast DNA sequences and target capture of approximately 1,000 low copy nuclear genes; estimate divergence times between major Rubus clades; and examine the historical biogeography of species diversification. Target capture sequencing identified eight major groups within Rubus. Subgenus Orobatus and Subg. Anoplobatus were monophyletic, while other recognized subgenera were para- or polyphyletic. Multiple hybridization events likely occurred across the phylogeny at subgeneric levels, e.g., Subg. Rubus (blackberries) × Subg. Idaeobatus (raspberries) and Subg. Idaeobatus × Subg. Cylactis (Arctic berries) hybrids. The raspberry heritage within known cultivated blackberry hybrids was confirmed. The most recent common ancestor of the genus was most likely distributed in North America. Multiple distribution events occurred during the Miocene (about 20 Ma) from North America into Asia and Europe across the Bering land bridge and southward crossing the Panamanian Isthmus. Rubus species diversified greatly in Asia during the Miocene. Rubus taxonomy does not reflect phylogenetic relationships and subgeneric revision is warranted. The most recent common ancestor migrated from North America towards Asia, Europe, and Central and South America early in the Miocene then diversified. Ancestors of the genus Rubus may have migrated to Oceania by long distance bird dispersal. This phylogeny presents a roadmap for further Rubus systematics research. In conclusion, the target capture dataset provides high resolution between species though it also gave evidence of gene tree/species tree and cytonuclear discordance. Discordance may be due to hybridization or incomplete lineage sorting, rather than a lack of phylogenetic signal. This study illustrates the importance of using multiple phylogenetic methods when examining complex groups and the utility of software programs that estimate signal conflict within datasets.

Using genomic data to develop SSR markers for species of Chresta (Vernonieae, Asteraceae) from the Caatinga

Chresta is a genus mostly endemic to Brazil that presents several rupicolous species with naturally fragmented distributions. Aiming to facilitate studies about genetic diversity and structure in these species, we developed a set of 22 nuclear and 6 plastid microsatellite markers that are transferable among different species of the genus. We used previously obtained genomic data from target capture and Illumina sequencing to identify putative repeat regions, designed and synthesized primers, and genotyped individuals from different populations of three species. All loci were successfully amplified in all three species and were overall variable, except for the plastid markers, which were monomorphic in two species. These newly developed microsatellites will be useful in studies focusing on the population genetics of Chresta.

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations.

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline—MoNaS (Mosquito Na+ channel mutation Search)—which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.

Targeted-Capture Sequencing Is a Useful Method for MRD Markers Screening in KMT2A (MLL) Rearranged Leukemia

Immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangement-based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with acute lymphoblastic leukemia (ALL). Infant ALL has a poor prognosis and requires therapeutic development based on accurate MRD assessment. However, there are some difficulties associated with the highly sensitive detection of MRD based on Ig/TCR gene rearrangements in infant ALL cases, compared to other B-ALL cases, because of the immature Ig/TCR gene rearrangement patterns and their oligoclonality. Since KMT2A(MLL) gene rearrangements are identified in infant ALL at a high frequency, the use of KMT2A rearranged sequences is an alternative method for MRD detection in KMT2A rearranged ALL cases. However, identifying the genomic breakpoints in KMT2A rearrangements by conventional methods is laborious and difficult in routine practice. To detect molecular MRD markers in infant ALL cases, we designed capture probes covering the coding and recognition signal sequences of the V, D, and J genes of the Ig/TCR loci and common genomic breakpoints in KMT2A, AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10 (AF10), and ELL. 88 infant ALL cases from the JPLSG MLL-10 study, including 73 cases with KMT2A rearrangements, were analyzed by high-throughput target-capture sequencing on the Illumina platform. The sequenced data were analyzed with the Ig/TCR recombination analysis tool Vidjil and the Genomon pipeline for detection of KMT2A rearrangements. Although 40% of cases in this study were marker-negative in the conventional marker screening analysis, clonal Ig/TCR targets for MRD measurements were identified in more than 80% of the cases by target-capture sequencing. Ig / TCR rearrangements detected in infant ALL were unique and related to age at diagnosis and type of KMT2A rearragement. KMT2A-rearranged cases showed a significantly higher frequency of incomplete rearrangements of IGH and TCRD compared to KMT2A-negative infant ALL cases. The target-capture method also showed an increased sensitivity of Ig/TCR-based MRD measurements even in cases where MRD measurement was possible. Genomic breakpoints of KMT2A rearrangements were detected in 80% of KMT2A-rearranged cases, in which KMT2A was most frequently rearranged with AFF1, followed by MLLT1, MLLT3, and MLLT10. Other rare fusion partners of the KMT2A gene were also captured by probes for KMT2A sequences. The determined patient-specific genomic breakpoint sequences could be used for monitoring the MRD of KMT2A-rearranged cases. Thus, target-capture sequencing is an easy and useful method for screening MRD markers in infant leukemia cases. Disclosures No relevant conflicts of interest to declare.

