Targeted Capture Sequencing Research Articles

Simulating the dynamics of targeted capture sequencing with CapSim.

MotivationTargeted sequencing using capture probes has become increasingly popular in clinical applications due to its scalability and cost-effectiveness. The approach also allows for higher sequencing coverage of the targeted regions resulting in better analysis statistical power. However, because of the dynamics of the hybridization process, it is difficult to evaluate the efficiency of the probe design prior to the experiments which are time consuming and costly.ResultsWe developed CapSim, a software package for simulation of targeted sequencing. Given a genome sequence and a set of probes, CapSim simulates the fragmentation, the dynamics of probe hybridization and the sequencing of the captured fragments on Illumina and PacBio sequencing platforms. The simulated data can be used for evaluating the performance of the analysis pipeline, as well as the efficiency of the probe design. Parameters of the various stages in the sequencing process can also be evaluated in order to optimize the experiments.Availability and implementationCapSim is publicly available under BSD license at https://github.com/Devika1/capsim.Supplementary information Supplementary data are available at Bioinformatics online.

Whole exome sequencing identified a pathogenic mutation in RYR2 in a Chinese family with unexplained sudden death

ObjectiveThis study aimed to identify the pathogenic mutation in a Chinese family with unexplained sudden death (USD) or occasional syncope. Materials and methodsWhole exome sequencing and target capture sequencing were respectively conducted for two related patients. The genetic data was screened using the 1000 genomes project and SNP database (PubMed), and the identified mutations were assessed for predicted pathogenicity using the SIFT and Polyphen-2 algorithms. ResultsWe identified a heterozygous mutation in the RYR2 gene at c.490C>T (p.P164S), highly conserved across all species, in three family members of USD, syncope and malignant ventricular tachycardias induced by treadmill exercise test, while another heterozygous de novo mutation in SCN5A at c.5576G>A p.R1859H was detected in one family member. Both variants were verified by Sanger sequencing. Importantly, RYR2 p.P164S is associated with the risk of sudden cardiac death, such as in catecholaminergic polymorphic ventricular tachycardia. ConclusionsA pathogenic mutation in RYR2 (p.P164S) is the likely cause of USD in a Chinese family associated with malignant ventricular arrhythmias. Whole exome and target capture sequencing can be useful for discovering the genetic causes of USD.

Repeat genetic testing with targeted capture sequencing in primary arrhythmia syndrome and cardiomyopathy.

In inherited primary arrhythmia syndromes (PAS) and cardiomyopathies (CMP), the yield of genetic testing varies between 20 and 75% in different diseases according to studies performed in the pre next-generation sequencing (NGS) era. It is unknown whether retesting historical negative samples with NGS techniques is worthwhile. Therefore, we assessed the value of NGS-based panel testing in previously genotype negative-phenotype positive probands. We selected 107 patients (47 PAS and 60 CMP) with a clear phenotype who remained genotype negative after genetic analysis of the main genes implicated in their specific phenotype. Targeted sequencing of the coding regions of 71 PAS- and CMP-related genes was performed. Variant interpretation and classification was done according to a cardiology-specific scoring algorithm ('Amsterdam criteria') and the ACMG-AMP criteria. Co-segregation analysis was performed when DNA and clinical data of family members were available. Finally, a genetic diagnosis could be established in 21 patients (20%), 5 PAS (11%) and 16 CMP (27%) patients, respectively. The increased detection rate was due to sequencing of novel genes in 52% of the cases and due to technical failures with the historical analysis in 48%. A total of 118 individuals were informed about their carrier state and either reassured or scheduled for proper follow-up. To conclude, genetic retesting in clinically overt PAS and CMP cases, who were genotype negative with older techniques, resulted in an additional genetic diagnosis in up to 20% of the cases. This clearly supports a policy for genetic retesting with NGS-based panels.

Next-Generation Sequencing-Aided Rapid Molecular Diagnosis of Occult Macular Dystrophy in a Chinese Family.

Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD) in a four-generation Chinese family with inherited macular dystrophy.Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Variants were filtered and analyzed to identify possible disease-causing variants before validation by Sanger sequencing.Results: Two heterozygous mutations—RP1L1 c.133 C > T (p.Arg45Trp), which is a hot spot for OMD, and ABCA4 c.6119 G > A (p.Arg2040Gln), which was identified in Stargardt’s disease were found in three patients, but neither of the mutations was found in the unaffected individuals in the same family, who are phenotypically normal or in the normal control volunteers.Conclusion: These results cannot only confirm the diagnosis of OMD in the proband, but also provide presymptomatic diagnosis of the proband’s children before the onset of visual acuity impairment and guidance regarding the prognosis and management of these patients. Heterozygous mutations of RP1L1 c.133 C > T (p.Arg45Trp) and ABCA4 c.6119 G > A (p.Arg2040Gln) are likely responsible for OMD. Our results further extend our current understanding of the genetic basis of OMD, and emphasize the importance of molecular diagnosis and genetic counseling for OMD.

Noninvasive prenatal diagnosis of 21-Hydroxylase deficiency using target capture sequencing of maternal plasma DNA

Here, we aimed to validate a noninvasive method using capture sequencing for prenatal diagnosis of congenital adrenal hyperplasia due to 21-Hydroxylase deficiency (21-OHD). Noninvasive prenatal diagnosis (NIPD) of 21-OHD was based on 14 plasma samples collected from 12 families, including four plasma sample collected during the first trimester. Targeted capture sequencing was performed using genomic DNA from the parents and child trios to determine the pathogenic and wild-type alleles associated with the haplotypes. Maternal plasma DNA was also sequenced to determine the fetal inheritance of the allele using hidden Markov model-based haplotype linkage analysis. The effect of fetal DNA fraction and sequencing depth on the accuracy of NIPD was investigated. The lower limit of fetal DNA fraction was 2% and the threshold mean sequence depth was 38, suggesting potential advantage if used in early gestation. The CYP21A2 genotype of the fetus was accurately determined in all the 14 plasma samples as early as day 1 and 8 weeks of gestation. Results suggest the accuracy and feasibility of NIPD of 21-OHD using a small target capture region with a low threshold for fetal DNA fraction and sequence depth. Our method is cost-effective and suggests diagnostic applications in clinical practice.

Evaluating allopolyploid origins in strawberries (Fragaria) using haplotypes generated from target capture sequencing

BackgroundHybridization is observed in many eukaryotic lineages and can lead to the formation of polyploid species. The study of hybridization and polyploidization faces challenges both in data generation and in accounting for population-level phenomena such as coalescence processes in phylogenetic analysis. Genus Fragaria is one example of a set of plant taxa in which a range of ploidy levels is observed across species, but phylogenetic origins are unknown.ResultsHere, using 20 diploid and polyploid Fragaria species, we combine approaches from NGS data analysis and phylogenetics to infer evolutionary origins of polyploid strawberries, taking into account coalescence processes. We generate haplotype sequences for 257 low-copy nuclear markers assembled from Illumina target capture sequence data. We then identify putative hybridization events by analyzing gene tree topologies, and further test predicted hybridizations in a coalescence framework. This approach confirms the allopolyploid ancestry of F. chiloensis and F. virginiana, and provides new allopolyploid ancestry hypotheses for F. iturupensis, F. moschata, and F. orientalis. Evidence of gene flow between diploids F. bucharica and F. vesca is also detected, suggesting that it might be appropriate to consider these groups as conspecifics.ConclusionsThis study is one of the first in which target capture sequencing followed by computational deconvolution of individual haplotypes is used for tracing origins of polyploid taxa. The study also provides new perspectives on the evolutionary history of Fragaria.