Molecular Characteristics That Predict Response to Azacitidine Therapy

Background DNA hypomethylating agents (HMAs), including azacytidine (AZA) and decitabine, have been established as key drugs for higher-risk myelodysplastic syndromes (MDS). Recently, a marked efficacy of decitabine has been reported in TP53-mutated myeloid malignancies, although it is not approved for MDS in many countries. Thus it is of an urgent interest whether the excellent response to TP53-mutated myeloid neoplasms is recapitulated with AZA and if so, what is the biomarkers reliably predicting response to AZA. Methods In this study we enrolled a total of 384 AZA-treated MDS patients, including 179 from a Japanese prospective study (JALSG MDS-212 trial), 163 from Karolinska Institute, and 42 from a Japanese multi-institutional registry, and analyzed mutations in 66 genes commonly mutated in myeloid neoplasms together with copy number abnormalities, using targeted-capture sequencing. For all patients, sequencing was performed on pre-AZA samples. Post-AZA samples were also analyzed for the majority (279/394, 73%) of patients. Results Analysis of pre-AZA specimens identified 1,085 driver mutations in 53 genes in 361 (94%) patients with a mean of 3.0 mutations/sample. CNAs were detected in 258 (67%) cases. Most frequently observed were mutations in TP53, RUNX1, TET2, SRSF2, and ASXL1, and other high-risk CNAs, such as -7/7q LOH, 5q LOH, and 17p LOH. TP53 lesions were found in 102 (27%) cases, of which 87 cases (85%) had biallelic lesions caused by either multiple mutations (N=28) or 17p LOH involving the TP53 locus (N=59). Sixty-seven cases (17%) achieved complete remission (CR) with a median observation period of 7.5 months. TP53 mutation, found in 48% of the cases, was the only lesion that was significantly associated with CR (OR=3.4, p&lt;0.0001) together with hematological improvements (HI-E: OR=2.32, p=0.011, HI-N: OR=2.29, p=0.038, HI-P: OR=1.88, p=0.039). Marrow CR (mCR), PR, SD with HI were also achieved in additional 71 (18%), 11 (3%), and 37 (9.6%) cases, respectively. However, 51% of the cases that achieved any response had disease progression with a median duration of only 313 days. Despite favorable initial response, TP53-mutation was still associated with dismal long-term outcomes (median OS 374 days versus 687 days in TP53-unmutated cases, p&lt;0.0001), which is in compatible with a short duration of remission period (median 199 days for CR cases and median 182 days for mCR/PR cases with TP53 mutation) in the former group. Given distinct characteristics in both genetic and clinical features in TP53 mutated cases, we subsequently inquired for additional biomarkers predicting outcomes according to the mutation status of TP53. In the TP53-mutated cases, platelet counts larger than median value of 7.1×104/μL was the only factor that predicted better survival (P=0.02). In the TP53-unmutated cases, higher age (&gt;72 years old, hazard ratio: HR 1.36, p=0.03) and high karyotype risks (Very High/High according to IPSS-R-based classification system, HR 1.31, p=0.04) were associated with a shorter OS, while mutations in DDX41 predicted a significantly better OS (HR 0.62, p&lt;0.001). Notably, those who received transplantation following AZA therapy (N=14) had a significantly prolonged survival with a median OS of 758 days for TP53-mutated cases, where TP53 mutations had disappeared after AZA treatment in six cases and decreased to &lt;10% in VAF in other 4 patients. Clonal dynamics was evaluated by measuring change of copy number-adjusted variant allele frequency (aVAF) between pre- and post-AZA samples. Both post-AZA mutational burden (p&lt;0.0001) and the size of reduction in aVAF during treatment (p&lt;0.0001) significantly correlated with clinical response (trend test). Conclusions Our study revealed distinct response profile to AZA treatment according to TP53 mutation status, underscoring the need of different tools to estimate treatment outcomes for them. High remission rate in TP53-mutated cases does not lead to long-term outcomes due to short remission duration, if ever obtained. Successive transplantation to AZA treatment during remission phase might rescue patients from this intractable disease. Disclosures Takaori: Novartis: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Papaemmanuil:Celgene: Research Funding. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Qiagen Corporation: Patents &amp; Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy.