Efficiency of ddRAD target enriched sequencing across spiny rock lobster species (Palinuridae: Jasus)

Double digest restriction site-associated DNA sequencing (ddRADseq) and target capture sequencing methods are used to explore population and phylogenetic questions in non-model organisms. ddRADseq offers a simple and reliable protocol for population genomic studies, however it can result in a large amount of missing data due to allelic dropout. Target capture sequencing offers an opportunity to increase sequencing coverage with little missing data and consistent orthologous loci across samples, although this approach has generally been applied to conserved markers for deeper evolutionary questions. Here, we combine both methods to generate high quality sequencing data for population genomic studies of all marine lobster species from the genus Jasus. We designed probes based on ddRADseq libraries of two lobster species (Jasus edwardsii and Sagmariasus verreauxi) and evaluated the captured sequencing data in five other Jasus species. We validated 4,465 polymorphic loci amongst these species using a cost effective sequencing protocol, of which 1,730 were recovered from all species, and 4,026 were present in at least three species. The method was also successfully applied to DNA samples obtained from museum specimens. This data will be further used to assess spatial-temporal genetic variation in Jasus species found in the Southern Hemisphere.

Telomere length and somatic mutations in correlation with response to immunosuppressive treatment in aplastic anaemia

We investigated the frequencies of cytogenetic aberrations and somatic mutations of prognostic relevance in 393 patients with aplastic anaemia (AA). Clonality was determined by G-banding/fluorescence insitu hybridization (FISH) (n=245), and targeted capture sequencing was performed for 88 haematopoiesis-related genes (n=70). The telomere length (TL) of bone marrow nucleated cells was measured at the single cell level by FISH (n=135). Eighteen (4·6%) patients showed disease progression, and monosomy 7 (50·0%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (32·9%) presented at least 1 mutation; the most frequently mutated genes were NOTCH1, NF1, SCRIB, BCOR and DNMT3A. The patient group with clonal changes (30·7%) showed an adverse response to immunosuppressive treatment (IST), compared to the non-clonal group, but this finding did not show statistical significance. The TL of AA patients was significantly shorter than normal control and patients with clonal changes showed significantly shorter TLs. Patients with TL>5·9 showed a higher response rate to IST (P=0·048). In conclusion, the patients with clonal changes or TL attrition showed a poor response to IST. Shorter TL can be used not only as a biomarker, but also as a predictive marker for treatment response to IST.

Recurrent SPI1 (PU.1) fusions in high-risk pediatric T cell acute lymphoblastic leukemia.

The outcome of treatment-refractory and/or relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis for this is not well understood. Here we report comprehensive profiling of 121 cases of pediatric T-ALL using transcriptome and/or targeted capture sequencing, through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), accounting for 3.9% (7/181) of the examined pediatric T-ALL cases, showed a double-negative (DN; CD4-CD8-) or CD8+ single-positive (SP) phenotype and had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell precommitment, establishment of T cell identity, and post-β-selection maturation and with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and, when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in a maturation block. Our findings highlight a unique role of SPI1 fusions in high-risk pediatric T-ALL.

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

Abstract 2255: SMAD4 loss is associated with poor prognosis in Middle Eastern colorectal cancer

Abstract Background: Colorectal cancer (CRC) is major cause of morbidity and mortality in cancer patients worldwide and it ranks as the most common cancer in males and the third most common among females in Saudi Arabia. Alteration in TGF-β pathway has been reported in many cancers. So In search for new molecular targets we analyzed the role of SMAD4, critical TGF-β pathway mediator, in Saudi colorectal cancers Methods: 426 CRC cases were analyzed for SMAD4 mutations by targeted capture sequencing and protein expression status by immunohistochemistry in a tissue microarray format. Results were analyzed for association with any clinico-pathological parameters and for prognostic significance. Results: SMAD4 mutations were detected in 11.9 %( 50/418) cases of Saudi colorectal cancers. CRC with SMAD4 mutations were significantly associated with NRAS mutations (p=0.0481). SMAD4 complete loss of expression by immunohistochemistry was seen in 12.8% (52/404) cases and this loss was associated with young age (p=0.0236) and inversely with MSI-high tumors (p=0.0005). SMAD4 expression loss by immunohistochemistry and mutation were significantly associated with each other (p=0.0028). In the multivariate analysis using the Cox proportional hazard model SMAD4 expression loss was an independent marker of poor prognosis (p=0.0119) Conclusion: SMAD4 alteration was found to be associated with a poor overall survival in the Saudi colorectal cancer patients. With the advent of targeted anticancer therapy, this study confirms the findings of other investigators and highlights a potential target which can be exploited to improve the survival of CRC patients in the Kingdom of Saudi Arabia Citation Format: Maqbool Ahmed, Abdul K. Siraj, Tariq Masoodi, Rong Bu, Fouad Al-Dayel, Khawla S. Al-Kuraya. SMAD4 loss is associated with poor prognosis in Middle Eastern 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 2255. doi:10.1158/1538-7445.AM2017-2255