A Fast and Accurate Diagnostic Method for Ph-like ALL Using the Ncounter System

Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions

Introduction Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is &gt;70. Approximately 5% of ATLL patients in Japan present at age &lt;50. Here, we hypothesized that ATLL in younger patients will have distinct genetic alterations. Methods DNA and RNA samples were extracted from frozen lymph nodes. Targeted capture sequencing for 98 genes and analysis of RNA sequencing (RNAseq) were performed in 8 samples of young patients with ATL (defined as ≤ 50 years in the current study) Results and discussion The most frequently mutated genes were CCR4 and CARD11, each in 3 of the analyzed cases (37.5%). Mutations of PLCG1, PRKCB and STAT3 were frequent in a previous genomic analysis (Kataoka et al. Nat Genet. 2015) that were not age-selected, but were not identified in our 8 cases. Three (37.5%) cases harbored concurrent CTLA4-CD28 and ICOS-CD28 fusions. This contrasts with previous reports, in which cases with both fusions were found in &lt;1% of PTCL and ATLL patients. We confirmed the presence of both fusions in all 3 cases by RT-PCR. The structure of the CTLA4-CD28 fusion suggests that the extracellular portion of CTLA4 is expressed on the cell surface where it can interact with CD80 and CD86 to activate signaling through the intracellular CD28 portion. In contrast to CTLA4-CD28, the ICOS-CD28 fusion links the N-terminal signal peptide of ICOS with the extracellular and intracellular portions of CD28. This fusion should simply result in overexpression of CD28 and haploinsufficiency of ICOS. To evaluate the function of CTLA4-CD28, we used Ba/F3 cells, which are strictly dependent on exogenous IL-3 for proliferation. We transduced Ba/F3 cells with CTLA4-CD28 or a CTLA4-CD28 mutant (mut) with three amino acid substitutions that abrogate CD28 signaling. We then cultured these cells in the presence or absence of Raji cells, which express CD80 and CD86. In the absence of Raji cells, neither CTLA4-CD28 nor CTLA4-CD28mut expression conferred IL-3-independent growth. However, Ba/F3 cells expressing CTLA4-CD28 cultured in the presence of Raji cells achieved IL-3 independence. This was not the case for Ba/F3 cells expressing CTLA4-CD28mut, indicating a requirement on functional CTLA4-CD28 signaling. Western-blot analysis identified activation of CD28 signal in Ba/F3 cells with CTLA4-CD28 co-cultured with Raji cells. These data indicate that ligand-bound CTLA4-CD28 can promote downstream signaling in the absence of an endogenous TCR complex. Gene set enrichment analysis (GSEA) comparing the 3 ATLL cases with CD28 fusions to the 5 cases that lacked fusions demonstrated enrichment of previously defined gene signatures associated with AKT and RAF signaling, both of which are downstream of CD28 activation. GSEA also identified enrichments of T-cell function-related signatures, including pathways involved in interferon responses, in cases with CD28 fusions Cases with CTLA4-CD28 and ICOS-CD28 fusions become haploinsufficient for CTLA4 and ICOS. As expected, these cases had lower expression of both genes by RNAseq. Interestingly, these cases also had higher expression of CD80. By immunohistochemistry, ATLL cells with CTLA4-CD28 and ICOS-CD28 fusions expressed CD80 and macrophages in the tumor microenvironment expressed CD86. Thus, both intra- and intercellular interactions could drive CTLA4-CD28 and ICOS-CD28 signaling in these cases. Treatment of Ba/F3 cells expressing CTLA4-CD28 and cultured in the presence of Raji cells with a CTLA4 blocking antibody suppressed proliferation in a dose-dependent fashion. A previous case report (Mol Genet Genomic Med, 2015) described a patient with Sezary Syndrome and CTLA4-CD28 fusion who had a deep but transient response to the anti-CTLA4 antibody ipilimumab. This study did not clarify the extent to which response resulted from blocking of cell-autonomous CTLA4-CD28 signaling versus activation of a cell non-autonomous immune response. Nonetheless, it strongly supports the testing of CTLA4 blockade in additional cases of T-cell malignancies, including ATLL, that harbor CTLA4-CD28 fusions. Disclosures Stevenson: Celgene: Research Funding. Ohshima:Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Weinstock:Verastem Oncology: Research Funding; Celgene: Research Funding.