Abstract 4641: Wild type APC is an independent marker of poor prognosis in late stage colorectal cancer

Abstract Background: Colorectal cancer (CRC) is one of the commonest cancer worldwide and ranks as the most common cancer in males and the third most common among females in Saudi Arabia. Wnt pathway alteration is well known in colorectal cancer. In this study we aimed to comprehensively study Wnt pathway alterations in a large cohort of Middle Eastern colorectal patients and to know its role in this unique ethnic group. Methods: 426 CRC cases were analyzed for APC, AXIN2 and CTNNB1 somatic mutations by targeted capture sequencing and protein expression status by immunohistochemistry in tissue microarray format. Results were analyzed for association with any clinicopathological parameters and for prognostic significance. Results: The incidence of APC, AXIN2 and CTNNB1 mutations in our patient cohort were 58.2%, 4.2% and 3.8% respectively. APC mutation was associated with grade 2 tumors (p=0.0494) and male sex (p=0.0349). AXIN2 mutation were associated with grade 3 tumors (p=0.0231), MSI-high tumors (p<0.0001) and AXIN2 protein expression loss (p=0.0056). CTNNB1 mutations were associated with MSI-high tumors (p=0.0076). AXIN2 and CTNNB1 mutation were significantly associated with each other (p=0.0263). Wild type APC was associated with poor overall survival and was an independent marker of poor prognosis in late stage CRC (p=0.0200). Conclusion: We reported the incidence of Wnt pathway alterations in this unique ethnic group on which such data is lacking. APC mutation plays major and other genes minor role in Wnt pathway activation. Incidence of APC mutation in this group is comparatively high compared to reported incidence worldwide. Wild type APC independently predicts poor prognosis in Middle Eastern late stage colorectal patients. Citation Format: Abdul K. Siraj, Rong Bu, Shaham Beg, Tariq Masoodi, Khawla S. Al-Kuraya. Wild type APC is an independent marker of poor prognosis in late stage 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 4641. doi:10.1158/1538-7445.AM2017-4641

31 - Targeted-Capture Sequencing of the Immunoglobulin Locus to Detect V(D)J Rearrangements and Recurrent Illegitimate Translocations in Multiple Myeloma

31 - Targeted-Capture Sequencing of the Immunoglobulin Locus to Detect V(D)J Rearrangements and Recurrent Illegitimate Translocations in Multiple Myeloma