Long Range Sequencing Shows Improved Resolution in the Detection of Beta Globin Cluster Variants

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.

Distinct, Ethnic, Clinical, and Genetic Characteristics of Myelodysplastic Syndromes with Der(1;7)

der(1;7)(q10;p10) is a recurrent chromosomal abnormality found in a wide variety of myeloid neoplasms observed in as high as 6% of myelodysplastic syndromes (MDS) in Asian populations, while rarely observed in Caucasian populations. It is thought to be generated by a recombination between two highly homologous centromere alphoid sequences which lead to an unbalanced abnormality of monosomy of 7q and trisomy of 1q. However, despite the presence of -7q, der(1;7) has been associated with a better prognosis compared to monosomy 7 or other del(7q) (-7/del(7q)). In addition to its association with +8 and del(20q), frequent RUNX1 mutations and a paucity of mutated TP53 have been reported in der(1;7) tumors, but otherwise, the molecular features of this abnormality have been poorly characterized in the literature. This is most likely because it is very rare in Caucasians, even though it represents one of the most prevalent lesions among Asian populations. The purpose of our study is to clarify the frequency and mutational landscape of der(1;7) in myeloid neoplasms on the basis of targeted-capture sequencing. A total of 1,707 MDS cases, including 944 German and 763 Japanese cases, were enrolled, from which we identified 73 (4.0%) cases with der(1;7). The prevalence was &gt;20 times higher in Japanese (9.0%) than German (0.43%) cohorts (p&lt;0.0001). We also identified a strong male predominance in der(1;7)-positive cases (90.4%) compared to negative cases. Also including an additional 22 cases, somatic mutations and copy number abnormalities in der(1;7) were interrogated in a total of 95 cases, which included 84 (88.4%) with MDS, 9 (9.5%) with AML, and 2 (2.1%) with MPN. Among MDS patients, 29 were low-risk, 47 were high-risk, and the rest were not specified. In mutation analysis, at least one mutation was detected in 98% of der(1;7) cases, most frequently affecting RUNX1 (42%), followed by EZH2 (26%), and ETNK1 (25%). Copy number analysis showed a high frequency of del(20q) and trisomy 8 in der(1;7) cases: 27.4% and 18.9% respectively. On the basis of mutant cell fractions, most of these mutations were present in subclones acquired within the major population harboring der(1;7). In particular, most of the EZH2 (7q35-q36) mutations were thought to be secondary events in der(1;7)-positive cases, while representing initial events acquired before UPD(7q) or -7/del(7q) in der(1;7)-negative cases. Of interest, der(1;7) was associated with a low frequency of TP53 mutations, which were seen only in 3% of cases with der(1;7), whereas highly prevalent in non-der(1;7) cases with -7/del(7q) (52%), which is concordant with a better clinical outcome was observed in der(1;7) cases compared with non-der(1;7) cases with monosomy 7 or other del(7q). Another unique feature of der(1;7) positive MDS was an extremely high frequency of RUNX1 mutations. However, the most prominent finding with secondary mutations in der(1;7) cases is the frequent hot spot mutation in ETNK1, which were originally reported in 8.8% of myeloid neoplasms with MPN features, like SETBP1 mutations. ENTK1 mutations were found in as many as 25% (23/95) of der(1;7) cases, while rarely seen in -7/del(7q) (1/89) (p&lt;0.0001) or amp(1q) (2/68) (p=0.0001). Despite the high frequency of trisomy 8 observed in der(1;7) cases, none were associated with ETNK1 mutations. In addition, all of the RAS pathway mutations (positive in 16 cases) were observed in der(1;7) cases with wild-type ETNK1, while none were in ETNK1-mutant cases. Morphologically, these ETNK1-mutated der(1;7) cases presented with an increased eosinophil count in peripheral blood (760.9/ul vs. 78.1/ul) (p&lt;0.001), compared to those without EKNK1 mutations, suggesting that ENTK1-mutated der(1;7) cases represent a novel disease entity within der(1;7), characterized by unique genetic features and increased eosinophils. In conclusion, der(1;7) is a genetically and clinically distinct subset of myeloid neoplasms, which showed unique features that are distinct from MDS cases in -7 and other del(7q). Especially, ETNK1 mutations subdivided cases with der(1;7) into two groups of genetically distinct subsets as shown in Figure 1. In the future, inhibition of the kinase activity in ETNK1 could be a novel therapeutic strategy in such a previously unrecognized subset as characterized by der(1;7) and eosinophilia. Figure 1 Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Handa:Ono: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Qiagen Corporation: Patents &amp; Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership.