Abstract DPOC-014: BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT

Abstract PURPOSE: Women with endometriosis, a painful condition caused by displaced endometrial tissue, have a 3-fold increased risk of developing endometrioid ovarian cancer (ENOC) and clear cell ovarian cancer (CCOC). How two distinct cancers arise from the same precursor lesion is unknown. Sensitive biomarkers are needed to identify women with endometriosis at risk of developing cancer. We performed whole genome sequencing on 29 ENOC and 36 CCOC cases and observed a highly frequent insertion event originating from an active LINE-1 (L1) retrotransposon in the TTC28 gene. L1 retrotransposons are mobile genetic elements that can take downstream DNA pieces and insert them into random genomic locations in a process called 3' transduction. L1s are epigenetically silenced in normal tissues, but are known to become activated in a variety of cancers. A recent study showed a stepwise loss of methylation across various L1 loci between normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and ENOC/CCOC tissues. We hypothesize that TTC28 L1 retrotransposon is an early event in the transformation of endometriosis into ENOC and CCOC and such events could be used as biomarkers for endometriosis with high cancer risk. METHODS: We compared the presence of TTC28 L1 3' transductions to six SNVs and frame shifts mutations in normal, endometriosis, and tumor tissues from different anatomical sites in four ENOC and four CCOC cases. PCR followed by Sanger sequencing was used to detect TTC28 L1 insertions, and micro-fluidic PCR assay followed by MiSeq sequencing used to detect SNV/frameshift mutations. To broaden the analysis we will use a target capture sequencing method to track novel TTC28 and other L1 transductions. In these experiments probes tiling 1kbp downstream of L1s will be used to capture DNA fragments containing L1 transductions; the captured fragments will be sequenced on the MiSeq. We will assess the difference in TTC28 L1 methylation status between normal, endometriosis, and tumor tissues via the sequencing of bi-sulfite treated DNA. RESULTS: TTC28 L1 retrotransposition insertion is present at all 5 tumor sites in 75% (6/8) of cases, and is present in 3/5 or 4/5 tumor sites in the remaining cases. Analysis shows that TTC28 L1 insertion preceded some SNV and/or frameshift mutations. Preliminary results show that TTC28 L1 promoters are unmethylated in tumors with L1 insertions. Future experiments involving additional cases with endometriosis tissues will be performed. We expect to see L1 promoter hypomethylation and L1 transductions in endometriosis tissues. CONCLUSION: TTC28 L1 promoter hypomethylation and TTC28 L1 transductions may be early events in the transformation of endometriosis to cancer that can be explored as a method to predict tumor development. The development of a target capture assay to detect novel L1 transductions will be crucial for investigating cases without whole genome sequencing data. Ultimately, we hope to detect L1 insertions in plasma samples, and use L1 insertions as a biomarker to identify high-risk endometriosis cases. Citation Format: Zhouchunyang Xia, Dawn Cochrane, Michael S Anglesio, Tayyebeh M Nazeran, Janine Senz, Amy Lum, Ali Bashashati, Yi Kan Wang, Sohrab P Shah, David Huntsman. BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-014.

A Pilot Study of Noninvasive Prenatal Diagnosis of Alpha- and Beta-Thalassemia with Target Capture Sequencing of Cell-Free Fetal DNA in Maternal Blood.

Thalassemia is a dangerous hematolytic genetic disease. In south China, ∼24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia. Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm. The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region. Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.

Phase II study of afatinib in recurrent and/or metastatic esophageal squamous cell carcinoma (R/M ESCC) (KCSG HN14-18).

4051 Background: Afatinib, an irreversible pan-ErbB kinase inhibitor showed anti-tumor activity against esophageal cancer in phase I trial. In this multicenter, open-label, single arm phase II study, we aimed to evaluate the activity and safety of afatinib in R/M ESCC. Methods: Patients (pts) who had ECOG PS 0-2 and had progressed on platinum-based chemotherapy for R/M ESCC were enrolled. Pts were treated with afatinib 40mg/day until disease progression, unacceptable toxicity, or patient’s refusal. Primary endpoint was objective response rate (ORR) per RECIST 1.1. The estimated sample size was 49, using a two-stage minimax design to evaluate incremental response rate from 5 to 15%. Secondary endpoints included progression-free survival (PFS), overall survival (OS), disease control rate (DCR), and safety profile. Additionally, we try to identify biomarker to predict efficacy of afatinib with target capture sequencing and gene expression profile as exploratory endpoints. Results: In a total of 49 enrolled pts (median age 60; range 44 -84), ORR and DCR were 14.3 % and 73.3%, respectively. With a median follow-up of 6.6 months, median PFS and OS was 3.4 months (95% CI 2.2-4.6) and 6.6 months (95% CI 5.2-8.0). Median treatment duration and duration of response were 2.8 months (range, 0.4-15.3) and 7.1 months (range, 2.5-13.9), respectively. Dose reduction and interruption occurred in 19 (38.8%) and 15 (30.6 %) pts. Treatment-related adverse events (TRAE) occurred in 33 pts (67.3%) with most common TRAEs being diarrhea (n=22, 44.9%) and acneiform rash (n=12, 24.5%). G3-4 TRAEs were rare, occurring in 7 pts (14.3 %). Conclusions: Afatinib demonstrated modest efficacy with manageable toxicity in platinum-resistant R/M ESCC patients. Given the modest response rate, identification of predictive biomarkers is essential for further clinical investigation of afatinib in R/M ESCC. Those biomarkers are being analyzed and will be presented in the conference (NCT02353936). Clinical trial information: NCT02353936. [Table: see text]

Using circulating tumor DNA analysis to depict genomic profiles and predict survival outcomes in patients with small-cell lung cancer.

8567 Background: Small-cell lung cancer (SCLC) accounts for approximately 15% of lung cancers. Most patients have extensive-stage disease with widespread metastases and poor survival. Understanding the molecular mutation profile of each SCLC patient would allow precision treatment and improved clinical outcome. However, tumor tissues from surgery are not available for most SCLC patients and biopsy specimens are often have limited quantities. Several studies have provided evidence of circulating tumor DNA (ctDNA) in detecting somatic variants of multiple solid tumors. This study evaluated utility of ctDNA to depict genomic profiles and predict survival outcomes in SCLC patients. Methods: 22 Plasma samples were obtained before initial treatment from 22 patients with SCLC enrolled between 2012 and 2016. Targeted-capture deep sequencing was performed to identify somatic variants in 465 cancer-related genes. Genomic mutation profiles were described and the clinical implications were further analyzed. Results: Tumor DNA can be detected in all 22 plasma samples collected from patients with SCLC. In total, 340 variants were identified, and the mean and median mutation rate were 6.3 and 6.6 per Mb. TP53 and RB1 are the most frequently mutated genes, detected in 90.9% (20/22) and 59.1% (13/22) patients, respectively. Further analysis showed that high ctDNA fraction in cell-free DNA (cfDNA) was associated with heavy tumor burden (R = 0.7, p = 0.0017). Moreover, patients with high ctDNA fractions (ctDNA fraction > = 18.3%) had poor progression free survival (PFS) (HR, 17.2; p = 0.0019). The median PFS of patients with high versus low ctDNA fractions was 5.2 months (95% CI 4.6 to 5.8 months) versus 10.0 months (95% CI 9.3 to 10.7 months), respectively. Conclusions: In this study, ctDNA analysis offers a promising way to depict the molecular profile in patients with SCLC. Moreover, these findings highlight the potential clinical utility of ctDNA to predicate clinical outcome in SCLC.

Landscape of somatic mutations in different subtypes: Advanced breast cancer with circulating tumour DNA analysis.

e23039 Background: It is particularly important to provide precise therapies and understand tumor heterogeneity based on the molecular typing of gene expression. Yet the landscape of somatic mutations in different subtypes of advanced breast cancer (ABC) is largely undetermined. Methods: Overall, 100 blood samples were obtained from 100 advanced female breast cancer patients who underwent therapy at Cancer Hospital, Chinese Academy of Medical Sciences from March 2015 to September 2016. Mutations in 1021 tumor-related genes in ctDNA was assayed by gene-panel target-capture next-generation sequencing. Results: Somatic genomic alterations in ctDNA including copy number variants and point mutations were identified in 96 of 100 patients (96.0%). The number of somatic mutations varied markedly between individual patients (mean 2.9, range1-31). No difference was found between four subtypes for the number of somatic mutations. However, the mean number of somatic mutations was higher in age at 40-50 year than patients over age 60 ( p= 8.46 vs 3.88; p= 0.01). Results from multivariate analyses showed that the number of somatic mutations was increased with the number of endocrine therapy line ( p= 0.007). TP53 and PIK3CA were two most frequently mutated genes detected in ctDNA of 100 patients which were recurrently detected in 43 (43.0%) and 32 (32.0%) patients, respectively. ESR1/PIK3CA were more prevalent in HR+ cancers ( p= 0.007, 0.025 respectively). NOTCH1 are more frequently detected in HER2- group than in HER2+ patients (15.63% vs 2.94%; p= 0.033). In multiple logistic regression analysis indicated that pathological grade, tumour size at diagnosis and PR statue were positive associated with PIK3CA mutations. Multiple regression analysis also revealed that ki-67, metastatic at diagnosis, number of metastatic sites, number of endocrine line were associated with ESR1 mutations. Conclusions: The results revealed that different subtypes ABC have their own genetic alterations features. Certain gene mutations may be related to clinical treatment especially endocrine therapy.