Preimplantation genetic testing for a family with usher syndrome through targeted sequencing and haplotype analysis

BackgroundPreimplantation genetic testing for monogenic defects (PGT-M) has been available in clinical practice. This study aimed to validate the applicability of targeted capture sequencing in developing personalized PGT-M assay.MethodsOne couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. Customized capture probe targeted at USH2A gene and 350 kb flanking region were designed for PGT-M. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A hidden Markov model (HMM) assisted haplotype analysis was performed to deduce embryo’s genotype by using single nucleotide polymorphisms (SNPs) identified in each sample. The embryo without paternal rare variant was implanted and validated by conventional prenatal or postnatal diagnostic means.ResultsFour embryos were diagnosed as free of father’s rare variant, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGT-M and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby’s genomic DNA sample. The auditory test showed that the hearing was normal.ConclusionsTargeted capture sequencing is an effective and convenient strategy to develop customized PGT-M assay.

P2.03-10 Comprehensive Molecular Profiling and Comparison of Common and Rarer Subtypes of Lung Cancer

Genomic profiling of tumours has become a crucial component of precision cancer medicine. In order to comprehensively characterize molecular alterations in different lung cancer subtypes, we analysed a total number of 327 samples by using Whole-Exome Sequencing (WES) and SNP genotyping arrays. Additionally, we used Targeted Capture Sequencing (TCS) for scanning a selected panel of genes (n=52) at high sequencing depth. We WES (McGill University Innovation Centre in Montreal) 153 paired tumour-normal samples, with a further 174 paired tumour-normal samples undergoing TCS. Sequencing data were processed and mutations identified using BWA (v.0.7.15), Picard (v.2.17.11), GATK (v.3.7), VarScan (v.2.4.2) and VEP (v. 92) softwares. Illumina OmniExpressExome (v1.6) arrays were used for genotyping all samples and copy number alterations (CNAs) were identified using ASCAT (v.2.5.2), DNACopy (v.1.56.0) and GISTIC (v.2.0) softwares. The analysed samples had a tumour content varying from 20 to 90%. The age range of patients was between 28 to 89 years. Out of 159 lung cancer patients, 89 patients had lung adenocarcinoma (LUAD), 36 squamous cell carcinoma (LUSC) and 34 lung neuroendocrine (NET), of which 22 were subclassified as lung carcinoid (LC), 6 small cell carcinoma (LSCLC), 5 large cell carcinoma (LCNEC) and 1 combined NET subtype. TP53 appeared as the most mutated gene in LUSCs (82%), non-carcinoid NETs (58%) and LUADs (47%), but not in the LC subtype, while the chromatin remodelling gene ARID1A was altered across all subtypes (9%). Other mutated genes in LUAD were KRAS (31%), STK11 (22%), RBM10 (15%), EGFR (14%) and KEAP1 (14%); in LUSC were PTEN (26%), CDKN2A (21%), KEAP1 (21%) and NF1 (15%); in NET, non-carcinoid top mutated genes included RB1 (42%), ENPP2 (33%), ERBB4 and STK11 (17% for each), while ARID1A and ACKR3 were each present in 9.5 % of LC. In LUADs, mutations in EGFR and KRAS appeared as mutually-exclusive (P=0.007), while gene pairs NFE2L2 - AKT1 (P=0.012) and STK11- ALK (P=0.029) were co-mutated in LUAD and LUSC, respectively. Deletions in exons 19 and 20 of EGFR correlated with longer survival time compared to patients with wild-type EGFR (P=0.058). In NET, patients with mutated RB1 showed lower survival time compared to patients with wild-type RB1 (P=0.022). Examination of CNAs showed TERT amplifications (5p15.33 cytoband) were commonly found at high frequencies across all subtypes, especially in non-carcinoid NET (71.4%). Other recurrent CNAs included amplifications in MYC in 37% of LUAD and 40% of LUSC, and in EGFR in 33% of LUAD and 14% of LUSC. Deletions in the CDKN2A locus were seen at frequencies of 31% and 28% in LUAD and LUSC, respectively. LC patients showed longer survival time compared to other tumours (P=0.015). COSMIC mutational signatures 18 (of unknown aetiology) and 24 (associated with exposure to aflatoxin) were exclusively found in LC. The results confirm that lung cancer is a group of heterogenous diseases. In addition to the known effects of EFGR mutations, possible therapeutic avenues could be suggested for TERT amplifications, for which nucleoside analogues have shown to promote cancer cell death or EZH2 inhibitors for ARID1A-mutated cancers.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas

Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Epigenetic modifier gene mutations-positive AML patients with intermediate-risk karyotypes benefit from decitabine with CAG regimen.

It remains unclear whether there is a relationship between therapeutic effects of hypomethylating agents (HMAs) and epigenetic modifier gene mutations (EMMs) in patients with cytogenetically intermediate-risk acute myeloid leukemia (IR-AML). Based on targeted-capture sequencing, we retrospectively analyzed the correlation between EMMs and prognosis in 83 IR-AML patients treated with decitabine in combination with cytarabine, aclarubicin hydrochloride and granulocyte colony-stimulating factor (DCAG, n = 35) or "7 + 3" induction regimens (n = 48). In the multivariate analyses, EMM (+) patients did not show any statistically significant difference in remission rates from EMM (-) patients in the DCAG group (p > 0.05), but achieved inferior complete remission (CR; p = 0.03) and overall remission rates (ORR; p = 0.04) after the first course of standard induction regimens (p < 0.05). In the EMM (-) cohort, the DCAG group showed the tendency of adverse total CR (p = 0.06). Besides, DCAG group with EMMs achieved the best survival outcome independent of baseline characteristics, whereas it was opposite in EMM (+) patients receiving standard induction regimens (p < 0.05). Additionally, in the EMM (+) cohort, the survival rate of isolated DCAG group was statistically similar to that of the combination of standard chemotherapies and allogeneic hematopoietic stem cell transplantation (allo-HSCT) (p > 0.40), whereas patients who received only standard regimens had the worst survival rate (0.0%, p < 0.01). It can be concluded that the EMMs might be regarded as the potentially predictive biomarkers of better response to DCAG in IR-AML patients.

Putting the genome in insect phylogenomics

Next-generation sequencing technologies provide a substantial increase in the size of molecular phylogenetic datasets that can be obtained for studies of insect systematics. Several new genome reduction approaches are leveraging these technologies to generate large phylogenomic datasets: targeted amplicon sequencing, target capture, and transcriptome sequencing. Although cost effective, these approaches provide limited data for questions outside of phylogenetics. For many groups of insects, sequencing the entire genome at modest coverage is feasible. Using these genomic reads, an automated Target Restricted Assembly Method (aTRAM) can use the results of blast searches to assemble thousands of single copy ortholog genes across a group of interest. These locally assembled genes can then be compiled into very large phylogenomic datasets. These genomic libraries have the advantage in that they also contain reads from the mitochondrial genome and symbiont genomes, as well the entire insect genome, and can be leveraged for additional studies beyond phylogenetics.

Noninvasive prenatal detection of hemoglobin Bart hydrops fetalis via maternal plasma dispensed with parental haplotyping using the semiconductor sequencing platform

Thalassemia is one of the most common monogenetic diseases in the south of China and Southeast Asia. Hemoglobin Bart's hydrops fetalis syndrome was caused by a homozygous Southeast Asian deletion (-/-) in the HBA gene. Few studies have proved the potential of screen for Bart's hydrops fetalis using fetal cell-free DNA. However, the number of cases is still relatively small. Clinical trials of large samples would be needed. In this study, we aimed to develop a noninvasive method of target-captured sequencing and genotyping by the Bayesian method using cell-free fetal DNA to identify the fetal genotype in pregnant women who are at risk of having hemoglobin Bart hydrops fetalis in a large-scale study. In total, 192,173 couples from 30 hospitals were enrolled in our study and 878 couples were recruited, among whom both the pregnant women and their husbands were detected to be carriers of Southeast Asian type (-/αα) of α-thalassemia. Prenatal diagnosis was performed by chorionic villus sampling, amniocentesis, or cordocentesis using gap-polymerase chain reaction considered as the golden standard. As a result, we found that the sensitivity and specificity of our noninvasive method were 98.81% and 94.72%, respectively, in the training set as well as 100% and 99.31%, respectively, in the testing set. Moreover, our method could identify all of 885 maternal samples with the Southeast Asian carrier and 36 trisomy samples with 100% of sensitivity in T13, T18, and T21 and 99.89% (1 of 917) and 99.88% (1 of 888) of specificity in T18 and T21, respectively. Our method opens the possibility of early screening for maternal genotyping of α-thalassemia, fetal aneuploidies in chromosomes 13/18/21, and hemoglobin Bart hydrops fetalis detection in 1 tube of maternal plasma.

Abstract 4286: TGFβ induced SMAD4 dependent apoptosis proceeded by EMT in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading cause of cancer-related deaths Worldwide. In Saudi Arabia, CRC is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in initiation and progression of CRC is well documented. Current study examined the role of TGFβ/Smad4 signaling pathway in a large cohort of Saudi CRC, followed by in vitro analysis to dissect the dual role of TGFβ on inducing epithelial to mesenchymal transition (EMT) and apoptosis. In this study, we investigated Smad4 alterations and their association with clinicopathological outcomes in a large cohort of CRC samples using targeted capture sequencing, Fluorescent in-situ hybridisation and immunohistochemistry. Our study demonstrated high frequency of Smad4 alterations with low expression of Smad4 protein identifying a sub-group of aggressive CRC to be an independent marker for poor prognosis. Functional studies using CRC cells show that TGFβ induces Smad4 dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1 was essential for TGFβ-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in CRC cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4 alterations were found in the Saudi CRC patients. Functional study results indicate that TGFβ induces Smad4 dependent EMT followed by apoptosis in CRC cells. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Divya Sasidharan Padmaja, Sandeep Kumar Parvathareddy, Rong Bu, Tariq Masoodi, Yan Kong, Saravanan Thangavel, Saeeda Omer Ahmed, Nasser Al-Sanea, Luai H. Ashari, Alaa Abduljabbar, Samar Alhomoud, Fouad Al-Dayel, Khawla S. Al-Kuraya. TGFβ induced SMAD4 dependent apoptosis proceeded by EMT in colorectal cancer [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 4286.

Abstract 738: Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability

Abstract Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=5), whole-exome sequencing (WES) (n=44), targeted-capture sequencing (n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequence-based CN analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Among 84 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=64, 76.2%), RB1 (n=29, 34.5%), CDKN2A (n=25, 30.0%), and ATRX (n=18, 21.4%). A fusion gene involving TRIO was newly identified by RNA sequencing in a single case. A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%)) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in as many as 83.3% of the MFS cases (n=70). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. Finally, an analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of initial diagnosis, while most of the other lesions were subclonal and acquired during the clinical course. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Teppei Shimamura, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Tetsuichi Yoshizato, Kosuke Aoki, Yoichi Fujii, Yasuhito Nannya, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability [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 738.