Distinct patterns of somatic mutation clearance and association with clinical outcome in patients with AML.

7005 Background: Persistence of somatic mutations at the time of complete remission (CR) was associated with poor outcome in patients (pts) with AML. Methods: We studied 95 pts with AML who were treated with frontline induction and subsequently achieved CR. We sequenced pre-treatment and CR bone marrow samples by targeted capture sequencing of 295 genes (median 280x coverage). We defined 3 levels of mutation clearance (MC) based on variant allele frequency (VAF): MC2.5, persistent mutation with VAF<2.5%; MC1.0, persistent mutation with VAF<1%; and complete mutation clearance (CMC). Results: In the pre-treatment samples, we detected 597 mutations in 78 genes in 87 (92%) patients. In the matching CR samples, 62 (10%) and 82 (14%) mutations persisted at VAF≥2.5% and ≥1%, respectively, which corresponded to 43 (49%), 34 (39%), and 30 (34%) patients achieving MC2.5, MC1.0 and CMC, respectively. Table 1 shows the differential patterns of MC based on the mutations and pathways. Mutations associated with clonal hematopoiesis of indeterminate potential (CHIP), DNA methylation, and splicing pathways had low rate of MC, whereas mutations in transcription factors or receptor tyrosine kinase (RTK) had high rate of MC. Pts who achieved MC1.0 (median 31.2 vs. 12.5 months, P = 0.04) or CMC (median 31.2 vs. 12.5 months, P = 0.049) had significantly better relapse-free survival (RFS). Conclusions: Somatic mutations associated with CHIP, DNA methylation, and splicing pathways persisted frequently in CR samples suggesting preleukemic origin. Pts with deeper MC had significantly better RFS. Somatic mutation clearance may help risk prediction of AML. [Table: see text]

Genomic landscape of adult mixed phenotype acute leukemia (MPAL).

7023 Background: MPAL is a rare subgroup of acute leukemia characterized by both myeloid and lymphoid phenotypes. Genetic basis of MPAL is not well understood. Methods: We studied 31 patients (pts) with MPAL (median age 53) that met 2008 WHO criteria. Bone marrow samples were studied by targeted capture sequencing of 295 genes (median 393x), RNA sequencing, and Infinium methylation EPIC array (Illumina). Mutational landscape was compared to 194 AML, 71 B-ALL, and 6 T-ALL cases. Promoter methylation pattern was compared to the data from 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (Nordlund et al. Genome Biology. 2013). Results: Eighteen (58%) pts had myeloid-T and 13 (42%) had myeloid-B phenotype. Four pts had t(9;22), 1 had 11q23 rarrangement, and 8 had complex karyotype. MPAL had similar number of mutations with AML but had higher number of mutations than B-ALL or T-ALL. Both AML-type and ALL-type mutations were detected in MPAL, supporting the mixed phenotypic features. However, NPM1, CEBPA and GATA2 mutations were specific to AML and were not found in MPAL. Myeloid-T and myeloid-B showed distinct patterns of mutations, in which DNMT3A, IDH2, NOTCH1, IL7R, and FBXW7 mutations were enriched in myeloid-T whereas RUNX1 mutations were enriched in myeloid-B. Myeloid-T and myeloid-B also showed distinct patterns of promoter methylation. Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B. Genes that have essential role in T-cell receptor (TCR) pathway ( CD3D, CD7, CD247, LCK, PRKCQ, CCR9, and TCL1A) were differentially methylated and differentially expressed between myeloid-T and myeloid-B. RNA sequencing revealed several known translocations such as, NSD1-NUP98, and KMT2A-MLLT4, in addition to the novel fusion proteins such as FOXP1-DNAJC15, RUNX1-NAP1L1, and BCL2-TM9SF3. Unbiased hierarchical clustering of MPAL, AML, B-ALL and T-ALL by promoter methylation revealed that myeloid-T had consistent similarity with T-ALL, while myeloid-B showed random similarity with either B-ALL or AML. Conclusions: MPAL is genetically heterogeneous and myeloid-T and myeloid-B shows distinct patterns of mutations, methylation and gene expressions. Therapy for MPAL may need to be tailored based on the genetic profiles.